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Electron Microscopy:

A Handbook of Techniques for the Biologist

Microscopy: A Handbook of Techniques for the Biologist Preface Acknowledgments INTRODUCTION TO ELECTRON

Preface

Acknowledgments

INTRODUCTION TO ELECTRON MICROSCOPY

UNIT 1 - PREPARATION OF BIOLOGICAL SAMPLES FOR TEM Chapter 1 - Chemical Fixation Chapter 2 - Ultrathin Sectioning & Ultramicrotomy Chapter 3 - Post-Staining Chapter 4 - Grids & Grid Supports

UNIT 2 - PREPARATION OF BIOLOGICAL SAMPLES FOR SEM Chapter 5 - Hard Tissue Preparation Chapter 6 - Soft Tissue Preparation Chapter 7 - Alternative SEM Specimen Preparation

UNIT 3 - BLACK & WHITE PHOTOGRAPHIC PRINCIPLES Chapter 8 - Film and Paper Composition Chapter 9 - Processing (Films and Papers) Chapter 10 - Negative Handling and Exposure (TEM & SEM) Chapter 11 - Enlargement Printing

A Handbook of Techniques for the Biologist Stephen J. Beck Nassau Community College Electron Microscopy:

A Handbook of Techniques for the Biologist

Stephen J. Beck Nassau Community College

Electron

Microscopy:

A Handbook of Techniques for the Biologist Stephen J. Beck Nassau Community College Electron Microscopy:
Electron Microscopy: A Handbook of Techniques for the Biologist
Electron Microscopy: A Handbook of Techniques for the Biologist

Electron Microscopy:

A Handbook of Techniques for the Biologist

Electron Microscopy: A Handbook of Techniques for the Biologist
Electron Microscopy: A Handbook of Techniques for the Biologist

Electron Microscopy:

A Handbook of Techniques for the Biologist

Stephen J. Beck Nassau Community College

TABLE OF CONTENTS

CONTENTS

Preface

Acknowledgments

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INTRODUCTION TO ELECTRON MICROSCOPY

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UNIT 1 - PREPARATION OF BIOLOGICAL SAMPLES FOR TEM Chapter 1 - Chemical Fixation

Additives Fixation Times & Temperatures Preparation of Fixatives Methods of Fixation “Routine” Biological Soft Tissue Protocol Tissue Processing Note Embedding Media Fixation Schedule Fixation Schedule Worksheet Final Chemical Fixation Considerations

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Chapter 2 - Ultrathin Sectioning & Ultramicrotomy Processing of Embedded Blocks - Block Trimming Glass Knife Making Diamond Knives Ultramicrotomy MT-2B Ultramicrotome Sectioning Procedure Troubleshooting Guide to Ultramicrotomy Materials Required for Ultrathin Sectioning

Chapter 3 - Post-Staining Uranyl acetate Lead Citrate Post-staining Procedure

Chapter 4 - Grids & Grid Supports Grids Grid Supports Formvar Films Carbon Coating

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CONTENTS

UNIT 2 - PREPARATION OF BIOLOGICAL SAMPLES FOR SEM Chapter 5 - Hard Tissue Preparation

Chapter 6 - Soft Tissue Preparation Critical Point Dryer Operation Alternatives to Critical Point Drying Fluorocarbon Drying Organo-Silicon Compounds Conductive Coating Vacuum Evaporation Sputter Coater Denton Desk II Operation Comparison of TEM and SEM Soft Tissue Protocols Fixation Schedule Fixation Schedule Worksheet

Chapter 7 - Alternative SEM Specimen Preparation Uncoated Specimens Cryofracture Technique Microbial Specimen Preparation Microbial Fixation Schedule Worksheet

UNIT 3 - BLACK & WHITE PHOTOGRAPHIC PRINCIPLES

Chapter 8 - Film and Paper Composition Emulsion Film Speed (ISO/ASA) Supports (Base) Routine Photographic Films and Papers Used For TEM and SEM

Chapter 9 - Processing (Films and Papers) Development Stop Bath Fixer Washing Drying

Chapter 10 - Negative Handling and Exposure (TEM & SEM) TEM - Hitachi HS-8 HS-8 Camera System and Film Exposure SEM - Hitachi S-2400 S-2400 Camera System and Film Exposure

SEM - Hitachi S-2400 S-2400 Camera System and Film Exposure Chapter 11 - Enlargement Printing Photographic

Chapter 11 - Enlargement Printing Photographic Paper Grades Process of Enlargement Printing Enlargement Printing Variables Printing Tricks

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PREFACE

The development of electron optics has had a profound effect on the study of life; its structure and function. Without the high technology tools known as the transmission (TEM) and scanning (SEM) electron microscopes, we would understand little about the world of the cell. As proof, I offer any typical college level introductory and advanced biology course, the textbooks these courses utilize, and any number of biological periodicals/journals. Biology professors will lecture on a variety of cellular structures and their related functions. Textbooks at all levels are crammed with TEM and SEM photomicrographs in an effort to illustrate any number of rudimentary concepts. Even to this day, cutting edge research relies on high resolution images as can be seen in many biological publications. In today’s high technology environment, the tools of scientific inquiry are easily overlooked and often taken for granted. Of course, one must understand that these instruments are tools; a means to an end. It will require a scientist to make sense of the images presented, someone who understands how the images were produced, even to the point of anticipation of a given result.

The novice in electron optics must first learn technique and the theory behind it. The purpose of this handbook is to provide a detailed explanation and procedural guide to the many tedious procedures of biological electron microscopy, TEM and SEM. It can be used in the laboratory as a step by step guide and outside of the laboratory as the student attempts to comprehend the many concepts of biological electron optics. This handbook is intended for introductory college level courses in TEM and SEM. Depending on the college, this could mean the undergraduate (two and four year institutions) or even the graduate level. At Nassau Community College, the specific relevant courses are Transmission Electron Microscopy (BIO 221) and Scanning Electron Microscopy (BIO 222). In my ten years of teaching these courses at NCC, I have had the pleasure to introduce this valuable discipline to students with varied backgrounds and experiences, from traditional two year college students to those with earned Ph.D.’s. Regardless of prior education, students begin the EM courses at essentially the same level. This handbook was created to assist any individual in the attainment EM skills so that they will be able to utilize these important tools as they strive to answer the questions of life processes.

The book is divided into three main units; Unit 1 covers the many topics of TEM biological sample preparation and is an excellent starting point in your understanding of biological EM. Many later topics in the book refer back to concepts introduced in this unit. Unit 2 involves the preparation of biological samples for the SEM. Even if you are only taking a course in SEM, much of the chemistry of chemical fixation is found in Unit 1, therefore, the SEM student is urged to refer back to the pertinent points. At NCC the ideal course sequence begins with the TEM course and is followed by the SEM course, which is logic I have followed in organizing this handbook. The final Unit 3 covers the concepts of black and white photography relative to both TEM and SEM. Here you will find general concepts of silver based photography followed by specific treatments of both TEM and SEM photomicrography and image capture using the Hitachi HS-8 TEM and the Hitachi S-2400 SEM, both of which are easily related to other common instruments in use today.

As you progress in your education, I expect that these skills you will acquire and the discipline that it takes to master them will serve you well, whether you actually use electron microscopy in the future or not.

vii

S.J.B.

August, 1996

ACKNOWLEDGMENTS

I would like to thank a number of individuals who have helped to make this book possible. Firstly, my wife and lifelong partner Mary and my children, Jonathan, Bradley and Jessica, for allowing me the precious time to write the manuscript. To my parents, Jack and Mary for providing me with the opportunity for an education and serving as such positive role models.

In addition, I acknowledge the influence of my mentors, Dr. Kenneth Erb and Dr. Gary W. Grimes, both of Hofstra University. As my graduate advisor, Ken Erb guided my development as a scientist capable of conducting original research. I must confess that most of the information in this manual is taken from the electron microscopy experiences provided by Gary Grimes, a true expert and innovator in the field of electron microscopy.

Finally, I would like to thank any of the faculty of Nassau Community College who have supported my endeavors over the past ten years, especially Dr. Dudley Chin, who as biology department chair, always encouraged even my failed attempts. His vision has initiated the technological revolution in the department as we approach the turn of the century. In addition, I thank Dr. Baruch May and Dr. Patricia Cassin, who as co-authors of two successful NSF grants, have brought an awareness of technological innovation to the college and our students. I also acknowledge the 1993/94 NCC Sabbatical Committee members who approved the sabbatical which made it possible for me to write the bulk of this manuscript. I finally acknowledge the support and foresight of the NCC administration, President Sean Fanelli and Vice President Jack Ostling for their continuing support of electron optics and other high technology endeavors at the college.

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INTRODUCTION TO ELECTRON MICROSCOPY

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Introduction to Electron Microscopy

When Max Knoll and Ernst Ruska were designing the original Transmission Electron Microscope (TEM) in Germany in the late 1920’s, they envisioned no biological applications for their instrument, provided it would even function as theoretically conceived. Today, the impact of electron optics, Transmission (TEM), Scanning (SEM), and a variety of others, is apparent. Our ability to directly resolve the microanatomy of the cell using these instruments has revolutionized our very understanding of life and its requisite processes. We take it for granted when a classroom instructor or a textbook describes the cristae of a mitochondrion, the 9+2 ciliary microtubule arrangement, ribosomal subunits and the phospholipid bilayer of a unit membrane. Where many cellular processes have been elucidated using a biochemical “grind & spin” approach (cell homogenization and cell fractionation via ultracentrifugation) followed by characterization of bio-molecules by isolation and purification using techniques such as gel electrophoresis, the cellular biochemist will often finally desire an image to support their chemical findings. The old maxim applies here, “a picture is worth a thousand words”.

Even though the operating environment of the EM is a high vacuum (10 -5 Torr) and biological samples must be preserved or fixed for examination, we have a unique insight to the molecular architecture responsible for life sustaining functions through the high resolving power of these instruments. Since structure begets function, and the EM can produces images of fixed cellular ultrastructure, it follows that we can come to a better understanding of cellular physiology through such images/electron micrographs. Of course, it becomes vital to comprehend how electron images are produced and how samples are handled in order to derive valuable data from a micrograph. This handbook will provide the novice with the procedural and theoretical information they need in order to make sense of the final product - the electron photomicrograph.

The Transmission Electron Microscope (TEM), as the name implies, transmits a high energy electron beam through a specimen in a high vacuum environment. The vacuum environment is required to prevent electron interactions with air molecules which would serve to randomly scatter the electrons. In order for the electron beam to penetrate a sample, it must be extremely thin, approximately 600-900Å thick. Most samples will have to be sectioned using an instrument known as an ultramicrotome. Soft tissues will need to be mechanically strengthened by epoxy resin embedment to withstand the forces of cutting.

The TEM can be viewed as an inverted light microscope (LM), with the source at the top of the instrument. The source of the TEM is a high voltage gun (50,000 volts or higher) which contains a pointed tungsten hairpin filament across which the high potential (voltage) is applied. The filament is housed in a metal cylinder with a central aperture (circular opening). This cylinder is known as the Wehnelt cylinder or Bias Cap since it is held at a slightly negative potential (the “bias”) with respect to the filament. This causes initial electrostatic repulsion of electrons coming off of the filament and the saturation of the Wehnelt cylinder. An anode, also with a central aperture, is held at ground potential. Due

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

to the large potential difference between the cathode, now saturated with electrons, and the anode, the electrons are accelerated through the distance between these two points - this is known as the accelerating voltage. Wide-angle electrons will be grounded out and some will continue at high velocity through the anode aperture in order to contribute to image formation. The electrons will continue to the first lens in the TEM, the condenser. This lens will refract or bend the source electrons to the specimen. It should be noted that the lenses of an electron microscope are electromagnetic consisting of an iron shroud with a central bore and external copper windings. By varying the current through the copper windings, the focal point of the lens can be modified. This effect can allow for adjustments to brightness, magnification and focus.

Once the electrons are focussed by the condenser lens, they will encounter the ultrathin specimen which is mounted on a grid and placed in a finely machined mechanical stage (with fine micrometer X,Y movements). Based on the electron density of various regions of the sample, some electrons will be backscattered while others will continue to transmit through the specimen. These transmitted electrons will next encounter the short focal length objective lens assembly which serves as the main imaging lens in the TEM and is critical to final magnification and focus of the image. This region of the TEM also includes an electromagnetic stigmator which is designed to reshape objective lens asymmetry arising from imperfect lens bores and lens and aperture contamination. Astigmatism (stigma meaning spot) results in stretched images of poor resolution. The octupole (8-pin) stigmator reshapes lens asymmetry by creating an asymmetric elliptical field to counter the lens distortion. The electron beam finally passes through a magnifying/demagnifying intermediate lens and then a magnifying projector lens which also functions to project the final real image on a fluorescent viewing screen. Since humans are not sensitive to electrons, the fluorescent material of the view screen is necessary to form an image that we can see. Electrons which pass through the electron transparent regions of the specimen will strike the fluorescent material causing it to emit photons which our eyes are sensitive to. Such region will appear bright. Specimen regions which are stained (usually with heavy metals) are electron dense and will not allow the transmission of electrons. They will not come in contact with the fluorescent screen and these regions will appear dark. This provides the contrast vital to image formation. A piece of film can be introduced via a camera mechanism beneath the view screen and a permanent exposure recorded. This will be explained in Unit 3.

In the TEM, a direct image is formed by the differential absorption of a transmitted electron beam. Electron dense versus electron transparent specimen regions are ultimately responsible for contrast. Resolving power (RP), the ability to distinguish two points as two separate and distinct points, is based on the wavelength of the transmitted electron source. Resolving power and source wavelength are inversely proportional. As wavelength decreases, the resolving power increases as given by the Abbe equation:

0.61λ

RP = ———— n (sin α)

INTRODUCTION TO ELECTRON MICROSCOPY

3

where λ is the source wavelength, n is the refractive index of the medium through which the source passes and α is one-half the objective lens acceptance angle. The relation n (sin α) is also known as the numerical aperture (NA) of the lens and under most ideal situations is usually equal to approximately 1.0, making the numerator of the equation most significant. In simple terms, the resolving power is essentially equal to approximately one-half of the source wavelength. For visible light, the shortest violet range wavelength is on the order of 400nm (equivalent to 4,000Å or 0.4µm). Given the above equation, the highest resolving power attainable using visible light is about 0.2µm (bacterial cell range). Electron wavelength is based on the de Broglie relationship stated as follows:

h

λ = ————

mv

where λ is the source wavelength, h is Planck’s constant, m is the mass of the particle (such as an electron) and v is the velocity of the particle. Substituting electron mass and velocity at one-third the speed of light (achieved using a 50kv electron gun/accelerating voltage, the wavelength of the electron is approximately 0.05Å. Given the Abbe relationship, the theoretical resolving power of a 50kv TEM would therefore be 0.025Å. Unfortunately, due to lens aberrations (spherical aberration, chromatic aberration and astigmatism) which cannot be totally corrected for in an EM, the actual resolving power limit for a modern TEM is about 2Å - easily molecular resolution, approaching the atomic level (for example, the naked DNA double helix is 20Å in width). It becomes clear that the higher the accelerating voltage at the gun, the greater the electron velocity will be and the shorter the electron wavelength leading to a higher resolving power. Most modern TEM’s have maximum accelerating voltages exceeding 100kv.

In order to achieve the highest resolving power, a single constant electron wavelength is required otherwise, chromatic aberration will degrade resolution. Voltage stabilization circuitry is critical with the absence of a source spectrum and therefore, the absence of “color” images - unless one uses computer enhancements or Dr. Martin’s dyes applied directly to photographic prints.

In the year 1938, Knoll and von Ardenne constructed the first Scanning Electron Microscope (SEM) prototype. They suggested that secondary electrons could be collected from the tops of opaque surfaces, the resultant signal then amplified and used to modulate the grid of a cathode ray tube (CRT). It took many years of refinement to produce a commercial SEM (1963) - the Cambridge Stereoscan. By comparison, the first commercial TEM (1938) - the Siemens Elmiskop, was available soon after the initial TEM development.

At first glance, the SEM appears to have a simpler design than the TEM with a noticeably shorter column. The TEM and SEM have an identical electron gun/anode design, however,

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

the SEM maximum voltage is approximately 25-30kv when using a tungsten hairpin filament. What follows is simply a series of 2-3 condenser lenses which serve to demagnify the primary electron beam diameter. In the SEM, resolving power (~30-40Å) is dictated by the diameter of the primary electron beam which scans the specimen surface in a raster pattern, much like the electron gun(s) in your TV scan the phosphorus pixels (picture elements) coated on the inside of the picture tube, in order to form an image. The SEM scan generator is responsible for the raster scan of the sample surface by the primary electron beam.

When the primary electron beam contacts the sample surface, a variety of energetic phenomena arise which can be detected and collected (provided the SEM is outfitted with the appropriate detector). The most common type of energetic phenomenon emitted is the secondary electron signal. This secondary electron signal is collected to form the typical, virtually three-dimensional image that is usually encountered in textbooks and publications. Other types of signal include backscattered electrons (BSE), characteristic x-rays (which can be used to create an elemental surface map, and photons or cathodoluminescence. Once again, each require a specific detector which are easily added to a modern SEM.

The weakly negative secondary electrons are emitted after surface contact with the primary electron beam. These secondary electrons are collected by a scintillator- photomultiplier detector. The signal is then amplified and routed to the electron gun of a black & white viewing CRT. Whether the pixels remain dark or light up is related to the level of signal arising from a given specimen area. Maximum signal gives rise to a bright/ white pixel, whereas, minimum signal results in a dark/black pixel. The final result is contrast and a black and white image on the viewing CRT. The amount of signal emitted from the specimen surface is a function of its topography or relief. High points of relief, in direct line of sight with the detector and primary electron beam, will produce the maximum signal and appear brighter than low lying areas, which will appear dark. The final image is indirect, based on point by point differential contrast due to the yield of secondary electrons from the sample surface.

Magnification is a function of the length of a line scanned on the sample as compared with the length of a line scanned on the viewing or photo CRT. Since the CRT dimensions never change, a magnification increase is effected by simply modifying your scan generator control to scan a smaller area/shorter line on the sample. Samples are mounted on aluminum stubs which are placed into the finely machined stage of the SEM. The stage has provisions for movements in the X, Y, Z (also known as working distance) directions, including T (tilt) and R (continuous 360˚ rotation). A benefit of the SEM image is its high depth of field and focus which leads to the striking, almost three-dimensional images.

In addition to the components described above, a stigmator is required in the design of a SEM and is one of the most difficult adjustments to teach the novice. Once again, the X, Y stigmation is required to compensate for lens asymmetry which arises from lens imperfections and contamination. One must learn to see the image stretching as you go

INTRODUCTION TO ELECTRON MICROSCOPY

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through fine focus in order to correct it with the stigmator.

The following illustration compares the design of the LM, TEM and SEM:

Light Microscope (inverted)

illuminator/bulb

Light Microscope (inverted) illuminator/bulb Transmission Electron Microscope (TEM) source + - electron gun Scanning

Transmission Electron Microscope (TEM)

source

+

Transmission Electron Microscope (TEM) source + - electron gun Scanning Electron Microscope (SEM) + glass:

-

electron gun

Scanning Electron Microscope (SEM)

+

source + - electron gun Scanning Electron Microscope (SEM) + glass: fixed condenser lens Demagnifies

glass: fixed

glass: fixed condenser lens Demagnifies electromagnets: variable

condenser lens

Demagnifiesglass: fixed condenser lens electromagnets: variable

electromagnets:variable

variable

focal length

(focus source at specimen level)

TEM double condenser

beam from 50,000 Å to 100 Åfocal length (focus source at specimen level) TEM double condenser focal length via variation in lens

focal length via variation in lensfocal length (focus source at specimen level) TEM double condenser beam from 50,000 Å to 100

(focus source at specimen level) TEM double condenser beam from 50,000 Å to 100 Å focal
 

(increases brightness)

current

iris

iris aperture (reduces extraneous source) condenser aperture
iris aperture (reduces extraneous source) condenser aperture

aperture

(reduces extraneous source)

iris aperture (reduces extraneous source) condenser aperture

condenseraperture

aperture

diaphragm

First Condenser Lensbrightness) current iris aperture (reduces extraneous source) condenser aperture diaphragm camera

camera
camera
aperture diaphragm First Condenser Lens camera glass Second Condenser Lens specimen slide grid

glass

Second Condenser Lens

First Condenser Lens camera glass Second Condenser Lens specimen slide grid insertion mechanism
First Condenser Lens camera glass Second Condenser Lens specimen slide grid insertion mechanism
First Condenser Lens camera glass Second Condenser Lens specimen slide grid insertion mechanism

specimen

specimen slide grid insertion mechanism Magnification Control Third Condenser Lens objective aperture (TEM) (grounds

slide

grid

insertion mechanism

specimen slide grid insertion mechanism Magnification Control Third Condenser Lens objective aperture (TEM) (grounds
Magnification Control
Magnification Control

Magnification

Control

Magnification Control
Magnification Control
slide grid insertion mechanism Magnification Control Third Condenser Lens objective aperture (TEM) (grounds stray

Third Condenser Lensslide grid insertion mechanism Magnification Control objective aperture (TEM) (grounds stray electrons which

objective aperture (TEM)

(grounds stray electrons which increases contrast)

intermediate lens (1-3)

intermediate lens (1-3) Final Aperture
intermediate lens (1-3) Final Aperture
intermediate lens (1-3) Final Aperture
intermediate lens (1-3) Final Aperture

Final Aperture

(TEM only - magnify)

lens (1-3) Final Aperture (TEM only - magnify) Signal (secondary electrons)   projector lens

Signal (secondary electrons)

 

projector lens

  projector lens
  projector lens
  projector lens

ocular(s)

(magnify & focus final real image on retina of eye [LM] or fluorescent view screen [TEM])

Detector

eyes/retina

film
film

fluorescent screen (TEM)

(direct imaging)

camera

(to record permanent, high resolution image)

film
film
objective lens (magnify & focus)
objective lens (magnify & focus)

objective lens

(magnify & focus)

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

UNIT 1 - PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

The high vacuum environment (10 -5 Torr) of the transmission electron microscope presents a major and obvious obstacle for the study of biological samples. Since death is the inevitable result of introducing a living organism into a vacuum environment, we are limited to the examination of dead specimens. Another problem that prevents the study of live organisms is the requirement that samples be sectioned ultrathin. Unless samples are thin enough, between 600-900Å, the electron beam cannot transmit or pass through it. Once cut, a cell cannot typically be expected to live. Unfortunately, as a result of these two limitations, we are not able to study actual processes occurring directly within the cell, even though the TEM has the resolving power to do so.

Chapter 1 - Chemical Fixation

Chemical fixation involves killing and preserving the organism/organ/tissue/cell in as life- like conditions as possible. The biological structures and their functions are fixed or “frozen” in time and space. Our ability to observe fixed biological ultrastructure using the TEM allows us to infer and come to understand function. The true goal of fixation is to enable the investigator to examine the structure(s) they are interested in (to see what you want to see!).

Since we now understand that structure and not direct function is studied using the TEM, we must determine the major factors which influence cellular structure. Cellular organelles such as ribosomes, mitochondria, etc. are a variety of molecules arranged in a specific three-dimensional architecture, with the TEM capable of resolution at the molecular level. What is responsible for this cellular ultrastructure that we can distinguish with the TEM? Firstly, is the importance of the most abundant molecule in the living cell, water, and its effect on other cellular molecules. Water is a charged dipolar molecule. Being charged, it easily interacts with other charged molecules including the charged “R” groups of the protein’s amino acids (proteins being the second most abundant molecule of the cell). Polar water is incapable of interacting with neutral and non-polar molecules/groups which are designated as hydrophobic (“water fearing”). The charged groups are known as hydrophilic (“water loving”). The primary order of protein structure is its amino acid sequence which includes a number of both hydrophobic and hydrophilic units. Aside from many types of interactions between amino acids of the protein (hydrogen bonding, ionic interactions, disulfide bridges, etc.), the interaction of water with these hydrophobic and hydrophilic units is crucial to the higher order protein structure, meaning, how it folds into a three-dimensional protein structure. In this scenario, hydrophilic amino acids would be found to the outside of the protein molecule, while the hydrophobic units would be found at the center of the molecule, “hiding” from the external, much more numerous, water molecules. These interactions also explain the orientation of phospholipid molecules which makeup cellular membranes (phospholipid bilayer). Secondly, we must recognize that cellular physiology and metabolic processes are responsible for maintaining cellular conditions favorable to the continuity of molecular,

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

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hence, organelle structure. Examples of some conditions which must be maintained (homeostasis) would include temperature, pH, and osmolarity.

Any changes in these conditions during fixation could lead to structural distortions mainly through the process of denaturation. Denaturation, the alteration of the 3-D shape of a molecule (i.e. protein), must be considered when selecting fixatives for TEM specimen preparation. With the high resolving power of the TEM, any minor alteration in the three- dimensional conformation of molecules is undesirable and would lead to the formation of artifacts (structures not normally present in the cell which are produced by some external intervention or agent). You might ask what chemical fixatives are doing and what distinguishes a good fixative from a poor fixative, relative to TEM preparation of biological samples. The main requirements of a fixative are that it stabilizes cellular ultrastructure without inducing distortions/artifacts. In stabilizing cellular ultrastructure, the fixative must prevent an undesirable process known as autolysis (meaning self-dissolution/self-digestion) from occurring. When an organism dies, cellular lysosomes begin to burst open, releasing a flood of hydrolytic, digestive enzymes into the cell. These autolytic enzymes would obviously degrade the very cellular microanatomy which the TEM has the power to resolve. Since autolytic changes proceed rapidly after death, it becomes important to introduce the fixative to the tissue as soon after death as is possible, ideally within 30 minutes.

In summary, fixation must kill and simultaneously stabilize cellular components through the prevention of autolysis upon death of the organism (whether it be unicellular or multicellular). As noted earlier, it is critical that the fixatives do not denature cellular molecules as a result of their stabilizing components and preventing autolysis.

There are a variety of fixation methods that are utilized world-wide in order to kill and preserve living materials. Most of these methods are not suitable to the fixation of samples for TEM due to the structural distortions/denaturation which result. A list of these methods is presented below with an explanation of their value or uselessness to TEM fixation. It should be pointed out that each method is capable of halting autolysis associated with the death of the organism. This fact qualifies each as a preservative.

Air Drying This method is employed for the preservation of harvested grains (wheat, etc.). Drying eliminates the water necessary for autolytic enzyme shape, and therefore, activity. Since all cellular proteins are denatured, this method is poor for TEM fixation.

Pickling The pickling process is used to preserve cucumbers (pickles), beets, etc. Preservation is made possible through a change in pH which denatures autolytic and other cellular proteins. The pH is usually lowered (acidic) through the introduction of acetic acid (vinegar). Due to denaturation, this method is also poor for TEM

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

fixation.

Alcohols Alcohols, such as ethanol (CH 3 CH 2 OH) generally make good fixatives since they are all dehydrating agents. Removal of water denatures autolytic enzymes along with other cellular proteins making alcohols unsuitable for TEM fixation if used alone. In TEM (and SEM) fixation, dehydration is an important step in the protocol, however, this process is carried out only after the tissues have already been stabilized with the primary and secondary fixing agents.

Heat The addition of heat energy causes the breakage of numerous bonds which are responsible for the 3-D conformation of cellular proteins, including the autolytic enzymes. The enzymes are denatured and the organism is preserved. Heat is used in the canning process. An example would include the cooking of vegetables prior to vacuum packing in the cans. Due to the resulting denaturation caused by heat, TEM samples cannot be simply “cooked” in order to preserve cellular ultrastructure.

Oxidizers/Precipitants Powerful oxidizing agents such as Osmium Tetroxide (OsO 4 ) and Potassium Permanganate (KMnO 4 ) were the first solitary fixatives used for TEM biological sample preparation. Both react at the double bonds present within unsaturated lipids, such as those found in abundance in the composition of biological phospholipid membranes. In addition, and as a result of their reaction with unsaturated lipids, they introduce an electron dense, heavy metal (Os/Mn precipitate) to those redox reaction sites. This resultant staining enhances contrast of TEM samples. It should be noted that OsO 4 was the primary and only TEM fixative prior to 1963. Since that time it has been discovered that these violent oxidizers can destroy delicate, labile (changeable) cellular structures such as microtubules. In 1963 a different primary fixative was proposed for TEM samples which eliminated such redox reaction damage, of especially, proteinaceous components. KMnO 4 is such a powerful oxidizer that all of the internal cellular cytoplasmic components are destroyed, with the exception of the membrane systems. If your goal is the study of a particular membrane system, you might wish to try KMnO 4 in conjunction with other protocols. Currently, OsO 4 is used as a secondary or postfixative due to its ability to react with and stain membranes of the cell. It may also react with and stain some protein elements of the cell which have been previously stabilized using aldehydes.

CAUTION: Osmium tetroxide is a highly reactive and potent fixative. The vapors alone can fix exposed epithelial surfaces of the mouth, nose, and especially, eyes (cornea) resulting in temporary blindness. Crystalline or in aqueous or buffer solution, osmium tetroxide should be handled with extreme care. Gas tight goggles, double gloves and a fume hood capable of 150 cfm flow

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

9

rate are the minimum safety recommendations. Polyunsaturated oil, such as corn oil, should be at hand in the case of a spill. It has been advised that approximately twice the volume of oil be added to an osmium tetroxide spill in order to neutralize it. If spilled in the open, the area should be vacated immediately and the proper authorities notified. No one should be allowed to reenter without a proper respirator. Waste osmium tetroxide should not be put down the sink but rather, stored in a clearly labeled waste bottle for later disposal by environmental carting firms.

Aldehydes Aldehydes, containing the reactive carbonyl group (C=O), have been used for years in the preservation of biological specimens (embalming, light level histology, etc.). It is somewhat surprising that they were not considered for TEM fixation prior to 1963 when Sabatini, Bensch and Barrnett introduced them as the primary fixative in TEM sample preparation. The role of aldehydes in fixation is their stabilization of the cellular protein matrix into a somewhat gelatinous state. The carbonyl groups react with any reactive amino acid “R” group which in turn results in the methylation (-CH 3 ) of the protein molecules. Neutral methyl groups interact between adjacent protein molecules resulting in velcro-like linkages and stabilization. Since the aldehydes do not denature the proteins, their active sites remain intact and histochemical and immunocytochemical (ICC) localization of proteins/enzymes can be performed. The fact that they do not denature make them ideal primary fixatives for TEM biological samples. The most common types of aldehydes used in TEM fixation are formaldehyde (HCHO), acrolein/acrylic aldehyde (CH 2 • CHCHO) and the dialdehyde known as glutaraldehyde (CHO-(CH 2 ) 3 -CHO). While both formaldehyde and acrolein are smaller molecules which diffuse more rapidly into tissue blocks, glutaraldehyde, the dialdehyde, is doubly reactive and allows for increased stability of the cellular protein matrix through the cross-linking of protein molecules. Although glutaraldehyde is the most commonly used single aldehyde fixative, an examination of the literature often reveals the use of a combination aldehyde primary fixative. In this case, glutaraldehyde is usually used in combination with one or both of the other aldehydes mentioned above. Conventional formalin solutions are not suitable for TEM fixation. Since formaldehyde is a gas in nature, it must be prepared as an aqueous solution. Through reaction of formaldehyde with water, formic acid formation results. This lowers the pH and serves to denature proteins. In addition, formalin contains methanol which is a dehydrating agent and also denatures. Paraformaldehyde, a purified crystalline trimer of formaldehyde, is the compound of choice for TEM fixation. Under low heat for 30min duration, paraformaldehyde goes into aqueous solution without formic acid formation and without the addition of methanol. Although acrolein is an excellent primary aldehyde fixative, it is difficult to work with since it is highly explosive and a potent lachrymator (tear gas).

10

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

CAUTION: As fixatives, all aldehydes should be handled with gloves, goggles and under a fume hood, especially acrolein since it is a lachrymator. Acrolein is also highly explosive and should be kept away from direct light, heat and flames. All used aldehydes should be stored in clearly labeled organic waste bottles for later safe disposal. Never put toxins down the drain!!

Additives

In the literature, you will notice that most fixatives are carried in a vehicle known as a buffer and may also include other additives such as salts (CaCl 2 ) or even sucrose. The main purpose of these additives is two-fold. Firstly, the buffer is important to the maintenance of the natural physiological pH of the tissue being fixed. Buffer solutions can react with and counteract the release of excess H + and/or OH - ions from the tissue being fixed. As discussed earlier, a change in pH would denature and is therefore not desired. Many types of buffers have been used in TEM fixation including veronal-acetate, PIPES, chromate, phosphate and cacodylate. Even though it has a short shelf life due to the eventual growth of bacteria, phosphate buffers are a good choice since they are non-toxic and therefore, easy to work with out in the open. Once prepared (there are many recipes such as Sorensen’s), phosphate buffers should be refrigerated at 4˚C to reduce bacterial growth. Cacodylate buffer should be handled with care under a fume hood since it contains arsenic. Fixatives are typically made up in the buffer solutions just prior to use. Buffers should be adjusted to the physiological pH levels of the organism’s internal (or external - as in the case of unicellular protozoa) environment. Mammals and a number of other animals range between pH values of 7.2-7.5 with botanical samples approximately 6.8.

The use of additional salts and/or inert substances such as sucrose are for osmolarity/ tonicity considerations. The fixative should be isotonic with the internal/external fluid environment of the tissue under study. Although trial and error is a common practice in determining the proper osmolarity, reference sources that list the osmolarity of specific animal blood/body interstitial fluids are available. Some labs also have access to an osmometer in order to determine precise tonicity requirements for the fixative vehicle. Another additive involves the use of dimethyl sulfoxide (DMSO) which is a penetrant used to enhance infiltration of fixatives into the tissue block.

Fixation Times & Temperatures

In the EM literature, you will find a wide range of fixation times and temperatures used. Who is correct? Can they all be right? Perhaps. One must consider the quality of the final micrograph that is being produced. Relative to the duration of each step in a TEM fixation protocol, there are optimum times of exposure and times which work around technician schedules for the sake of convenience. In the specific fixation of soft mammalian tissues which follows, the indicated times are optimal for ideal preservation with the minimum of

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

11

tissue component extraction and artifact production. In the literature you may see primary fixation in glutaraldehyde for 1hr to overnight. Under ideal circumstances, fixation through the tissue block will occur within 1hr, provided it is small enough in dimension (0.5 cu.mm.). Optimal times should be used whenever possible and practical.

There are two schools of thought on the proper temperature of fixation. One suggests that fixation should be conducted in the cold, on ice, at 4˚C to slow autolytic enzyme activity and reduce extraction of cellular components. The other suggests that room temperature fixation hastens fixative infiltration and the actual biochemical process of fixation. In the cold, we are actually slowing the desired process and in some cases, preventing it (if fix time is inadequate) which can lead to the production of artifacts. The best recommendation is to begin the process/protocol at 4˚C and allow it to come to room temperature (probably by the time you reach the ethanol dehydration series).

Preparation of Fixatives

Phosphate Buffers: Used as a vehicle to carry the various fixatives and as a wash. The solution is prepared using monosodium (NaH 2 PO 4 ) and disodium (Na 2 HPO 4 ) phosphate. Solutions are usually prepared with molarities that are consistent with the tonicity of the sample, such as 0.02M, 0.05M, 0.1M, 0.2M, 0.5M. The molecular weight in grams should be added to one liter of distilled water to yield a 1.0M solution. Simple calculations can be performed to reduce the molarity from 1.0M and to prepare 100ml of solution vs. 1,000ml (divide by 10). Take note of whether the sodium phosphate is hydrated since the addition of one or more water molecules will increase the gram molecular weight. By way of example, to prepare a 1.0M solution of monohydrated monosodium phosphate (NaH 2 PO 4 • H 2 O), the gram molecular weight, 138g, is added to 1,000ml of distilled water. A 0.1M solution would be prepared by adding only 13.8g to 1,000ml of DH 2 O. To prepare 100ml of the 0.1M solution, only 1.38g would be added to 100ml of DH 2 O. In order to adjust the pH of the buffer solution, the proportions of monosodium relative to disodium phosphate must be varied according to the chart below.

pH

6.0

6.2

6.4

6.8

7.0

7.2

7.4

7.6

7.8

8.0

NaH 2 PO 4 (in ml)

87.7

81.6

73.5

51.0

39.0

28.0

19.0

13.0

8.5

5.3

Na 2 HPO 4 (in ml)

12.3

18.4

26.5

49.0

61.0

72.0

81.0

87.0

91.5

94.7

Phosphate buffers should be freshly prepared and stored in the refrigerator at 4˚C to reduce the growth of bacteria. The pH should be checked with a pH meter and adjusted as necessary.

Buffer solution(s) should obviously be prepared first, in advance of any fixative since the fixatives are mixed with the buffer.

12

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Glutaraldehyde: This primary fixative is available in bottles in aqueous solutions of

25% and 50% and in ampoules sealed under nitrogen gas in 8% aqueous solution. Due to polymerization of the glutaraldehyde in high concentrations, the 8% sealed ampoules afford increased shelf life. Whatever concentration is used, it must be added to an appropriate amount of buffer solution to yield the final concentration (approximately 3% is ideal for most purposes). For example, to prepare 3.2% glutaraldehyde in buffer, 20ml of 8% glutaraldehyde is added to 30ml of phosphate buffer. The buffered glutaraldehyde should be made just before use and stored in the refrigerator in the dark.

Osmium Tetroxide: OsO 4 is available as crystals in 0.5g and 1.0g quantities, in sealed ampoules, or in aqueous solution (such as 2% and 4%) in sealed ampoules. Crystalline OsO 4 is ideal for making larger quantities (50-100ml) of the working solution which is usually set at 1-2%. A meticulously cleaned and dry ground glass stopper bottle with a teflon seal should be used to prepare the working solution. Since crystals of OsO 4 are large and dissolve slowly, it is recommended that the ampoules be held under running hot water which allows the crystals to melt. The ampoule is then gently rolled between the gloved hands to permit recrystallization of the OsO 4 in a thin layer inside the ampoule. The ampoule is inserted into the ground glass bottle and the stopper introduced. The bottle is shaken which causes the ampoule to break inside the bottle (Note: EM supply companies currently use pre-scored ampoules, some in conjunction with a plastic external cylinder to prevent a cutting injury. The ampoule should be opened, placed in the bottle, and the ampoule filled with buffer using a pipette. The bottle is then filled with the remaining buffer. It is important that the ampoule sink to the bottom of the bottle in order for the OsO 4 to go into solution). Once the ampoule is broken, the appropriate amount of buffer is added to yield the final working solution. To prepare a 1% OsO 4 solution, use 1.0g of crystalline OsO 4 in 100ml of buffer (or 0.5g in 50ml of buffer). When using the solution, be careful not to pipette near the bottom of the bottle since you may pick up some glass shards and introduce them to your tissue samples causing physical/mechanical damage. Once again, it must be cautioned that osmium tetroxide is extremely toxic and should be handled under the fume hood wearing gas tight goggles and double gloves. Cooking oil/ corn oil should also be available for possible spills. As with all fixatives, OsO 4 is prepared just before use. It is ideal to prepare the working solution one day before use and leave it out at room temperature overnight. After use, the solution should be stored in the refrigerator. It should be tightly stoppered and placed inside another container of glass or metal to prevent escape of vapors which will also react with and blacken the inside of the refrigerator.

Ethanol Dehydration Series: For TEM specimen dehydration, 70%, 95% and 100%

ethanol are required. Ethanol concentrations of 95% and 100% are available. It is ideal to maintain the volume of absolute (100%) ethanol since air in the storage vessel contains water vapor which mixes with the ethanol and reduces the concentration. Small pint bottles are available for one time use since it is imperative that no water remain in the tissues. Another storage technique involves standing the 100% ethanol over a layer of anhydrous cupric sulfate which serves to absorb water. It should be changed when it begins to turn blue which indicates that it is hydrated. Do not mix up the cupric sulfate

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and EtOH prior to use. The cupric sulfate must be allowed to settle on the bottom of the container.

In order to prepare ethanol dilutions of less than 95%, one should not use the more expensive 100% ethanol. In this case, 95% ethanol is used as follows:

1. The amount of 95% ethanol in ml, equal to the desired final concentration, is measured

out (for example, a 30% EtOH solution begins by measuring out 30ml of 95% EtOH).

2. Distilled water is added to make up a total of 95ml of solution (to complete the above

30% EtOH solution, 65ml of DH 2 O is added to the 30ml of 95% EtOH - total volume is

95ml).

Ethanol solutions can be refrigerated in the dark until ready for use.

Propylene Oxide: Propylene oxide is available from EM supply companies usually in 250ml quantities. It should be used undiluted. The addition of water would defeat the purpose of the prior ethanol dehydration series and prevent the infiltration of epoxy resin into the tissues. It is important to know that propylene oxide is extremely volatile. If you are not careful in the solution exchange process, your tissues will dry down leading to artifacts.

Propylene oxide (1,2 epoxy propane) is a small, rapidly diffusing, molecule which introduces the epoxy monomer into the tissues thus aiding infiltration. It serves as a transitional solvent as it is miscible with both ethanol and the mixed, unpolymerized epoxy resin.

CAUTION: Propylene oxide is extremely flammable and should be stored in a cool dark environment. Never expose it to direct heat or flame. It should be handled with gloves, goggles and under a fume hood since it is a suspected carcinogen.

Epoxy Resins: The use of epoxy resins is ideal for the embedment of biological samples for ultrathin sectioning for the TEM. Due to its three-dimensional polymerization, samples are provided with excellent mechanical strength capable of tolerating the forces of sectioning. The preparation of epoxy resins involves mixing the epoxy compound(s) such as “Epon” 812 and/or Araldite 6005, with specific curing agents including acid anhydrides (DDSA) and tertiary amines (DMP-30). Additionally, depending on the epoxy resin used, a plasticizer such as dibutyl phthalate (DBP) may be necessary. It is critical that all components be thoroughly mixed. The Epon-Araldite mixture used below has proven excellent for the embedment of soft mammalian tissue samples. The stock solution and the final working solution should be hand mixed for at least 15 minutes each. The stock solution can be frozen for future use. A plastic syringe can be used to store the stock solutions in exact volumes and with minimal trapped air which could lead to condensed water vapor contamination of the stock solution as it is allowed to come to room temperature. Stock solutions must be allowed to come to room temperature prior to their use in the preparation of the working solution.

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Mixing of epoxy resins introduce tremendous amounts of air which should ideally be removed. Air molecules present in the resin mixture would prevent proper infiltration of the resin into the tissues. Due to the viscosity of the mixed resin, outgassing through standing at atmospheric pressure would be a lengthy and incomplete process. Therefore, mixed resin should be put under a low vacuum environment in order to remove air. Care should be used in the rate of achievement of vacuum since the resin mixture will overflow the container, carried out by air bubbles, and contaminate the bell jar. The vacuum should be achieved slowly by regulating the air inlet valve.

Contamination of the work area with unpolymerized resin should be a definite concern. Be careful not to touch doorknobs, refrigerator handles, etc. with contaminated gloves. Never pour unpolymerized resins into the sink. Resins should be mixed in disposable plastic beakers using glass rods. Excess resins should be polymerized which renders them safe for disposal.

CAUTION: Epoxy resins and curing agents can cause contact dermatitis and may be carcinogenic. All resin components should be handled in a fume hood and with double gloves (immediately discard the outer pair if they become contaminated). When outgassing mixed resins, the low vacuum setup should be put in a fume hood. Ovens for polymerization of resins should be vented to the outside or placed in a fume hood.

Although the Epon-Araldite mixture below is prepared volumetrically, the most accurate way to measure it is by weight. Due to the viscosity of the resins, a quantity is certain to remain behind in the serological pipette used to measure it.

Epon-Araldite Mixture

Stock Solution (can be frozen)

 

Small Volume

Large Volume

Araldite 6005

12.5ml

25ml

Poly/Bed 812

15.5ml

31ml

Dibutyl phthalate

2ml

4ml

Final Working Solution

Stock Solution

4ml

8ml

DDSA

10ml

20ml

DMP-30

14 drops*

28 drops*

(*Drops introduced with a Pasteur Pipette)

Another useful formulation is an Epon mixture ideal for embedding directly in plastic or glass containers such as tissue culture plates, petri dishes, microfuge tubes, etc. This

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15

mixture introduces nadic methyl anhydride (NMA) in addition to DDSA. Note the slight difference in the volume of NMA used in conjunction with original Epon 812 resin vs. the “812” resins available today from EM supply houses (Poly/Bed 812-Polysciences, Inc., EM Bed 812-EMS, Inc., etc.)

Epon for Glass/Container Work

 

Component

Volume using Epon 812

Volume using “812” substitute

Epon 812 or 812 substitute

12.5ml

same

DDSA

6.5ml

same

NMA

6.25ml

9.0ml

DMP-30

1.0ml

same

DBP

0.25ml

same

Mixed resins will polymerize at 60˚C for 48 hours. If heat is a concern, epoxy resins will also cure under ultra-violet (UV) light.

Methods of Fixation

Immersion: In this type of fixation, the tissue of interest is removed/excised from the organism (or if small enough, the entire organism) and placed into the primary buffered fixative such as glutaraldehyde. The buffered primary fixative can be put into petri dishes or as large drops on a card of dental wax. When a complex animal (mammal, etc.) is used, it must be sacrificed quickly, followed by opening the body cavity, identifying the organ(s)/ tissue(s) of interest, and rapidly excising them using a razor blade or scalpel. Once the tissues are transferred to the fixative, they must be minced to blocks which are less than 1.0mm 3 in dimension (0.5mm 3 is optimal) using clean single-edged razor blades. Mincing should be done with care since mishandling will result in mechanical damage artifacts. Artifacts will also arise if the tissue pieces are allowed to dry. It is imperative that they be kept submerged in the fixative solution at all times. Once minced, the tissues are transferred to small vials containing the primary fixative and allowed to remain for the allotted time (usually about one hour). All subsequent steps in the protocol (with the exception of epoxy resin embedding) can be carried out in these vials through decanting the current solution and quickly, yet carefully, introducing the next solution in the protocol.

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

In-situ: In-situ fixation, meaning ‘in the original place’, is accomplished by bringing/

applying the primary fixative to the specimen of interest. By example, fixation of a thin plant leaf is conducted by placing a ring of petroleum jelly on the leaf surface and filling the ring with the primary fixative. Once fixed, the area of interest is cut out, minced and placed into vials to continue with the protocol. Another example involve the fixation of tissue culture cells. The fixatives are simply administered to the culture dishes through pouring them on and off of the cellular monolayer. In the animal fixation described above under immersion fixation, once the animal is humanely sacrificed and the body cavity cut open, primary fixative should be poured into the cavity in order to initiate fixation.

Vascular Perfusion: This technique uses the vascular system of an animal to deliver

the primary fixative deep into the tissues and directly on target. This is probably the most optimal means for administering the fixative. Delicate tissues subject to rapid post- mortem change must be fixed in this manner. A good example would be nervous tissues. In this procedure, the animal is anesthetized and the heart and major blood vessels exposed. Typically, a canula is inserted into the aorta and a balanced saline solution is allowed to gravity feed into the blood vessel. Eventually, the saline solution is cut off by a clamp and the fixative is allowed to flow into the vascular system. The tissues of interest can be later excised, minced, and placed into vials in order to continue the process.

Fixation by Vapors: Small delicate surfaces, such as membranes, may be fixed in this

manner. The specimens are simply suspended over a solution of osmium tetroxide, usually overnight. The samples will blacken to indicate that fixation has occurred. Samples can be dehydrated and embedded in resin for ultrathin sectioning.

In our lab, we use a combination of in-situ and immersion fixation of biological samples as described above. Once again, care and common sense should be used in the handling of all fixatives!

“Routine” Biological Soft Tissue Protocol

In fact, no single “routine” protocol for the fixation of soft biological tissues exists. There are as many protocols as there are investigators, with no one better than the other. As long as your protocol enables you to observe the cellular features that you are interested in, it is valid. Of course, protocols will vary based on the particular organism being studied. It is apparent that botanical samples should be fixed using different protocols than animal samples, however, protocols will vary even among related organisms. By way of example, metazoans of different classes, orders and even genera will have varied physiologies which give rise to a wide range of body/interstitial fluid characteristics. As discussed earlier, in order to prevent artifacts, the buffer pH and tonicity would have to be considered for each animal investigated. In addition, conditions of fixation should be considered. Are you fixing under ideal laboratory conditions or are you in a tropical field setting? How much time will you have to mince the tissues after excision? You may have to store tissues in tropical climates for days to weeks without the ability to precisely mince the tissue blocks until you return to the lab. Even under these unfavorable conditions, you

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

17

can modify your fixation protocol to yield quality results. In a study of neo-tropical bats, Nagato, Tandler and Phillips used a trialdehyde-DMSO primary fixative which consisted of 1% glutaraldehyde, 1% paraformaldehyde, 0.5% acrolein, 2.5% dimethyl sulfoxide, and 1

mM CaCl 2 in 0.05 M cacodylate buffer (pH 7.2). The tissue was stored for 14 days in

tropical conditions before being transferred to 3% glutaraldehyde at 4˚C. Given the

circumstances, the resulting TEM micrographs were of extremely good quality.

The protocol which shall now be outlined relates primarily to soft mammalian tissue

samples prepared under ideal laboratory conditions. The solutions are initially at 4˚C and are allowed to come to room temperature. The steps in this “routine” protocol are as follows:

Primary Aldehyde Fixation - used singly, buffered glutaraldehyde (usually 3-4%) is

the most common choice for TEM fixation of biological tissues. Using methods of fixation described earlier, the primary fixative is delivered to the tissue of interest. In this lab, the most common method employed for soft mammalian tissues is in-situ followed by immersion fixation in petri dishes. The tissue is minced carefully using two clean single- edged razor blades into pieces no larger than 3.0 cubic mm, with the ideal tissue block size no larger that 1.0 cubic mm. If you wish to complete mincing in the primary fixative, you

will need to reduce the tissue blocks to 0.5 cubic mm, however, this can wait until the final

buffer wash just prior to osmium tetroxide (OsO 4 does not penetrate as well as the aldehydes). Once minced, the tissue blocks are transferred to small glass vials containing the primary fixative.

The

aldehydes will penetrate the tissue block to fix cellular proteins as described earlier.

The

ideal fix time for aldehydes is approximately 1 hour, however, tissues can be stored in

glutaraldehyde at 4˚C for a number of days. I recall having stored samples in glutaraldehyde for as long as 1 year without the development of major structural artifacts.

Buffer Wash - usually the same buffer is used as is used to prepare the aldehyde

fixative. In this lab, phosphate buffers are used. The buffer wash is done in order to wash

out any unbound aldehydes which would react with the OsO 4 in the next step, leading to a contaminating precipitate reaction. This wash is usually done a number of times (3 times) for ten minute intervals.

Osmium Tetroxide Postfix - buffered OsO 4 in 1-2% solutions is administered to the

tissues next. At this point, the tissue blocks must be on the order of 0.5 cubic mm in dimension. The OsO 4 primarily reacts with unsaturated fats in the cell and imparts an electron dense heavy metal staining to regions of the cell which are composed of these lipids (such as cell membranes). OsO 4 also reacts with some cellular proteins, however, because aldehydes were used first, the proteins should now be stable and not subject to denaturation by this powerful oxidizing agent.

OsO 4 postfixation is usually carried out for 1 hour with the tissue samples blackening to

indicate the activity of the fixative.

18

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Distilled Water or Buffer Wash (Optional) - many investigators use a brief wash of

either distilled water or buffer to remove excess OsO 4 . The wash is performed since the OsO 4 may react with the ethanol in the following step. At times, a cloudiness may develop in the fixation vials if this step is avoided. In this lab, we usually dispense with this wash since the cloudy result is rare and short-lived, usually lasting for the initial ethanol change. If this step is performed, usually 2-3 changes of ten minutes each is sufficient.

Dehydration Series (Graded Ethanol/Acetone Series) - the tissues are passed

through an ascending dehydration series of either ethanol or EM grade acetone. To prevent distortion and shrinkage, many individuals ascend slowly to 100% ethanol or acetone, starting at 10% and ascending in 10% steps (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%). In order to preserve delicate, internal labile structures, it is also important to get the tissue out of an aqueous environment as soon as possible, therefore, we start dehydration of samples for TEM at 70% (two changes for ten minutes each). We then proceed to 95% (two changes for ten minutes each) and then to 100% (two changes for ten minutes each), typically using ethanol. The 100% ethanol vials must be completely filled to prevent air (containing water vapor) from reducing the concentration. The absolute ethanol should be standing over anhydrous cupric sulfate before use to ensure it is not hydrated. This is a critical step since any water remaining in the tissue will prevent epoxy resin infiltration. The dehydration series is performed since water and most embedding media (epoxy resins) are not miscible.

Transitional/Intermediate Solvent (Propylene Oxide) - this is an optional step in

the protocol which will ensure successful epoxy resin infiltration. Propylene oxide (1,2 epoxy propane) is a highly volatile, flammable, small molecule which rapidly penetrates the tissue blocks. It serves to enhance infiltration of the tissues by epoxy resin since it carries in the reactive epoxy monomer. If used, three changes for ten minutes each is adequate.

Propylene Oxide : Mixed Epoxy Resin (1 : 1 ratio) - in this step, an equal volume of

propylene oxide and mixed (unpolymerized) epoxy resin is introduced to the tissues. The purpose is to make a gradual transition to pure epoxy resin and enhance its infiltration into the tissue blocks. This step is carried out in vials for 1 hour.

Vacuum Infiltration - after the pure mixed epoxy resin has been placed under a low

vacuum (bell jar and rotary pump setup) to eliminate air introduced through the mixing process, a small volume is poured into a small diameter petri dish (enough to fill the bottom and produce a thin uniform layer). Tissue blocks are selected and placed into the resin using toothpicks or bamboo sticks. The labelled petri dishes are placed into the low vacuum setup and the bell jar is pumped out. This process is carried out for 1 hour to prevent gas from impeding epoxy resin infiltration.

Embedding - During the 1 hour vacuum infiltration process, BEEM (Better Equipment

for Electron Microscopy) capsules (which have a preformed truncated pyramid tip of 1.0 square mm) are stuffed with an identification label and filled with epoxy resin mixture to

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

19

a slight negative meniscus. Allow air bubbles to break naturally and further fill capsules as needed.

Identification labels should identify the investigator and the tissue type. Write the labels in pencil only since pen and other inks can react with the resins. Once written, the labels are wrapped around a wooden stick (bamboo) and placed into the BEEM capsule. Don’t wrap the label too tightly around your stick since you want the label to spring against the inner walls of the capsule. When viewed from above, the labels should be barely visible. If they project into the center of the BEEM capsule, try to push them against the sides with two sticks. You do not wish the labels to impede the descent of the tissue blocks to the tip of the capsule.

With the BEEM capsules labelled and filled with the epoxy resin mixture, individual tissue blocks can be transferred from the petri dishes to the center of the filled BEEM capsules using a pointed bamboo stick. The tissue blocks are heavy as a result of osmium impregnation and will sink to the capsule tips prior to the polymerization of the resin.

BEEM capsules should be supported in some type of holder. In this lab we use old micropipette tip holders. Some EM supply companies manufacture BEEM capsule holders and some individuals simply punch appropriate sized holes into cardboard squares.

With the tissues transferred to the BEEM capsules, the holders are placed into an oven at 60˚C for at least 48 hours in order that the epoxy resin polymerize. All containers (plastic beakers, petri dishes, etc.) which came in contact with the epoxy resin should also be put in the oven and be allowed to polymerize for safe disposal. Vials which held the 1:1 propylene oxide/resin mixture should be left open under a fume hood for a number of hours to overnight to allow for the evaporation of the propylene oxide (never put propylene oxide near a heat source/oven). The vials can later be placed in the ovens to polymerize the epoxy resin and finally discarded.

After 48 hours, the BEEM capsules can be removed from the ovens and stored at room temperature. The tissues are ready for trimming and ultrathin sectioning.

Tissue Processing Note

From the mincing of tissues in the primary fixative and up to the point of vacuum infiltration, tissue blocks are handled and contained in small glass vials with plastic snap- caps (8ml capacity). Chemical agents are simply decanted using a Pasteur pipette and the replacement agent added with a different pipette. Do not allow your tissues to dry down during processing. This will lead to denaturation and artifacts. When decanting, always leave a small amount of the original solution to cover the tissue blocks, especially when decanting the highly volatile ethanol (especially 100%), acetone and propylene oxide.

20

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Embedding Media

In order for electrons to pass through a sample in the TEM, it must be reduced in thickness to between 600-900Å. Therefore, the sample must be sectioned ultrathin. Ultrathin sectioning would be impossible unless the biological soft tissues we study are strengthened to withstand the incredible forces encountered during sectioning. Unless a suitable embedding media is used, the soft tissues would simply disintegrate at the cutting edge along with the fine structures we wish to see.

An adequate embedding media for TEM samples must meet certain requirements which follow:

1. consistency - a formulation must yield the same results for each mix.

2. availability - components must be readily available.

3. purity - components must be identified and characterized to avoid artifacts.

4. solubility - in common solvents.

5. miscibility - with other embedding media/curing agents/dehydrating agents (e.g.

alcohol).

6. viscosity - low is ideal from a standpoint of convenience.

7. polymerization - must be controllable, uniform, and occur in a reasonable amount of time.

8. transparent - to light (to view tissue in blocks and sections) and electrons.

9. stability - under the electron beam.

10. sublimation - avoids liquid interface and resultant surface tension forces.

11. cross-linked polymer - for good ultrastructural preservation / strengthens tissues.

12. stainability - allows for heavy metal (TEM), pigment (LM) and histochemical staining.

13. no shrinkage - little volume change during polymerization.

14. stores well - long shelf life (some preliminary mixtures can be stored in the freezer).

In general, resin components should be mixed thoroughly to avoid uneven polymerization and embedment. Hand mixing with a clean glass rod for 15-20 minutes is usually sufficient. Of course, this mixing will introduce a great deal of air to the mixture. Since the mixture is viscous and lengthy staining times may cause separation of the components, a low vacuum (bell jar / rotary pump) setup can be used to eliminate gas from the mixture. Such a setup is best run under a fume hood to avoid resin fumes.

Another important consideration is that the hardness of the resin should closely match the hardness of the tissue in question. This will be considered in the discussions of the various embedding media which follows.

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

21

Methacrylates

The first embedding media to provide good results for the embedment and sectioning of TEM samples were the methacrylates which were introduced by Newman, et al. in 1949. These resins are low in viscosity and their hardness can be adjusted by varying proportions of methyl versus butyl methacrylate. The catalyst typically used to polymerize the methacrylates is benzoyl peroxide. Initially, they were hailed as the ideal TEM embedding agent until it was demonstrated that they polymerize unevenly. Since they are linear polymers, it was noted that they shrink nearly 20% during the polymerization process. This leads to a flowing of cellular components and gross distortions known as “explosion artifacts” due to the presence of large, seemingly vacuolated regions. In addition, the methacrylates are very unstable under the electron beam. This clearing of the resin produces an increase in contrast (since only tissue components are left behind). However, due to surface tension which arises at the liquid resin/tissue interface and the flowing and subsequent collapse of cellular structures, distortion artifacts are a certainty. Formvar (plastic) coated grids were typically used to support methacrylate sections.

Epoxy Resins

Introduced in Denmark for TEM by Maaløe and Birch-Anderson (1956) and later in England by Glauert, et al. (1956), the epoxy resins are and continue to be the best embedding media for biological samples. The first quality epoxy resin developed for use in TEM sample embedments was “Araldite”, an extremely viscous compound which was used for many years in England by model makers. Later, the epoxy resin known as “Epon” 812 was developed and eventually marketed by the Shell Oil company. Today, many “812” epoxy resins are available from EM supply companies (PolyBed 812-Polysciences, Inc., EM Bed 812-EMS, Inc., etc.). As discussed earlier, in this lab, we use an Epon-Araldite mixture which is suitable for a variety of tissues.

Epoxy resins are transparent, yellowish, cross-linked, thermosetting (with the application of heat) polymers which are highly viscous in their unpolymerized form. The modern epoxy is a diepoxy which has an epoxy group at each end of the molecule. The epoxy group is a strained, three-membered ring (C-O-C) which when ruptured, provides the energy 22Kcal/ mol) to drive the polymerization forward. Spaced along the organic molecular chain between the two epoxy groups are 1-5 hydroxyl (-OH) groups. Both the epoxy groups and the hydroxyl groups react with a variety of “curing agents” and either heat or ultra-violet (UV) light to yield the three-dimensional, cross-linked polymer. The final mechanical properties of the embedment are based on the type of epoxy resin and curing agents used.

The epoxy group reacts with any reactive hydrogen atom which leads to its rupture and subsequent release of energy to drive the polymerization. A good source of reactive hydrogen atoms is the organic molecule class known as tertiary amines. The tertiary amines such as Benzyldimethylamine (BDMA) and Tridimethyl amino methyl phenol (DMP-30) are also known as catalysts or accelerators and are responsible for linear (end-to-end) polymerization. They should be added last and just before use of the epoxy mixture. Another class of organic molecules known as acid anhydrides react with the hydroxyl groups of the epoxy resin molecule to provide cross-linking. Acid anhydrides such

22

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

as Dodecenyl succinic anhydride (DDSA) and Nadic methyl anhydride (NMA) are also known as hardeners.

The mixture of epoxy resin (Epon/Araldite), acid anhydride hardener (DDSA), tertiary amine catalyst (DMP-30) and heat (or UV) yields a strong three-dimensional polymer which is resistant to solvents and heat. The three-dimensional polymerization provides excellent mechanical strength for ultrathin sectioning without shrinkage and resultant explosion artifacts. The ultrathin sections are extremely stable under the electron beam, so much so that uncoated grids can be used to pick up the sections.

As mentioned earlier, it is important to match resin and tissue hardness. Hard tissue embedded in a soft resin would break out of the resin block. Soft tissue in a hard resin would be distorted or disintegrate as it is moved across the cutting edge. Much of this matching is a trial-and-error process of working with different resins and curing agents. By example, when using Araldite with the hardener NMA, the resultant polymer is too hard and brittle. When using DDSA, the block is still too hard for biological tissues. In this case a modifier such as Dibutyl phthalate (DBP) is used. This compound is nonreactive, however, it increases elasticity to produce a softer embedment. DPB is also known as a “plasticizer”. In another example, when DDSA is used with Epon, the block is too soft. Conversely, when NMA is used with Epon, the block is too hard. This situation is ideal since final hardness can be adjusted by varying the proportions of DDSA and NMA. One should be reminded that most epoxy resins are immiscible with water and that complete dehydration must be carried out prior to the introduction of epoxy. Any water in the tissue will result in a poor embedment. The transitional solvent, propylene oxide, aids in the infiltration of epoxy resins into the tissue and its use is recommended when possible.

Other Embedding Media Other types of embedding media for TEM samples exists. Polyester resins such as Vestopal W and Rigolac provide an improvement over methacrylates since they are not subject to shrinkage due to three-dimensional polymerization. They are polymerized by heat, light and oxygen and should be protected from such sources by keeping mixtures refrigerated in the dark.

Another useful resin mixture is known as Spurr’s resin (vinylcyclohexene dioxide-VCD) which is a low viscosity resin ideal when infiltration of the sample may be difficult. Botanical samples are often embedded with Spurr’s resin since the cell wall may provide a barrier to the penetration of high viscosity resins.

Water soluble resins are available such as Aquon which is prepared by extraction of the water soluble fraction of Epon. Aquon is completely miscible with water at 15˚C and below. Glycol methacrylate (GMA) is also relatively water soluble for sample preparation where dehydration would be impractical or detrimental to the tissue.

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

23

Fixation Schedule Mammalian Soft Tissue Protocol - In-situ and Immersion fixation - 0.5mm3 tissue blocks Initial Fixation at 4˚C - Ascending to Room Temperature

Schedule

Duration

3% Glutaraldehyde (0.2 M phosphate buffer, pH 7.4)

1

hour

Buffer Wash

3

x 10 minutes each

1% OsO4 (buffered as above)

1

hour

Buffer or DH2O Wash (optional)

2

x 10 minutes each

70% Ethanol 95% Ethanol 100% Ethanol (fill vials completely)

2

x 10 minutes each

2

x 10 minutes each

2

x 10 minutes each

Propylene Oxide

3

x 10 minutes each

1 Propylene Oxide : 1 Mixed Resin

1

hour

Vacuum Infiltration (in small petri dishes)

1

hour

Embed (in pure Epon/Araldite mixture) Place labels into BEEM capsules, then add degassed resin, then tissue blocks. Tissue blocks will descend to the tip of the EEM capsules before polymerization of resin.

48 hours at 60˚C

24

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Fixation Schedule Worksheet Mammalian Soft Tissue Protocol - In-situ and Immersion fixation - 0.5mm3 tissue blocks Initial Fixation at 4˚C - Ascending to Room Temperature

Schedule

Time In

Time Out

3% Glutaraldehyde (0.2 M phosphate buffer, pH 7.4)

   

Buffer Wash

   

1% OsO4 (buffered as above)

   

Buffer or DH2O Wash (optional)

   

70% Ethanol

   

95% Ethanol

   

100% Ethanol (fill vials completely)

   

Propylene Oxide

   

1 Propylene Oxide : 1 Mixed Resin

   

Vacuum Infiltration (in small petri dishes)

   

Embed (in pure Epon/Araldite mixture) Place labels into BEEM capsules, then add degassed resin, then tissue blocks. Tissue blocks will descend to the tip of the BEEM capsules before polymerization of resin.

   

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

25

Final Chemical Fixation Considerations

Throughout the process of chemical fixation, the electron microscopist must be reminded that this is not a natural event and that the final result must be considered artifact. All images of fixed biological samples produced by the TEM are artifactual, not “true-to-life”. Does this mean that the cellular structures which have been previously described truly do not exist? Do double membrane bound structures known as mitochondria, which possess internal infolded cristae, really exist? Are they simply an artifact of preparation? One can answer those questions from an examination of the scientific literature. When you see a recurring feature which possesses the identical structural intricacies and occurs in regions of the cell with specific requirements, in this case energy, we can be confident as to the existence of this structure. At the same time, one must be extremely careful not to induce artifacts of preparation which can arise from improper handling of the samples. Poor technique throughout the entire fixation protocol will lead to glaring defects and artifacts when finally viewed under the high resolution of the TEM.

Finally, while conducting a fixation protocol one must be cautioned that each step may involve your exposure to highly toxic chemical agents. Remember that fixatives kill and preserve living tissues, including yours! A healthy respect for all potentially hazardous chemicals should be developed by the student of science/biology. Use common sense in working with all chemicals! Assume that all chemicals you are unfamiliar with are hazardous and use appropriate precautions such as the use of disposable gloves (two pairs if necessary), goggles, and the fume hood. The ideal course of action is to consult the Material Safety Data Sheet (MSDS) for the chemicals you will be exposed to and follow the listed safety guidelines. MSDS should be available in any location that involves the handling of chemicals. Specific precautions will be found both in association with any chemical listed in any fixation protocol in this book. Whether a chemical agent has an immediate effect (contact dermatitis, blindness, death) or a latent and/or cumulative effect (cancer), the goal is to eliminate exposure of both yourself and future students to the agent. Be careful not to touch bare tabletops, door handles and knobs, etc. with contaminated gloves. You will be contaminating the work area for unsuspecting personnel that would come into bare-handed contact with these surfaces, possibly for years to come (especially in the case of resin components). Also, be aware of the location of emergency equipment in the lab, such as fire blankets, fire extinguishers, eye wash and shower station(s). The phrase “Look before you leap”, meaning, think before you do anything, is most appropriate when dealing with dangerous chemicals in the laboratory.

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Chapter 2 - Ultrathin Sectioning & Ultramicrotomy

Unless the electron beam produced by the electron gun of the TEM can pass through the specimen (or transmit - hence the name Transmission Electron Microscope), achieving an image of high quality and resolution, or any image at all, is impossible. In order for the electron beam to pass through a sample, it must be sufficiently thin. This level of thinness required has been determined to be between 600-900Å, what is conventionally known as ultrathin. Since most samples, biological or otherwise, which one would consider viewing under the TEM are much thicker than this, a process known as ultrathin sectioning is required. It wasn’t until the early 1950’s that quality ultrathin sections were cut routinely. It is for this reason that biological TEM lagged behind the development and commercial availability of the TEM; the first commercial TEM was marketed by the Siemens corporation in 1939 ( an instrument designated the Elmiskop I).

The precision instrument which was developed and makes it possible to cut ultrathin sections is the ultramicrotome. The following information covers the theory and use of the ultramicrotome along with the related requirements which are necessary for its use, including block trimming and glass knifemaking/diamond knife usage.

Processing of Embedded Blocks - Block Trimming

Prior to ultrathin sectioning, polymerized blocks must be trimmed freehand under a dissecting microscope. This process is conducted since tissue blocks rarely descend completely to the tip (truncated/flat face) of the BEEM capsule. This results in an embedded tissue block which is somewhere beneath the blockface. When you consider how little material is removed from the face through ultrathin sectioning (600-900Å), it obviously becomes necessary to expose the actual tissue sample at the surface of the block, otherwise, you will be sectioning epoxy resin only. Unlike in the preparation and sectioning of samples for the light microscope, TEM sections should be completely filled with tissue sample. In LM sectioning, a considerable portion of the section periphery is the embedding material, such as paraffin, with the actual tissue centrally located.

Block trimming is a vital step which will ensure that your sections will contain a maximum amount of tissue. After considerable effort is taken to properly fix your tissue samples, careful block trimming is critical to success at the ultramicrotome.

Block trimming is conducted entirely by hand under a dissecting microscope, preferably with a bright overhead light source. Since the process is entirely manual, many students find this to be one of the most difficult procedures to learn in TEM specimen preparation. The ideal setup is an older style Bausch & Lomb stereoscope with a “pod” head that is fit with 15X oculars and yields a maximum magnification of 45X. A bright external B&L illuminator is used in conjunction with the stereoscope, a setup which is far superior to a newer model B&L stereoscope with its less intense light source focused from overhead by a mirror.

UNIT 1 – PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

27

Besides the microscope setup described above, block trimming also requires an embedded block (in a BEEM capsule), some type of chuck (such as a collet chuck) for holding the block, a trimming base or stage for supporting the chuck, a pair of pliers for removing the block from the BEEM capsule, a package of double-edged razor blades, a beaker of acetone (with a cover) to remove oil from the razor blades, Ross lens tissue to dry the acetone dipped blades, and a metric ruler in order to determine the dimensions of the trimmed blockface. It should be noted that although most individuals in the field do not recommend the use of double-edged blades, they are by far sharper than single-edged razor blades. The minor inconvenience of learning to use these highly flexible blades can be translated into much less difficulty at the ultramicrotome. The more rigid single-edged blade is not nearly as sharp and results in trimmed blocks with what we refer to as a “frosted” face and sides. These frosted areas appear smooth, however, they are actually irregular and can lead to sectioning problems such as sections which do not completely detach from the blockface as they are cut.

Prior to block trimming, the polymerized block must be removed from the BEEM capsule.

A simple way to proceed would be to slit the side of the capsule with a single-edged razor

blade. If one is not very careful, this technique often results in numerous cut fingers. The

recommended method involves using a pair of pliers to force the block out of the capsule using pressure. The BEEM capsule is first squeezed on its side which results in a

noticeable separation of the resin from the capsule’s plastic. The pliers are then positioned at the capsule tip, just above the circular ring, superior to the truncated BEEM pyramid. Slow and even plier pressure is applied which results in the block moving outward from the capsule. Do not bring the jaws of the pliers together as you might damage the blockface. Instead, reposition the pliers slightly higher on the BEEM capsule and continue. Once this has been done enough, at least one-half of the block will project from the BEEM capsule. You should be able to twist and pull the block from the capsule at this point where it will be inserted about two-thirds of the way into a chuck. Empty BEEM capsules can be saved to place over the tip of a trimmed block and protect it until the time

of sectioning.

For the Sorvall-Porter Blum MT-2B ultramicrotome, collet chucks, with circular openings which are reduced in diameter upon tightening (as found in the design of a drill) are employed. It is best to load the blocks horizontally, from the side so that you don’t have to combat gravity. As stated earlier, blocks should be loaded about two-thirds of the way into the chuck. The threaded chuck is then screwed into the trimming base which is placed onto the stage of the stereoscope. The blockface is focused on at maximum magnification (45X) and brightest overhead illumination. The 1.0mm square blockface formed by the BEEM capsule is then set square in the field of view by rotating the trimming base.

A double-edged razor blade is placed for a few seconds in a 50ml beaker of acetone. It is

removed and wiped dry by pulling a sheet of Ross lens tissue away from the each edge. The blade should be held with the index fingers of each hand on top of the blade and the thumbs underneath. Fingers of each hand should be in close proximity with a slight pulling against each other to increase blade tension and rigidity. The other three fingers of

28

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

each hand are anchored on the trimming base with the block and chuck between. Cutting should occur away from the individual, however, some cut the top (across the blockface) towards themselves with success.

Initially, the blockface is cut down to the tissue. Thin sections are cut which are as parallel to the original blockface as possible. The blade should be held level or even better, at a very slight downward angle which ensures that the blade will dig in and produce a uniform and smooth new face. A slight blockface angle may result which can be compensated for on the ultramicrotome. A sharp blade results in a characteristic striated appearance on a reflective blockface. If “frosting” appears, the region of the blade being used has dulled and should be moved to a sharp area. For routine work, your goal is to trim the face down to the tissue block. You can be sure that the block has been reached when black material appears in your hand trimmed sections. This black material is your osmium fixed sample. At this point, your blockface is much larger than the original 1.0mm square and must be reduced by trimming the sides of the block. Sides should be trimmed parallel to the angle formed by the original BEEM capsule (~45˚) and not very deeply. The ideal pyramid is short and broad-based. Deep, steep sides leads to a poorly supported pyramid which vibrates when sectioned leading to a problem known as “chatter”.

As the sides are reduced, so is the blockface. The ideal final dimensions of the blockface are 0.25mm on the longest side(s); not to exceed 0.5mm, with the longest sides parallel to each other. If the longest sides are not parallel, the ribbon of sections that come off the knife edge during sectioning will curve back to the edge instead of moving out in a straight line. A number of final blockface shapes can be trimmed such as a square, rectangle, and trapezoid. The trapezoid is the best shape to trim since it is most probable that sections of this shape will form what are known as ribbons. The following illustration (fig. 1) shows a block before and after trimming.

Fig. 1

Tissue

Tissue

1.0mm

0.25mm Tissue Side View 1.0mm Block Trim 1.0mm Tissue 0.25mm Top View } Razor Blade
0.25mm
Tissue
Side View
1.0mm
Block Trim
1.0mm
Tissue
0.25mm
Top View
} Razor Blade Hand Trim Lines

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

29

In closing a discussion of block trimming, it must be noted that most ultramicrotomes can be used to trim blocks using glass knives since the knife stage and specimen arm angles can be manipulated through a wide range. Additionally, automatic block trimming devices and even gadgets to remove blocks from BEEM capsules are marketed. Although such instruments exist, for a price, most labs still trim blocks by hand.

Glass Knife Making

A suitable cutting edge for use in ultrathin sectioning is mandatory. Metal edges cannot be

sharpened adequately to cut sections on the order of 600-900Å. The use of free fractured

glass knives, introduced by Latta and Hartmann, was an important advance to the

eventual production of ultrathin sections. The sharpest cutting edge that can be produced

is that of free fractured glass. Even our ancestral cave dwellers knew the value of broken

glass as they fashioned obsidian (volcanic glass) into tools for cutting. Today some surgeons will use obsidian scalpels to reduce “hamburgerization” of the integumentary (skin) tissues which can lead to scars. Only a handful of such courageous surgeons will use obsidian scalpels since they risk cutting their own valuable and skilled hands.

In glass knifemaking (see fig. 2 on opposite page), plate glass strips are used. In this lab, LKB glass strips are used. LKB (now Leica) manufactures fully stress flown glass strips in which lines of stress are virtually eliminated. These high quality glass strips produce the sharpest eventual cutting edges. When a piece of glass is free fractured, it is precisely scored using a carbide scoring wheel or diamond tipped scribe. The score line must not run entirely across the length of glass you are trying to break, from one edge to the other. On the contrary, the score must be along the midline of the length of glass to be broken, at or near the center. Pressure is then applied beneath the score line and the glass free fractures to yield the sharpest cutting edge known. The simplest and least expensive way to apply this pressure is through the use of glazier’s pliers. The only problem here is inconsistency in the quality of the glass knives. In the early days of electron microscopy, one individual would be hired for the job of making glass knives. This person would become proficient at glass knifemaking through repetition and could routinely make quality edges for ultrathin sectioning.

For many years, instruments have been available to somewhat automate the process of glass knifemaking. These instruments, such as the LKB 7800 series knifemakers, allow anyone to manufacture quality glass knives. The LKB knifemaker has provisions for

precisely aligning, clamping, scoring and breaking glass strips of various dimensions. The most common dimensions of the glass strips purchased to make glass knives are 9-18" long

x 1.0" wide x 1/4" deep. Shorter glass strips (9-10") are easier to handle and the LKB

knifemaker can be used to cut longer strips in half. To cut a long strip in half, the strip is

laid on top of the two spring loaded posts and the end of the strip is lined up even with the small black dot mark on the right-hand side of the machine’s surface. The strip is scored and broken as per the procedure for making a 1.0" square described below.

30

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Fig. 2 – Glass Knifemaking: 1/4 inch score line free break path
Fig. 2 – Glass Knifemaking:
1/4 inch
score line
free break path

1 inch

1 inch

score line 1 inch 1 inch
score line
1 inch
1 inch
1 inch 1 inch score line 1 inch 1 inch frilling cutting edge conchoidal fracture plane
1 inch 1 inch score line 1 inch 1 inch frilling cutting edge conchoidal fracture plane

frilling

cutting edge

conchoidal fracture plane

1 inch 1 inch frilling cutting edge conchoidal fracture plane base (1 mm) Boat/Trough Attachment: tape

base (1 mm)

Boat/Trough Attachment:

tape seal

water level

1 inch 1 inch frilling cutting edge conchoidal fracture plane base (1 mm) Boat/Trough Attachment: tape

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

31

Before making a free fractured knives, the glass strip must be carefully cleaned in a non- filming soap such as Liquinox. It should be held at one end only, the end which will not be used to make the knives, and thoroughly rinsed by holding the end used to make the knives upright. This prevents any soap on your hand which holds the glass from running down the strip and contaminating it. A thorough rinse in tap water followed by a final rinse in distilled water is recommended. The strip is then rested at an angle against lint free cloth, with the useful end upright, and allowed to air dry. Don’t wipe the glass dry, even with so-called lint free cloth, as you will produce a static charge on the glass and attract dust. Once dry, you can proceed with glass knifemaking on the LKB 7800. Initially, the LKB knifemaker should be set up with the breaking knob, the large black knob on the lower right of the machine which is used to apply pressure, fully counter- clockwise. The clamping/scoring head should be elevated in place using the ball-ended clamping lever and the silver scoring adjuster should be on the “lines” setting (the three lines should be facing up - the other positions are 2.5 and 3.8).

Holding the clean glass strip by one end, it is carefully lowered to the surface of the LKB 7800 with its scored/frilled edge down. The strip can be touched on the upper 1.0" wide surface but not on the 1/4" deep edge. Touching the strip on its surface, it is pulled down flush with the white plastic guide (which is set at 90˚) and slid to butt up against the first of two metal posts. The clamping/scoring head is lowered using the clamping lever until it contacts the glass strip surface. Upon contact of the clamping head, immediately release the glass strip surface with your hand. Failure to release with your hand at this point will result in the scored glass not breaking. Lower the clamping lever so that the ball end is just above the surface of the machine (finger width space). With the score adjuster set on “lines”, score the strip by smoothly pulling the white scoring lever out. When the scoring lever is pulled, a carbide wheel descends and ascends in an arc to avoid scoring the glass from edge to edge. This score is made perpendicular to the length of the strip, exactly 1.0" in from the end. The large black breaking/pressure knob is then turned slowly and consistently until the glass free breaks. When the glass breaks, the breaking knob is turned back full counter-clockwise and the clamping head is elevated using the clamping lever while supporting the head using the extended scoring lever. At this point, a separate 1.0" glass square has been produced. The remaining glass strip can be removed and placed on lint free cloth.

The glass square can be slid to the top of the pair of black pressure feet and carefully rotated counter-clockwise into a diamond formation, by touching only the top surface of the square. The lower edge of the diamond should be slid between the two white plastic supports with the upper edge aligned across from the inverted “U” of the upper metal holder. The upper holder is brought into place by pushing in and rotating the small black control knob on the rear left side of the machine. The clamping head is lowered as before and the scoring adjuster is set to 2.5. Before scoring, the small black damper is moved to just touch the lower point of the diamond using the small metal lever and the fork-like knife holder is slid under the diamond (this can be done before scoring or after the knives break). The glass is scored at 2.5, once again not from edge to edge and this time slightly off the perpendicular, and broken using the slow and steady movement of the breaking

32

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

knob. Once the glass breaks, the clamping head is elevated, the damper is reset (dot to thin line) and the upper holder is released by pulling and rotating the holder knob. The two knives will rest on the fork-like holder which can be removed. They should be left on the holder and carried to a dissecting microscope. Do not allow them to rest on their sides on a tabletop as the edge may pick up contamination. Caution: Never leave upright glass knives unattended. Unsuspecting individuals can come along and rest their hands down on top of the extremely sharp cutting instrument. A clean plastic (tri-pour) or glass beaker is recommended to cover and protect both your knives and colleagues.

After preparation of the knives, they must be evaluated for quality. Obviously, poor knives should never be used for ultrathin sectioning. Handle the knives carefully so that the cutting edge will not become damaged or contaminated, especially with oil from your skin. Initial evaluation is performed by visual inspection of both knives without a microscope. From the side you will see the shape of a right triangle. The base of the knife will have a 1.0mm raised heel. The heel should be an even rectangle with its longest sides parallel. The cutting edge should be relatively straight and level, however, a convex or concave edge is not uncommon. The convex edge often has the longest usable cutting area. Looking directly at the angled side of the knife, you will notice the sloping conchoidal fracture plane. It is believed that the further to the right this fracture plane exists before descending, the longer the usable cutting edge will be. The only way to evaluate a glass knife edge’s quality is to examine it under a dissecting microscope with an overhead light source. The setup used for block trimming is ideal. For the best view, a dark background is recommended. In this case a small square of black photograph mounting board can be cut and placed on the microscope stage. The magnification should be adjusted to allow for viewing of the entire knife edge (~ 30X). The knife should be held with the angled edge facing outward and the knife set in the shape of a “V”. While looking under the stereomicroscope the glass should be rocked so as to concentrate the overhead light on the very edge of the knife. The knife edge should lie near the center of the field of view. When the knife edge is focused you should see a broken bright line on the right side of the cutting edge. The broken line is indicative of typical imperfections in the edge which is known as “frilling”. The left edge of the knife should appear as an unbroken bright line which indicates its sharpness and high quality for sectioning. In order for the knife to be usable, at least 1/3 to 1/2 of the left edge should be unfrilled. Knives with quality edges less than this should be discarded (in an appropriate sharps container to minimize risk to the maintenance staff). The knife edge can also be evaluated for debris which would lead to the appearance of sectioning artifacts. Do not try to clean the edge of debris using a duster can (compressed air) since particulate and organic matter in the can will damage the edge. Dirty knives must be discarded with the student exercising greater caution in the preparation of additional knives. Always prepare more than one good knife in the event that the knife is damaged or contaminated in transit to the ultramicrotome. Once at the ultramicrotome, you will not want to backtrack and make more knives.

Once the knives are evaluated, they must be “boated” through the application of a tape trough/boat. This boat will hold floatation fluid, typically double distilled water, which will

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

33

allow the sections to come off the knife edge and float on a liquid surface as opposed to sticking to the dry glass edge. The boat is usually made of mylar adhesive tape and is available through some EM supply companies. A suitable substitute would be electrical tape strips which are cut in half (on a clean piece of glass) to reduce the width.

Once a length of tape is cut, the knife glass is placed to it. You will have more control in touching the knife to the tape versus touching the tape to the knife. You should orient the knife to the tape in such a way that only a short length of free tape extends to the left of the placed knife. This will minimize the chance of the eventual two free sticky tape ends touching and transferring adhesive to the knife edge. In terms of orientation, the tape must be exactly even with the upper cutting edge. The lower edge of the tape must be parallel to the lower base of the knife. Once the knife is placed to the tape, the knife can be picked up and the long free tape end can be wrapped around to fashion a symmetrical boat having a small gap at the bottom (where the tape meets the angled edge of the knife). If the orientation is not correct, the tape can be carefully pulled off an another attempt can be made. The more attempts one makes, the greater the risk of contaminating the knife edge. Excess tape projecting from the back of the knife is cut off using a razor blade and being careful not to touch the knife edge. While boating the knife one should be careful not to touch the tape in the region which will actually form the boat. When water is introduced, oil from the skin will serve to contaminate the floatation fluid and ultimately, your sections. After the tape boat is attached, it must be sealed to prevent leakage using nailpolish (or molten paraffin wax). The polish should be applied all along the lower tape edge, especially at the gap on the angled edge of the knife. Excess polish should be removed from the brush on paper toweling and a thin line run up the two back edges of the knife. Do not apply polish to the knife edge. The polish is allowed to dry for about 20 minutes at which point, the knife can be used to section on the ultramicrotome.

Since glass is a supercooled liquid, it tends to flow away from the sharp edge and become dull over time. Ideally the glass knife should be used within 24 hours of manufacture, however, if kept in a storage box and desiccator, it should be useful for a few days. Another problem with the glass knife is that it rapidly dulls with use. Only between 15-30 ultrathin sections may be cut before the appearance of visible knife marks which are noticeable dark, vertical, irregularly spaced lines in the section. These unmarked sections will be picked up followed by moving the knife to an unused cutting region. An alternative to the glass knife is the diamond knife described below.

Diamond Knives

Diamond knives are extremely delicate and expensive instruments which can cost as much as $5,000.00 based on the length of the edge. Obviously, only experienced microtomists should handle a diamond knife since simply touching the edge or cutting a section greater than 1.0µm may permanently damage it. The benefits to using a diamond lie in its hardness. Proper care results in a knife that is sharp for years and can cut numerous sections without having to stop and move the knife. The entire edge is sharp which results from taking a quality gemstone and cleaving it into smaller fragments which are polished

34

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

using diamond dust (the actual process is a trade secret of the few companies which manufacture diamond knives). The diamond is mounted into a metal holder which is glued into a metal trough using epoxy. The need for making glass knives and attaching tape boats is eliminated.

A common problem in using a diamond is that it is quite hydrophobic which prevents

wetting of the knife edge. The remedy involves carefully running an eyelash along the inside edge of the diamond with the boat full. If the diamond still won’t wet, a bit of saliva or dilute wetting agent (such as Photoflo 200:1) can be used on the eyelash.

Routine cleaning of the diamond knife involves a gentle stream of distilled water from a squirt bottle to clean the boat and knife edge. On occasion and if sections adhere to the edge, a cleaning stick made of styrofoam or pithwood can be run along the edge, in one direction only. Never apply forward or backward pressure on the edge. Cleaning sticks can be dipped in ethanol or even saliva to remove adherent material from the diamond edge.

A recent addition to the types of knives used in ultrathin sectioning is the sapphire knife.

As with the diamond knife, the sapphire is permanently mounted in a trough and is superior to the glass knife. It is however inferior in quality to the diamond knife and is incapable of cutting hard samples such as tooth and bone, without damage to the edge.

Ultramicrotomy

The development of the ultramicrotome has been critical to the formation of high quality images of biological material using the TEM. In order for the electron beam to pass through the specimen, it must be less than 1,000Å thick, ideally between 600-900Å. To produce sections thin enough for use in the TEM took years of research into mechanical engineering of the actual instrument in addition to fixation and embedding materials, blockface requirements and cutting instruments (glass and diamond knives).

When TEM development was taking place in the 1930’s, its use for the examination of biological samples was not envisioned. Early investigators who were interested in examining biological samples with the TEM would attempt to use whole mounts of fractured materials with poor result. In 1939, von Ardenne proposed cutting tapering, wedge shape sections whereby a portion of the section would be thin enough for TEM observation. Modification of a Spencer 820 microtome, used for paraffin embedded LM level sections, by Pease and Baker in 1948 resulted in a 1/10 reduction in block advance to the cutting edge. They also reduced the block face to 1.0mm2 and produced sections 0.3- 0.5µm thick. As detailed in the unit on embedding media, Newman et al. (1949) introduced methacrylates which became the first embedding media of quality for TEM samples. Using a metal knife which had been sharpened with a new technique, along with an attached trough which was used for the first time, Hillier and Gettner (1950) made further modifications to the Spencer 820 microtome and produced sections 0.2µm thick. A major advance in the field took place in 1950 with the introduction of glass fracture knives by Latta and Hartman. In 1952, Palade used methacrylate embedding, glass knives, a

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

35

blockface smaller than 1.0mm 2 on an ultramicrotome designed by Claude and Blum and was able to obtain sections at the useful thickness of 1,000Å. In 1953, the Sorvall “Porter- Blum” MT-1 ultramicrotome was introduced commercially. This instrument was hand- driven and allowed for reproducible ultrathin sectioning by anyone who had the patience to learn its operation. Needless to say, the first quality TEM photomicrographs of sectioned biological material appeared at this time.

Since the introduction of the MT-1, numerous improvements in design lead to the development of the Sorvall MT-2 and 2B with motorized drive, and eventually the MT- 9000, currently available from RMC, Inc. LKB (now Leica) was the first company to introduce an ultramicrotome using the advance principle of thermal expansion. The LKB Ultrotome series also had superior lighting and improved specimen blockface to knife adjustments for greater control in orienting knife to blockface. Current state-of-the-art instruments include the Leica Ultracut with fiber optic blockface illumination which make it almost impossible to chop off your blockface, a common problem of the inexperienced microtomist.

Other advances in the field of ultramicrotomy include the work of Peachey (1958) who suggested that interference colors of sections under a cold (fluorescent) light source can provide a good estimate of section thickness. He determined that the following section colors relate to their thickness:

60 nm

gray

60-90 nm

silver

90-150 nm

gold

150-190 nm

purple

190-240 nm

blue

240-280 nm

green

280-320 nm

yellow

Ultramicrotome Theory

In the design of an ultramicrotome (fig. 3), a rigid cantilever arm is employed to which a trimmed block can be mounted. The trimmed block is held within a chuck. The most common type is a collet which tightens down on the block by turning the threaded collar, usually clockwise, leading to a reduction in the diameter of the circular opening. Other chucks, such as those used in early LKB ultramicrotomes, resemble nosecones which are split in half lengthwise and held together by a hexagonal set screw. Vise-type chucks are also available with a pair of adjustable flat jaws to accommodate flat embedments. It is imperative that the chuck holds the block securely to avoid vibration during sectioning.

The ultramicrotome cantilever arm must be able to move in three degrees of freedom about various pivot points. Firstly, the arm must be able to move up and down in an arc; this is known as the cutting stroke since downward movement through a cutting edge

36

ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

will yield a section. Secondly, the arm must be able to move from side to side in an arc; this is known as the bypass stroke since it allows the blockface to bypass the knife as it is moved to the top of the cutting stroke. Without this motion, the blockface would be damaged through compression as it hit the back of the knife. In the early Sorvall “Porter- Blum” MT-1, the arm moved within a parallelogram shaped cutout. Modern instruments such as the MT-2 eliminate the side to side motion in favor of a retraction of the arm at the bottom of the cutting stroke. In the LKB/Leica instruments, the knife stage is retracted electromagnetically. Either way, contact between the blockface and the back of the knife is avoided. Finally, the arm must be able to extend linearly along its axis; this is referred to as the advance since it allows the arm to advance the required minuscule amount to the stationary cutting edge. The amount of advance will determine the ultimate section thickness. In the development of the ultramicrotome, two methods of advance have been used. The first, used in the Sorvall MT-1 and MT-2(B), is mechanical which is designed around a vertical pivoting arm which rests on a micrometer lead screw. As the lead screw turns, the lower portion of the vertical arm moves to the back of the ultramicrotome, while the upper portion advances out toward the front of the machine. Since the rigid cantilever arm is attached to the upper portion of this pivot arm, it will be moved forward toward the knife edge. The vertical pivot arm will eventually reach an end point and will have to be reset. One should be sure to reset the mechanical advance machine before mounting a block.

The second type of advance used is thermal which involves the uniform expansion of a metal under uniform heating. This method, used in LKB (now Leica) Ultrotomes for example, must have provisions for variable heating to control section thickness and a cooling fan in order to “reset” the mechanism, since there is a limit of thermal expansion for any given metal. The arm must be motor driven and electronically controlled. The mechanism must be able to compensate for changes in cutting speed while maintaining section thickness. By example, if silver sections are cut at 1.0mm/sec and you change the cutting speed to 2.0mm/sec, the machine will have to introduce a point of hesitation into the cycle to continue to yield silver sections. Without this hesitation, the cycle time would be shortened, therefore, heating time reduced leading to a thinner section.

In terms of cutting speed, the best results are obtained if the cutting speed is uniform (usually somewhere between 1.0-2.0mm/second). Variations in cutting speed was a common problem in producing good sections using the hand-driven Sorvall MT-1. Many labs designed a reduction gear to attach to the cycling wheel leading to greater uniformity in cutting speed. Whether mechanical or thermal advance, the introduction of motor drives to the ultramicrotome was a vast improvement.

Another important feature of the ultramicrotome is the stage to which the cutting instrument/knife is attached. In some cases the stage is permanently mounted to the ultramicrotome (LKB Ultrotome III) while others are removable (Sorvall MT-2). The stage must be finely machined which will allow for fine control in bringing the knife to the blockface. The stage must incorporate a knife holder (for both glass and diamond knives) and provision for setting the clearance angle, the most important angle of the knife

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

37

relative to the blockface, which is initially set at 4˚. There is usually a device/jig to set the proper height of the knife edge in the stage. The stage will also incorporate control for the lateral movement of the knife to select the appropriate cutting area (knife edge adjustment), rotation of the stage to allow for machine block trimming and most importantly, to establish that the lower edge of the blockface be parallel to the knife edge before cutting (knife rotation). If not parallel, one side of the blockface will be cut before the other. Many thick sections would have to be taken to yield a full blockface which would probably be too large for ultrathin sectioning. The final provision is the stage advance (knife advance) with both coarse and fine adjustments. This control is critical since the knife must be manually moved to the blockface for the first facing cut. Since the ultramicrotome arm advances in such small increments, it cannot be expected that the arm would ever reach a stationary knife located some distance from the blockface. Additionally, the cantilever arms are not capable of traveling large enough distances to reach the knife. The knife must be moved to the blockface using the fine controls of the stage. A common problem for the beginner is their lack of patience in advancing the stage to the blockface. The result is a chopped blockface which must be retrimmed.

It should be noted that each control on the stage will have a lock to reduce vibrations. Vibrations can lead to a sectioning artifact known as chatter. Chatter is a regular variation in thick and thin areas within a section. This produces a uniform dark and light banding pattern on the section. Fine order chatter, which cannot be seen under the light microscope, ruins the section by obscuring details. Reduction of vibration is a major concern to the microtomist. This can be accomplished by making sure all locks are engaged (chuck, stage, etc.), that the block trimmed is short and broad-based, and that the table which supports the ultramicrotome is sturdy (special anti-vibration tables are available but may be unnecessary depending on the room used for sectioning). In general, ultramicrotomes incorporate a massive baseplate in order to lessen the effects of vibration.

Another important feature of the ultramicrotome is lighting. As described earlier, a fluorescent light source is used to produce section interference colors for thickness determination. Good lighting is also important for the critical advance and initial alignment of knife edge to blockface. It is recommended that the position of the light be adjusted regularly during advance of the knife to the blockface for the best possible view. Modern instruments incorporate fiber optic lighting on the blockface which leads to a more accurate approach and fewer chopped blockfaces.

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Fig. 3

Collet Chuck Water Level Block Microtome Arm Clearance Angle Glass (2-5°) Knife
Collet Chuck
Water Level
Block
Microtome Arm
Clearance Angle
Glass
(2-5°)
Knife
Knife Edge Ultra-thin Sections in Ribbon Water Surface Collet Chuck Microtome ArmBlock
Knife Edge
Ultra-thin
Sections in
Ribbon
Water Surface
Collet Chuck
Microtome ArmBlock

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

39

MT-2B Ultramicrotome Sectioning Procedure

The following describes the procedure to follow when sectioning with the Sorvall “Porter- Blum” MT-2B ultramicrotome. Many of the general steps can be applied to the proper use of any microtome on the market with a noted improvement in lighting over the MT series. Failure to adhere to the procedure as outlined may result in frustration and many chopped blockfaces.

INITIAL SETTINGS & PROCEDURES

• Press Reset Button

• Upper Thickness Pivot Knob at 10 (10 x 10Å = 100Å)

• Thickness Wheel (front left side) at 12-14 (1200-1400Å)

• Cutting Speed at 1.0mm/sec

• STAGE: Fully Retracted with ALL LOCKS OFF - Do NOT Place on MT-2B at this time!

THICK SECTIONING PROCEDURE

• Mount trimmed block and orient with fluorescent light fully extended out! Firstly, obtain a fully reflective blockface using loosened “arc” adjustment and hand tighten thumbscrew (B&L binocs set at highest magnification). Secondly, orient bottom of blockface parallel to “ground” using chuck rotation and hand tighten (This adjustment is not critical since knife edge parallel can be obtained using stage knife rotation). Use tools to completely tighten chuck (NEVER OVERTIGHTEN ANYTHING).

• Mount Knife in Holder (Be sure front of knife is flush with aluminum guide plate).

• Mount Knife Holder in Stage and Lock at 4˚.

• Push Light Source back in and Mount Stage to MT-2B (use stage rotation lock on left).

• Stage should be pushed forward completely until it stops.

• Check Position and Duration and set if necessary (observe from side of setup).

• Using lowest magnification setting on B&L binocs, locate block pyramid and knife edge.

• Focus on knife edge and bring pyramid into focus by rotating cycling wheel clockwise. THIS MUST BE DONE OFTEN OR YOU WILL CHOP YOUR BLOCKFACE !!!

• Coarse advance the stage (knife) to the blockface. Increase the binocs mag as blockface and knife edge get closer. Always refocus on the knife edge, THEN, bring the blockface into focus by cycling (focus the “cutting” stroke and not the “retraction” stroke).

• Your goal is to get the knife edge and blockface in focus together in the same field of view at the highest binocs mag (“3” = 45X).

• Position your knife to face the block near the center of the knife edge and gently LOCK.

• Advance closer using the coarse control until you feel you might chop your blockface.

• After cycling the blockface to the lowest point in the cycle, fill your boat with clean distilled water using a drawn micropipette or syringe, to a slight positive meniscus. Level and clean the water surface using Ross Lens Tissue (the water should be of a uniform reflective gray without any dark shadows near the knife edge).

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

• Lock out coarse advance and go to fine advance using thumbscrew (NEVER TIGHTEN SCREW WITH STAGE IN FULLY RETRACTED POSITION). Only the large diameter advance barrel should turn with each line equal to 1.0µm increments.

• Critically focus on the knife edge and cycle the blockface back into sharp focus.

• Fine advance without cycling until extremely close to knife edge. Determine if bottom of blockface is parallel to knife edge. If not, carefully rotate stage until corrected and gently LOCK stage rotation.

• When critically close, begin to interleaf cycling with fine advance with a final reduction in advance to 1.0µm per cycle prior to your first section coming off.

• You may note a light reflection of the knife on the blockface (see diagram). The reflection (best seen in a faced block) indicates you are at least 10.0µm from the face. The reflection will shrink and finally disappear when you are 10.0µm from the face:

and finally disappear when you are 10.0 µ m from the face: • Your goal is

• Your goal is to take 4-6 full face thick sections (1.0µm green) by manual cycling.

• With the blockface at the lowest point in the cycle, pick up some thicks using your eyelash and place them in a drop of water at the center of a clean microscope slide.

• Allow to air dry and observe using the phase contrast microscope for the presence and quality of tissue.

• Proceed to thin section if all checks out.

ULTRA-THIN SECTIONING PROCEDURE

• With block in lowest point of cycle, refill and clean and level boat (Ross Tissue).

• Retract with fine advance (50µm at this step and 10-20µm thereafter).

• Cycle blockface into critical focus with knife edge (should see reflection).

• Unlock and move knife edge just inside of quality area of knife as was determined by prior evaluation. Gently RELOCK.

• Fine advance until reflection disappears then with cycling and 1.0µm advance until the first section is cut.

• Lock the stage advance and START the MOTOR (depress red button).

• Note section colors (probably purple) and reduce thickness using side (NOT PIVOT) thickness wheel until PALE GOLD/SILVER is obtained. WALK AWAY - LET MACHINE DO ITS JOB !!!

• After 15-20 sections are cut or until noticeable vertical knife marks appear, STOP MOTOR.

• Isolate floating sections using the eyelash and pick up on the dull side of 200-400 mesh GRIDS (use 200 mesh for ribbons, 300-400 mesh for loose sections (undesirable). Hold slightly bent grid in jeweler’s forceps and touch dull side down on top of floating sections. Turn grid dull side up and blot dry on filter paper. Unlock forceps and place grid on grid gripper or on edge in grid box.

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• Follow ultra-thin sectioning instructions above and obtain more silver sections. REMEMBER TO UNLOCK THE STAGE ADVANCE LOCK BEFORE RETRACTING THE STAGE AND MOVING THE KNIFE FURTHER LEFT !!!

BE PATIENT & GOOD LUCK !!

Final Ultramicrotomy Comments

Some additional comments are warranted with regard to the MT-2B procedure which was just outlined. Under “initial settings and procedures”, the required steps should be obvious. Recommended initial cutting speeds are usually in the 1.0 to 2.0mm/sec range. The stage should be carefully checked to ensure that it is fully retracted otherwise, you could easily ram the knife into the blockface when the stage is first mounted to the ultramicrotome.

Under “thick sectioning procedure”, the initial blockface orientation is critical. The goal is to obtain an initial full-face thick section manually. This is impossible unless the blockface is properly oriented. In order to accomplish correct orientation, the proper lighting on the face is important. With the MT series, the fluorescent light must be fully extended so that the blockface is illuminated from the front. After blockface orientation, the light must be placed in a rear position in preparation for advancing the knife to the face. When mounting the knife in its holder, be careful not to break the tape/nail polish seal. Make sure that the knife seats on top of the height adjusting set screw and not behind it. If the knife is not flush with the front aluminum guide plate, the clearance angle you set will be inaccurate. A feature of the MT-2B is position and duration controls (the MT-2 did not have these controls). They provide fine control over the ultramicrotome’s cutting range and are best demonstrated at a slow cutting speed (0.33mm/sec). At this slow speed you will notice the actual cutting range of the instrument. The actual range exists over the position and duration of this slow rate of cantilever arm travel. Above and below the cutting range, the arm moves faster. Obviously, you want the cutting range to coincide with the knife edge. Looking from the side of the stage/block-arm setup, one must ensure that the slower range of blockface/pyramid travel is coincidental with the knife edge. If not, the position of the cutting range can be raised or lowered and/or the duration (length of cutting range) can be lengthened or shortened.

Initial facing of the block should be done at or slightly to the right of the center of the knife edge. The left side should be reserved for ultrathin sectioning. When filling the boat, it is important that the blockface be positioned at the lowest point of the cycle. The indicator of

a proper water level is a uniform reflective silver-gray surface appearance. If the block is

at a high point in the cycle, it will cast a shadow on the water surface and you will be unable to determine the proper level. Relative to trough fluids, double distilled water in a sterile centrifuge tube works best. Some investigators use a low concentration of 1-3% acetone or ethanol to facilitate wetting of the knife edge. Although rarely seen with glass knives, failure of distilled water to wet the diamond knife edge is common. Carefully

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

running an eyelash along the inside edge of the diamond is often a solution, especially if the eyelash has been dipped in a wetting agent (Kodak photoflo) or even saliva.

Only experience and practice will enable you to determine when to begin fine stage advance and ultimate interleafing of advance with cycling in order to avoid ramming your blockface. The reflection of the knife edge in the blockface is the best indicator of distance, however, it is often difficult to see in a hand trimmed blockface. Once thick sections are obtained, picking them up becomes difficult for many beginners. An eyelash which is brought up quickly from underneath a group of sections works well. If the eyelash proves too difficult, a syringe tip or shaved toothpick can be tried. The toothpick should be dipped in acetone or you may introduce a large amount of debris into the boat. The thick sections will be used to verify the presence of tissue in the section along with its quality and orientation.

Under “ultrathin sectioning procedure”, you must be sure to first unlock the stage advance and then retract the stage before moving the knife to section in a new area. The knife/ stage will be fine advanced with a good reflection of the knife edge in the faced block. The reflection will also aid in the determination of parallel relative to the knife edge and bottom edge of blockface. Once the first semi-thick section is cut, the stage advance lock must be engaged to prevent chatter. Once the motor is started, ultrathin sectioning will begin. Reduce the side thickness control until silver sections are obtained. If the block was properly trimmed, a ribboning of sections will occur. Once knife marks appear, the motor is stopped with the blockface at the lowest point in the cycle and the sections are picked up on the dull side of a grid. Pickup is easily accomplished by simply touching the dull side of the grid to the floating sections. To protect the tape boat seal, a bend is put in the edge of the grid (do not attempt to bend formvar coated grids). With the edge of the grid locked into the jeweler’s forceps, it is laid flat, dull side down, on filter paper. The forceps are then raised to create an approximate 45˚ angle with the filter paper surface. The bent grid held by the forceps is put aside and the eyelash is used to separate long ribbons into ribbons of 15 to 20 sections and isolate them in the center of the boat (isolated silver sections can also be rounded up at the center of the boat if ribbons are not formed). The grid is picked up and using the unaided eye, positioned over the boat in a flat, horizontal orientation. Looking through the binocs, the grid can be arranged so that the sections/ribbon will be centrally located after pickup. The grid is gently lowered to the sections so as not to disrupt the surface tension forces and taken away. Do not submerge the grids! The dull side of the grid is used for a number of reasons. Since the dull side is rough in comparison to the polished (shiny) side and the sections themselves are rough, they adhere better to the dull side of the grid. Another advantage is that you establish uniformity and always know what side your sections are on and on the dull side, you can actually observe the reflective sections after you collect them (fig. 4).

After sections are collected, grids are blotted dry on edge (see post-staining procedure- Chapter 3) and stored dull side up on a “grid gripper” (also known as a grid mat) to air dry, after which they can be post-stained. The grid gripper prevents grids from attaching to the lid of the petri dish via static electricity. The safest place to store grids is on edge in

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

43

a grid box. Serial sections collected on slot or hole grids should never be put on a grid gripper or blotted flat down on filter paper.

Fig. 4

collected on slot or hole grids should never be put on a grid gripper or blotted
collected on slot or hole grids should never be put on a grid gripper or blotted
collected on slot or hole grids should never be put on a grid gripper or blotted

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Troubleshooting Guide to Ultramicrotomy

When an instrument capable of cutting sections on the order of 600-900Å is used, it becomes obvious that a number of problems may be encountered. The following section provides information on troubleshooting the most common problems associated with ultrathin sectioning. Although at times this process can be quite infuriating and frustrating, the key to mastery of the ultramicrotome is practice. The more problems encountered at the outset, the better equipped one becomes at finding solutions. Students may initially get lucky and achieve good results on their first attempt but it is unlikely that they will be able to produce consistent quality results unless they encounter some of the problems which are described below.

• Inability to cut any sections:

a. microtome advance not reset

b. specimen/blockface too large

c. poor embedding or soft block

d. poor knife (not evaluated correctly) or improper clearance angle setting

e. water level in trough too low

f. vibrations in room

g. blockface gets wet due to high water level

h. faulty ultramicrotome (check that all locks are tight)

• Variations in section thickness from one section to another

a. blunt/dull knife edge

b. incorrect clearance angle (usually needs to be increased)

c. incorrect cutting speed

d. soft block

e. drafts or temperature changes in the room

f. faulty ultramicrotome (check that all locks are tight)

• Knife marks

a. imperfections or contamination of the knife edge - knife poorly evaluated

• Chatter

a. resin and tissue hardness are not properly matched

b. poorly trimmed block - excessively high/tall, not well supported

c. locks not engaged - looseness leads to vibration

d. cutting speed may be too high

e. clearance angle may be too high

f. blockface too large (approaching 1.0 sq. mm)

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

45

• Compression (can use organic vapors of chloroform or heat to flatten sections)

a. cutting speed too high

b. incorrect clearance angle

c. poor knife edge

d. soft block

• Folded/Wrinkled sections

a. poor knife edge

b. cutting speed too high

c. water level too low

d. soft block

e. incorrect clearance angle

• Sections pulled down back of knife

a. water level too high

b. cutting speed too slow

c. clearance angle too small

• Ribbons not straight or not formed

a. poorly trimmed block

b. two sides of blockface parallel to knife edge are not parallel to each other

c. water level in trough not correct

d. incorrect cutting speed

e. blunt/dull knife

• Holes in sections

a. poor infiltration of resin into tissue

b. air bubbles in resin/tissue - vacuum infiltration is recommended to avoid this

• Cannot see sections as they come off the knife edge/cannot achieve proper trough level

a. adjust/move illumination

b. blockface not at lowest point in cycle

c. leaky boat seal

It becomes obvious from a glance at these troubleshooting tips that many problems can be solved by manipulating cutting speed and/or knife clearance angle. LKB/Leica has summarized a methodical approach to varying these parameters. They suggest starting at 4˚ and a speed of 2mm/sec. If the results are unsatisfactory, they recommend changing the speed to 1mm/sec, then 5mm/sec. If good results are still not obtained, a change in clearance angle to 1˚ is recommended (don’t forget to retract your knife sufficiently) starting once again at 2mm/sec and moving to 1mm/sec, then 5mm/sec. A final step would involve changing the clearance angle to 7˚ using the identical cutting speeds as previously noted.

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

At all steps of ultrathin sectioning, contamination is to be avoided. The most common problems arise when oil is not removed from razor blades used in trimming and glass strips are not adequately cleaned with a non-filming soap and rinsed well enough. Additionally, as glass knives are prepared they become contaminated from oily fingers and trough tape adhesive. Trough fluids must be pure double distilled water and one must be careful not to contaminate the fluid by introducing dirty instruments such as grids, forceps and eyelashes into the boat. If the trough fluid is dirty, section contamination is inevitable. Once sections are collected on grids, they must be stored in a dry, dust free environment such as a grid box. Care should be taken not to drop the grids. Keep a layer of Ross lens tissue under the work area as you handle the grids. If you should happen to drop the grid, at least it will be onto a non-linting paper. Be especially careful when post- staining which is a common source of section contamination.

Materials Required for Ultrathin Sectioning

The following is a complete list of all the materials required in the EM lab to conduct ultrathin sectioning and all related procedures such as block trimming and glass knifemaking.

• Block Trimming

a. Properly embedded tissue within resin block

b. Small (50ml) beaker containing acetone for the removal of razor blade oil

c. Double edged razor blades (cleaned with acetone prior to use)

d. Ross lens tissue (used to dry and clean blades before and during use)

e. Microtome chuck (collet, etc.) to hold block

f. Trimming base plate for supporting the chuck while trimming

g. Pliers (to squeeze block out of BEEM capsule)

h. Small metric ruler to estimate size of trimmed blockface

i. Dissecting microscope with overhead illuminator (such as Bausch & Lomb)

• Glass Knifemaking

a. Plate glass strips (LKB/Leica recommended)

b. Liquid soap (non-filming such as Liquinox)

c. Distilled water for final rinsing

d. LKB/Leica Knifemaker (7800 series or more recent model)

e. Dissecting microscope with overhead illuminator for knife edge evaluation

f. Mylar boat tape (or strips of electrical tape which are cut in half lengthwise after attachment to a clean strip of plate glass)

g. Nail polish for sealing the boats

h. Razor blade for cutting excess boat tape

UNIT 1 PREPARATION OF BIOLOGICAL SAMPLES FOR TEM

47

• Ultramicrotomy

a. Trimmed block (0.25 - 0.5mm on the longest side with two sides parallel)

b. Glass fracture knives with attached and sealed boats (2-4 knive are recommended with at least 1/3 to 1/2 quality cutting edge on left side as previously evaluated)

c. Ultramicrotome (complete with microscope head, stage, glass knife holder, cold light source, and any required accessories)

d. Double distilled water in sterile centrifuge tube (place in test tube rack)

e. Clean micropipette or tuberculin syringe to fill boat

f. Eyelash to pick up thick sections and to orient thin sections in boat

g. Ross lens tissue (to clean and regulate meniscus level in boat)

h. Acetone cleaned 200, 300, 400 mesh grids (on clean filter paper in a covered petri dish)

i. Jeweler’s forceps with an o-ring lock

j. Filter paper in covered petri dish to blot grid dry after section collection

k. Grid gripper in covered petri dish for temporary storage of grids (quality grids should later be moved to a grid box for permanent safe storage)

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Chapter 3 - Post-Staining

In order to form an image, enhancement of contrast (differences in optical or grain density) is necessary, especially when an ultrathin section is considered. In light microscopy, colored dyes/stains are utilized in order to improve contrast and impart color differences. In the preparation of biological samples for the TEM, electron dense, heavy metal stains are used.

The most common staining employed is double post-staining, meaning that two stains are used after the sections are collected on grids. Another technique which is readily found in the literature involves en bloc staining of tissue blocks before the blocks are embedded in the epoxy resin(s). Typically, tissue blocks are stained en bloc using aqueous 0.25% to 4% uranyl acetate solutions for 5-60 minutes just prior to dehydration in ethanol (after osmium tetroxide post-fixation, tissues should receive a buffer wash).

Post-staining involves the use of the salts of the heavy metals, lead and uranium. Caution should be used in handling these heavy metal stains since they are extremely toxic, especially in powdered form. Uranyl salts are low-level radiation emitters. Disposal of heavy metals should be done in accordance with local regulations.

The usual stains employed are uranyl acetate, followed by lead citrate. Uranyl acetate and lead citrate react will many cellular components including proteins and nucleic acids. If

one does not stain with uranyl acetate, the nuclei are most affected with a noticeable lack

of contrast due to weak or no staining of the nucleic acids contained within. Both stains

are said to be positive stains since they increase the density of the structures under consideration as opposed to the background. In negative staining, the background density is increased relative to the structures (as in the negative staining of virus particles using 1-2% phosphotungstic acid - PTA). The preparation of stain solutions is described below.

Uranyl acetate

A 0.5% aqueous solution of uranyl acetate is prepared by dissolving 0.2g of uranyl acetate

in 40ml of distilled water in an Erlenmeyer flask (50ml) and gently mixing over a 10 minute period. The solution will be clear and yellow with no notable sediment. The solution is photosensitive and should be stored in the dark at room temperature. The solution may be filtered or centrifuged just prior to use, however, this is usually not necessary so long as you take the solution from the center of the given volume. It should be noted that uranyl acetate solutions can also be prepared in methanol (saturated solution), ethanol or acetone and in volumes less than 40ml. Many investigators will prepare small volumes as needed to reduce the possibility of contamination.

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Lead Citrate

There are a number of formulations for lead citrate with the most widely used developed by Reynolds (1963) and Venable and Coggeshall (1965). In the Reynolds formulation, 1.33g of lead nitrate and 1.76g of sodium citrate are added to 30ml of CO 2 -free, double distilled water in a 50ml Erlenmeyer flask and mixed regularly over a 30 minute period. At this point, 8ml of 1N (4%) NaOH is added with an obvious “clearing” of the solution upon

mixing. Finally 12ml of the CO 2 -free, double distilled water are added with gentle mixing. The final working solution should be placed into sterile centrifuge tubes. The tubes should be filled to capacity to minimize CO 2 bearing air from coming in contact with the solution. The tubes containing the solution should be stored in the cold at 4˚C and centrifuged for a least 5 minutes just before use. The final pH of the solution should be checked and ideally be alkaline at 12 or higher.

The main problem associated with the use of lead citrate is contamination. Lead citrate readily reacts with atmospheric CO 2 to form a crystalline precipitate known as lead carbonate. On your sections, lead carbonate deposits are extremely electron dense leading to large, unsightly dark staining artifacts which obscure detail. One should avoid breathing directly on the lead citrate solutions. As you stain, you should breathe out of the side of your mouth and also minimize the time of lead exposure to the air.

The Venable-Coggeshall formulation allows you to prepare smaller quantities of the solution as it is needed. In this recipe, 0.01 to 0.04g of lead citrate are added to 10ml of CO 2 -free, double distilled water in a centrifuge tube along with 0.1ml of 10N (40%) NaOH and mixed.

In order to prepare CO 2 -free distilled water, it can be autoclaved and sealed right after removal or it can be boiled for at least 10 minutes and then sealed while hot. Allow the water to come to room temperature before solution preparation.

Post-staining Procedure

Grids can be post-stained within 15 minutes following section collection, after the grids have air-dried. The following procedure should be used with care taken not to contaminate work areas with toxic heavy metal stains (fig. 5).

• Using a squirt bottle, sprinkle water on the table surface.

• Take a precut 4" by 4" square of Parafilm and pull along the water surface to produce a smooth, flat sheet without air bubbles. The sheet should be spread smooth with the

protective cover (imprinted with the product name) in place as to prevent water from contacting the parafilm surface. Once flattened, the protective cover is removed and the film is covered with a Petri dish lid.

• Place 10-15 pellets of NaOH (caution - caustic, causes severe burns) at the top of the

parafilm sheet and recover with the petri dish lid. The NaOH should be wetted slightly and will serve to absorb atmospheric carbon dioxide, reducing the possibility of lead

carbonate formation and contamination.

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

• Using a Pasteur pipette, place as many drops of aqueous Uranyl acetate stain in a

horizontal row to correspond with the number of grids to be stained, not to exceed five (5) drops per setup. If one holds the tip of the pipette near the surface of the parafilm and has good control over the pipette bulb, the diameter of the drops can be adjusted. The drops should be just slightly larger in diameter that the 3.0mm grids themselves. The UA drops should be placed in a row just below the NaOH pellets without coming in contact with the NaOH.

• Using a different pipette, place an identical row of CO 2 -free distilled water drops below each drop of the UA without placing them too closely together. Then, skip a row (leaving room for a row of lead citrate) and form two (2) more identical rows of the CO 2 -free

distilled water.

• Without direct breathing on the solution or staining setup, lay out the row of lead citrate directly below the first distilled water row using a clean, new pipette. Recover with the petri dish lid.

STAINING: Place a grid section (dull) side down in a drop of UA using jeweler’s forceps

and stain for 15 minutes with the petri dish cover in place. From 1 to 5 grids can be stained simultaneously. After 15 minutes the first grid in UA can be carefully picked up on edge and blotted on edge with a filter paper circle. The filter paper circle should be placed between the tines of the forceps which hold the grid. The circle can now be more accurately controlled in its movement to contact the edge of the grid. A small amount of stain will be absorbed onto the filter paper. The blotted grid can be placed into the water drop below the UA drop for rinsing (30-60 seconds). Repeat for each grid as necessary (up to a total of five).

As blotting commences, the filter paper circle should be rotated to a clean area and eventually discarded. The tines of the forceps can be cleaned regularly throughout the process by blotting them on a clean filter paper region. After the first water rinse, blot each grid on edge as described and move them into the lead citrate stain for up to 5 minutes (DON’T BREATHE!!). After the lead citrate, the grids can be blotted and moved through two water rinses for approximately 1 minute each. After the final water rinse, the grid is blotted on edge and placed section (dull) side up on a grid gripper or stored on edge in a grid box to dry. After post-staining, grids can be observed using the TEM within 15 minutes, when they have dried.

Be extremely careful not to damage the grids during staining. Since the grids are handled quite often during staining, it is common for beginners to mangle their grids. This is not desirable considering all of the work that has come before the staining process.

If you encounter a large amount of staining artifact, you can try using a two setup method where one setup contains UA followed by three rows of water (without NaOH pellets) and the other contains NaOH pellets, a row of lead citrate and three rows of water. This setup minimizes lead citrate exposure to the air. Some investigators will use a rinse row of 0.01N NaOH below the lead citrate in this setup, however, some loss of lead staining may result.

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Fig. 5

Parafilm (4"x4")

SAMPLES FOR TEM 51 Fig. 5 Parafilm (4"x4") Petri Dish NaOH Pellets Uranyl Acetate (15 min)

Petri Dish

NaOH Pellets

Uranyl Acetate (15 min)

Distilled Water (30-60 sec)

Lead Citrate (2-5 min)

Distilled Water (30-60 sec)

Distilled Water (30-60 sec)

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Chapter 4 - Grids & Grid Supports

Grids

Grids are typically 3.0mm in diameter and can be made of any non-magnetic metal such as gold, platinum and nickel. The most common metal used in the manufacture of grids is copper. They come in a variety of mesh sizes (referring to the number of bars per inch) and mesh patterns. Common grid mesh sizes are 200, 300, 400, up to 1000. It is recommended that large sections in ribbons be collected on 200 mesh or larger size grids. The ribboning helps support the sections and larger mesh sizes translate into a larger viewing area (less section regions covered by grid bars). Isolated sections should be picked up on 300-400 mesh grids in order that they be supported and not wrap around grid bars. Square mesh grids are routine, however, hex-mesh and other shapes are available. Finder-grids are also available with reference coordinates to help you relocate previously examined sections. Open hole and slot grids, which must be coated (see grid supports below), are useful for serial sectioning.

Grids should be cleaned before use by sonicating them for 5 minutes in a small beaker of acetone. After sonication, the excess acetone is poured off and the beaker is placed upside down on a 9cm diameter filter paper circle in the large diameter lid of a petri dish. As the acetone evaporates, the clean grids will drop to the filter paper surface. Use the other half of the petri dish to cover the grids and label the their type and size with a marker.

Grid Supports

The use of modern epoxy resins in the embedding of biological samples for the TEM has reduced the need for grid support films. The three-dimensional polymerization of epoxy resins with the addition of the proper curing agents and heat (60˚C) or UV, results in an embedment which is resistant to damage from solvents and heat, including the electron beam. Ultrathin sections are therefore very stable under the electron beam. Embedding media which were used in the past for TEM, such as the methacrylates, were unstable under the primary beam with the resulting ultrathin sections requiring a support. Even with the use of epoxy resins, grid supports may be necessary. A good example involves the process of serial sectioning. Serial sections allow us to follow a particular structure through many ultrathin sections in order to develop an understanding of its three- dimensional architecture. Since grid bars could block the ability to view the structure(s) of interest, typical square-mesh or hexagonal-mesh grids are not employed for the collection of serial sections. Instead, an open slot or circle grid is used (see below). The large open area cannot support sections, therefore, a grid support must be prepared.

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Square Mesh Grid
Square Mesh Grid

Hole/Circle Grid

SAMPLES FOR TEM 53 Square Mesh Grid Hole/Circle Grid Slot Grid The most common type of

Slot Grid

FOR TEM 53 Square Mesh Grid Hole/Circle Grid Slot Grid The most common type of support

The most common type of support used today is either plastic - Formvar (polyvinyl formal) or carbon films or a combination of both.

Formvar Films

Working solutions of formvar in ethylene dichloride (0.25%) can be purchased directly from EM supply companies or you can prepare the solution yourself by placing 0.25g of formvar powder into 100ml of ethylene dichloride and gently mixing. The solution should be clear with no sediment and stored in a brown bottle out of direct sunlight and heat. CAUTION: Ethylene dichloride is flammable and is a potential carcinogen. When ready for use the solution should be transferred to a clean coplin jar.

Supplies:

0.25% Formvar solution in Coplin jar clean 400ml plastic beaker (tri-pour) Glass Microscope Slides Ross Lens Tissue Single-edged Razor Blade Liquinox soap (non-filming) Distilled water Grids (to be coated) Jeweler’s Forceps

The glass slides are cleaned using Liquinox soap, rinsed in distilled water and allowed to stand on end and air dry. The plastic beaker is then filled completely (about overflowing) with distilled water and returned to the work area. A clean glass slide is dipped slowly into the formvar solution and withdrawn. The end of the slide is placed on Ross lens tissue to remove the excess solution and allowed to air dry for a few minutes (some individuals gently wave the slide to hasten drying). The slide is now scored with a single-edged razor blade just inside the periphery of the glass slide and across the slide into three to four square regions (fig. 6). The slide is picked up (scored side up) and slowly introduced into the distilled water of the plastic beaker at a very shallow angle (20-30˚). Prior to the introduction of the formvar coated slide, the distilled water surface is cleaned by pulling a sheet of Ross lens tissue across the water surface, as was done to clean the water surface of the glass knife boat used in ultramicrotomy. Just before placing the formvar coated slide into the distilled water, one can breathe on the scored surface. “Frosting” the surface

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

in this way is believed to facilitate stripping of the formvar off of the glass slide. Under fluorescent lighting, you should observe floating formvar “sections” in the beaker. These floating films should appear of a uniform silver color under fluorescent lighting. If the films are dark gold, purple, etc., the solution must be cut with the addition of ethylene dichloride. If the films are too thin, you can add more powdered formvar to the solution. Formvar films which display a variety of colors (rainbow) may be contaminated and a new solution should be freshly prepared.

The greatest difficulty in working with these plastic films involves their stripping from the glass slide when placed into the distilled water. Exceptionally clean slides are not ideal for this purpose. You can also experiment with slides of different manufacturers as some prove better than others. To facilitate formvar stripping, a thin and even coating of oil from the skin (usually best when taken from alongside the nose) can be applied by index finger to one surface of the slide (make sure you know which surface is oil-coated since this is the side you will want to score). One can also use a thin coating of saliva on one side of the slide. Both techniques work well to strip the formvar from the slide and it should be noted that contamination of the formvar does not occur as a result.

Once the formvar films are cast on the water surface, the slide can be dropped to the bottom of the beaker. Using the fine forceps, grids can be carefully placed, dull side down, on the floating formvar film. Concentrate the grids at one end of the film without overlapping them. The final technique involves collecting the coated grids, dull side up (with the formvar surface on top). Some individuals use a large container such as a fishtank to submerge the films from above with a glass slide. The slide is swept through the water in a large “U” fashion and is removed with the coated grids on top of the slide. Others use a rapid “slap and flip” technique with a glass slide to collect the coated grids. The simplest method involves placing a clean glass slide or wax coated cardboard strip exactly perpendicular (90˚) to the floating formvar film, in the region without any placed grids and gently submerging the slide using a straight downward force (fig. 7). This film with the coated grids just rolls up on the slide with the grids in the correct orientation (dull side up). The slide is removed from the water and allowed to air dry. Once dry, the grids are picked up from the slide surface and either used immediately or stored for later use.

Fig. 6 - Formvar Coating on Glass Slide

Formvar Coating Glass Slide Razor Blade Score Lines
Formvar Coating
Glass Slide
Razor Blade Score Lines

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Fig. 7 - Collection of Formvar Coated Grids

Glass Slide Slot Grids (Dull Side Down)
Glass Slide
Slot Grids (Dull Side Down)

Clean DH2O Surface

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Carbon Coating

Carbon films are strong and extremely stable under the electron beam. Additionally, they can be made very thin and are essentially electron transparent. Carbon coating can be carried out alone or on top of plastic films in order to provide increased stability. Some investigators also “sandwich” various support films around the ultrathin sections (plastic- sections-plastic, plastic-sections-carbon).

Carbon coating is usually conducted in a high vacuum evaporator. The high vacuum created in the bell jar is required so that air molecules will not interfere with the evaporated carbon and lead to an uneven coating. Two pure carbon rods are prepared so that one is sharpened to a point and the other is flat on end. The rods are locked into the two suitable electrodes in the evaporator with the pointed end of one rod in direct contact with the flat end of the other. Beneath the carbon rods are the grids you wish to coat on a glass slide and a white porcelain plate with a drop of low vacuum oil or immersion oil at its center. The bell jar is evacuated to 10-5 Torr and voltage is applied to the electrodes. At the pointed carbon rod tip, it becomes white hot and carbon is evaporated. The carbon falls to evenly coat the grids. Thickness can be determined by the darkness of the porcelain plate in comparison to the central oil spot. Due to the presence of the oil, the area beneath it will not darken but rather, remain a bright white. The desired color of the indicator plate is described as a light tan/brown which results in a 400-700Å thick coat (fig. 8).

Since carbon does not adhere well to naked copper grids, an initial coating of plastic such as formvar is recommended. If desired, the plastic coat can later be dissolved away in chloroform and ethylene dichloride.

Carbon coating attachments are also available for low vacuum sputter coaters (see discussion of sputter coating in Unit 2 on SEM Sample Preparation). Carbon coating of SEM samples over metallic coating (gold, etc.) is required when elemental analysis is being conducted.

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Fig. 8 - Carbon Evaporation Setup

Bell Jar carbon rods electrode oil grids
Bell Jar
carbon rods
electrode
oil
grids

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Unit 2 - Preparation of Biological Samples for SEM

Since the same high vacuum environment (10 -5 Torr) is required in order to generate an electron beam in the conventional SEM as is required for the TEM, the observation of living samples is not usually possible (environmental ESEM’s and differentially pumped, low vacuum SEM’s may permit the observation of living samples). However, because the SEM is utilized primarily in the observation of surface features, the tedium of ultra-thin sectioning, necessary to TEM specimen preparation, is avoided. Depending on the nature of the surface of interest, a sample might be prepared simply by adhering it to an aluminum support or stub and examining it in the SEM. Of course, such a sample would have to be hard and conductive, characteristics rarely found together in biological specimens. Common examples of hard surfaces include chitinous insect and crustacean exoskeletons, lignified cellulose cell walls of plant material/wood, calcium carbonate shells of molluscs/bivalves such as oysters and clams, silicon frustules (shells) of diatoms, and complex calcium and phosphorus hydroxyapatites which comprise part of the non-living matrix of bone. A problem exists in that these and other biological samples are usually insulators which are incapable of emitting an adequate signal when contacted with the SEM primary electron beam. Lack of adequate signal leads to an inferior quality image (low SNR). In addition, there are many occasions when one wishes to examine a soft tissue sample which would readily degrade under the high vacuum operating environment of the SEM, not to mention, be subject to the forces of autolysis and decomposition. Another factor to consider is whether the visualization internal structure using a SEM is desirable or even possible.

Since ultra-thin sectioning is not required for SEM samples, they can typically be larger (recall that TEM tissue blocks had to be minced to a thickness no greater than 0.5mm in one dimension to ensure complete infiltration of fixatives and embedding media). When dealing with soft tissues, the tissue pieces should remain somewhat small (~2-3mm 3 ) to allow for complete penetration of the primary fixative and prevention of internal collapse and resultant artifacts in surface morphology (shape). Also of importance is the handling of small samples and microbes. The following chapters shall be divided into the preparation of a variety of samples including hard tissues/structures, soft tissues, internal structures, microbial samples and SEM samples prepared for the TEM for SEM/TEM correlation.

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Chapter 5 - Hard Tissue Preparation

As stated earlier, hard tissue samples can include chitinous insect and crustacean exoskeletons, lignified cellulose cell walls of plant material/wood, calcium carbonate shells of molluscs/bivalves such as oysters and clams, silicon frustules (shells) of diatoms, the non-living matrix of bone and teeth. Such samples may have adherent soft tissue and/or surface debris associated with them. This material should be removed using as delicate a technique as possible. Surface lint and dust may be easily removed with compressed air (care should be taken since some compressed air sources contain particulates which could induce surface damage). In addition, surface debris can be rinsed away using distilled water or an isotonic physiologic buffer solution (the need for an isotonic solution is questionable since hard tissues are unlikely to shrink or swell osmotically). In the case of soft tissue adhering to samples such as bone, the soft tissue can be dissolved away by boiling in 28% ammonium hydroxide followed by subsequent rinses in distilled water.

CAUTION: Ammonium hydroxide must be opened and used in a fume hood. Due to its extremely high vapor pressure, a bottle of 28% ammonium hydroxide opened in even a well ventilated room will quickly saturate the atmosphere with toxic ammonia. Inhalation could prove to be fatal.

Following cleaning, which may not be necessary, hard tissue samples are mounted to a standard 15mm diameter, aluminum specimen mount or stub, using an appropriate adhesive (types of suitable adhesives will be covered later in this unit).

Depending on specimen conductivity, or the usual lack of it when dealing with biological samples, and the desired SEM imaging voltage (low 5kv or high > 5kv to 25kv), the mounted samples will then be given an ultra-thin (100 - 200Å) conductive coating of a metal such a gold, platinum, palladium, or gold-palladium. Since most samples are generally insulators and high voltage is usually selected for maximum signal generation and the resultant high quality images, the norm involves the application of this conductive coating. The techniques and procedures of sputter coating and vacuum evaporation will be covered in detail later in this unit, in conjunction with soft tissue preparation.

It should be noted that even though hard samples are inherently more durable than their soft tissue counterparts, care should be exercised in handling these samples so as not to induce artifacts. This is especially true of samples such as small insects which should be gently attached to the specimen stubs using fine forceps. The use of liquid adhesives for small samples should be avoided since the liquid has a tendency to creep up the sides of the specimen and envelope it. This is not a problem if one wishes to study glue!

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

Chapter 6 - Soft Tissue Preparation

In order to examine soft tissue samples in “as near to life-like condition as possible”, chemical fixation is required. Without chemical fixation, post-mortem autolysis and decomposition would distort even the surface of the specimen of interest. Even with the surface “stabilized” through conductive coating, internal degradation would lead to glaring artifacts of surface morphology. In addition, chemical fixation will permit observation of internal structures using cryo (cold) techniques and allow for the correlative preparation of SEM samples for the TEM via epoxy resin embedment and ultra-thin sectioning.

The preparation of soft tissue samples for examination in the SEM begins in an identical manner to the preparation of samples for the TEM (see Unit 1 - Preparation of Biological Samples for TEM). A generalized protocol follows:

Primary Aldehyde Fixation Buffer Wash Secondary Osmium Tetroxide Postfixation Optional Buffer/DH2O Wash Dehydration Series (Ethanol/Acetone) Intermediate Fluid Series (Freon TF/113) Critical Point Drying (in Transitional Fluid - liquid CO2 or Freon 13) Mounting (adhesive on 15mm dia. aluminum stub) Conductive Coating (Sputter Coater or Vacuum Evaporator)

Firstly, the tissues of interest must be excised and placed into the primary fixative. As for TEM preparation, we typically use a combination method of in situ and immersion fixation. The tissue blocks for SEM preparation can be somewhat larger since the aldehydes (primary fixative) can penetrate through at least 3mm of sample. Tissue blocks are therefore best minced to the dimensions of 2-3mm 3 . If strips of tissue are cut, they should be no thicker than approximately 2-3mm on one of the sides.

Minced tissue blocks can be transferred to vials containing the primary aldehyde fixative and remain for one hour. Once again the best single aldehyde choice is the doubly reactive, cross-linking, glutaraldehyde. As for TEM sample primary fixation, we use a 3% concentration of glutaraldehyde carried in the proper isotonic buffer vehicle (for most soft mammalian tissues, 0.2M phosphate buffer, pH 7.2-7.4 is ideal). The chemical action of aldehydes (stabilization of the cellular protein matrix) and other fixatives along with the purpose of buffer solutions and their preparation are covered in the chapters on TEM specimen preparation.

Once the primary fixation is complete, three buffer washes of ten minutes duration each are conducted to remove unbound aldehyde and prevent the undesirable precipitate reaction with the secondary fixative, namely, osmium tetroxide.

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Following the buffer washes, the samples are placed into the secondary fixative, osmium tetroxide. The OsO 4 will react mainly with unsaturated lipids and impart an increase in conductivity to the biological sample. The OsO 4 is also carried in the same buffer vehicle as for the glutaraldehyde. You will recall that the benefit to osmium tetroxide fixation for TEM was the introduction of an electron dense stain (for phospholipid membranes and other osmophilic structures) for added contrast. In SEM, the generation of a signal from the surface is of paramount concern. Incorporation of the heavy metal osmium to the sample allows for increased surface signal emission when contacted by the primary electron beam of the SEM. Later in this unit, I will discuss how osmium incorporation can be enhanced by the addition of agents such as tannic acid and thiocarbohydrazide (TCH), so much so, that conductive surface coating may be avoided.

An optional buffer or distilled water wash of two changes for ten minutes duration each may be performed, although, I have not observed any artifacts arise due to the omission of this step.

Next, the tissue must undergo complete dehydration, but not for the same reason as TEM sample dehydration. Residual water in the tissue would cause surface tension collapse as it dried. For this reason, the common process of critical point drying - CPD (or some alternative) is employed in the preparation of samples for the SEM. The CPD process, which is carried out under liquid carbon dioxide, would be ineffective if water remained in the tissue block. A complete description of CPD theory and procedure is found later in this unit. By contrast, you will recall that samples are dehydrated for TEM in order that the epoxy resins infiltrate the tissue blocks (epoxies are not miscible with water). Both acetone and ethanol (EtOH) are common dehydrating agents used. Since shrinkage of tissue blocks and the resultant surface distortion is undesirable for SEM specimens, the dehydration schedule is more gradual for SEM, starting at 30% EtOH. The usual ascending series is 30%, 50%, 70%, 95% EtOH for ten minutes each, followed by 100% EtOH for two changes of ten minutes each with the vials being filled to capacity as usual. The preparation of such a dehydration series using 95% (not 100%) ethanol has been described in the unit on TEM specimen preparation.

The final steps in the soft tissue protocol will involve the preparation of tissue blocks for critical point drying which has traditionally been performed in either liquid carbon dioxide or liquid freon (Freon 13). Usually, before the critical point drying process, tissue blocks are passed through an ascending intermediate fluid series of Freon TF/113 since it is miscible with both ethanol and the transitional fluid used in critical point drying. The freon is diluted with 100% ethanol, not water, in order to produce the concentration series. The usual ascending series is 30%, 50%, 70%, 95% Freon TF/113 for ten minutes each, followed by 100% Freon TF/113 for two changes of ten minutes each.

As the scientific community has become more aware of the environmental impact of freons on the degradation of the protective ozone layer (which partially shields the earth from harmful UV radiation), alternatives to its use have been discovered and will be considered following the discussion of critical point drying. A common past alternative to Freon TF

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ELECTRON MICROSCOPY: A HANDBOOK OF TECHNIQUES FOR THE BIOLOGIST

was amyl acetate with its characteristic strong, banana-like odor. It was typically used as an intermediate fluid between ethanol and liquid CO 2 . Due to its toxicity, it should always be purged out of a critical point dryer into a fume hood. A benefit to its use was that one always knew it had been completely purged from the critical point dryer be the marked absence of its trademark odor.

The process of chemical fixation of biological soft tissues have been carried out in a liquid environment. If in the final step, these tissues are simply allowed to air dry, tremendous surface tension distortion will occur leading to obvious surface artifacts. It should be noted that water can exert a surface tension force of over 2,000 psi. In order to prevent this surface tension damage, the technique of critical point drying (CPD) has been utilized since 1968.

The principle of CPD involves an understanding of phase (solid-liquid-gas) boundaries, especially, for our purposes, the boundary between liquid and vapor. Every fluid possesses what is known as a critical density (D c ) at which the boundary or interface (the actual liquid surface) between the liquid phase and the vapor phase becomes indistinguishable. At first, when a fluid is introduced to a sealed container, an equilibrium exists between the liquid and vapor phases. In order to attain critical density, this equilibrium must be shifted to the vapor phase through heating and the related increase in pressure. Critical density (D c ) is attained at the critical temperature (T c ) and critical pressure (P c ) of the given fluid. Therefore, to eliminate the interfacial boundary between liquid and vapor and avoid the associated surface tension force, the fluid must be elevated to its critical temperature and pressure.

Since the critical temperature and pressure of water is excessive, at 374˚C and 3,184 psi respectively, and would damage delicate biological soft tissues, other liquids typically serve as transitional fluids (so named because they make the transition between liquid and vapor phases). The most common of these is liquid carbon dioxide (LCO 2 ) and liquid freon 13. Due to its higher price and negative impact on the environment, liquid freon has become the less popular of the two transitional fluids. Liquid CO 2 has a critical temperature and pressure of 31˚C and 1,073 psi, respectively, compared with liquid freon 13 at 28.9˚C and 561 psi. The liquid CO 2 is introduced to the critical point dryer at the high pressure (600 -800 psi) of its storage tank. The storage tank is specially ordered with a siphon tube which takes the liquid CO 2 from the bottom of the tank since as the tank is emptied, CO 2 gas rises and collects at the top of the tank.

CAUTION: Liquid carbon dioxide is under high pressure and is very cold. Use care in handling the tanks and opening the tank valve. The use of a regulator is not required, however, the tank pressure is between 600-800 psi. Make sure the hose between the CPD and the tank is threaded and tightened properly, and is a hose rated for high pressure applications (a minimum burst pressure rating should be printed on the hose - the hose used in this lab is rated at 17,000 psi). In addition, the storage tank

UNIT 2 – PREPARATION OF BIOLOGICAL SAMPLES FOR SEM

63

should be chained or strapped to a wall or a tabletop to prevent it from accidentally falling and rupturing. Under its high pressure, the tank could act as a missile causing serious injury. Finally, CO2 is a colorless, odorless gas and should be vented from the CPD into a fume hood to prevent asphyxiation as it displaces the normal atmosphere.

Critical Point Dryer Operation

The CPD is a thick walled metal chamber which can withstand pressure in excess of 2,000 psi. The chamber is constructed of a metal of excellent heat conductive properties such as brass, copper or bronze. The chamber is usually machined to accommodate some type(s) of specimen holder(s) which will retain the tissue blocks throughout the procedure. The chamber also includes three high pressure needle valves, the inlet valve (for the introduction of the LCO 2 ), the drain valve (to drain off the intermediate fluid - freon TF/ 113) and the vent valve (to vent the CO 2 gas). Additionally, the CPD chamber must have some provision for heating the transitional fluid. In the more expensive models, there is an electrical heater. In the least expensive models, the entire CPD chamber is simply lowered into a container or bucket of hot water. The main drawback to this method is that residual external water on the unit might contact the tissues as they are removed, rehydrating them. Our Pelco Jumbo Dryer (same as the Polaron Jumbo) is designed with a water jacket just external to the drying chamber. Hot and/or cold water can be routed through the jacket by means of plastic tubing attached to a faucet. A temperature gauge is added in order to monitor the water temperature, although, the only required gauge is a chamber pressure gauge. The Pelco CPD is equipped with a safety valve which is designed to rupture at 2,000 psi.

CAUTION: The CPD is a high pressure device which if used improperly can lead to serious injury or death. Follow all manufacturer directions carefully. If equipped with a thick quartz view window (as in the case of the Pelco Jumbo Model), it MUST BE EXAMINED FOR CRACKS