OPTICAL MICROSCOPE

1.0 Introduction

The light microscope, so called because it employs visible light to detect small objects, is probably
the most well-known and well-used research tool in biology. Yet, many students and teachers are
unaware of the full range of features that are available in light microscopes. Since the cost of an
instrument increases with its quality and versatility, the best instruments are, unfortunately,
unavailable to most academic programs. However, even the most inexpensive "student"
microscopes can provide spectacular views of nature and can enable students to perform some
reasonably sophisticated experiments.

A beginner tends to think that the challenge of viewing small objects lies in getting enough
magnification. In fact, when it comes to looking at living things the biggest challenges are, in
order,

obtaining sufficient contrast

finding the focal plane
obtaining good resolution
recognizing the subject when one sees it

The smallest objects that are considered to be living are the bacteria. The smallest bacteria can be
observed and cell shape recognized at a mere 100x magnification. They are invisible in bright field
microscopes, though. These pages will describe types of optics that are used to obtain contrast,
suggestions for finding specimens and focusing on them, and advice on using measurement
devices with a light microscope.
2.0 Type

There are two basic types of optical microscopes: simple microscopes and compound microscopes.
A simple microscope is one which uses a single lens for magnification, such as a magnifying glass.
A compound microscope uses several lenses to enhance the magnification of an object. The vast
majority of modern research microscopes are compound microscopes while some cheaper
commercial digital microscopes are simple single lens microscopes. Compound microscopes can
be further divided into a variety of other types of microscopes which differ in their optical
configurations, cost, and intended purposes.

(a) Simple microscope

A simple microscope uses a lens or set of lenses to enlarge an object through angular
magnification alone, giving the viewer an erect enlarged virtual image. The use of a single
convex lens or groups of lenses are still found in simple magnification devices such as the
magnifying glass, loupes, and eyepieces for telescopes and microscopes.

Figure 1: Diagram of a simple microscope

(b) Compound microscope

A compound microscope uses a lens close to the object being viewed to collect light (called
the objective lens) which focuses a real image of the object inside the microscope (image
1). That image is then magnified by a second lens or group of lenses (called the eyepiece)
that gives the viewer an enlarged inverted virtual image of the object (image 2). The use of
a compound objective/eyepiece combination allows for much higher magnification.
Common compound microscopes often feature exchangeable objective lenses, allowing
the user to quickly adjust the magnification. A compound microscope also enables more
advanced illumination setups, such as phase contrast.

Figure 2: Diagram of a compound microscope

 3.0 Components

Figure 1: Components of optical microscopy

Eyepiece Lens: the lens at the top that you look through. They are usually 10x or 15x power.
Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has
a mirror, it is used to reflect light from an external light source up through the bottom of the stage.
Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If your
microscope has a mechanical stage, you will be able to move the slide around by turning two
knobs. One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can
be rotated to easily change power.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost
always consist of 4x, 10x, 40x and 100x powers.
Rack Stop: This is an adjustment that determines how close the objective lens can get to the
slide. It is set at the factory and keeps students from cranking the high power objective lens down
into the slide and breaking things.
Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen.
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has
different sized holes and is used to vary the intensity and size of the cone of light that is projected
upward into the slide. There is no set rule regarding which setting to use for a particular
power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast
you desire and the particular objective lens in use.

4.0 Operation

Figure 3: Principle of optical microscopy.

The optical components of a modern microscope are very complex and for a microscope to
work well, the whole optical path has to be very accurately set up and controlled. Despite this,
the basic optical principles of a microscope are quite simple.
The objective lens is, at its simplest, a very high powered magnifying glass i.e. a lens with a
very short focal length. This is brought very close to the specimen being examined so that the
light from the specimen comes to a focus about 160 mm inside the microscope tube. This
creates an enlarged image of the subject. This image is inverted and can be seen by removing
the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing
a rather dim specimen, a highly-enlarged image can be seen. It is this real image that is viewed
by the eyepiece lens that provides further enlargement.
In most microscopes, the eyepiece is a compound lens, with one component lens near the front
and one near the back of the eyepiece tube. This forms an air-separated couplet. In many
designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first
lens bringing the real image to a focus and the second lens enabling the eye to focus on the
virtual image.
In all microscopes, the image is viewed with the eyes focused at infinity (mind that the position
of the eye in the above figure is determined by the eye's focus). Headaches and tired eyes after
using a microscope are usually signs that the eye is being forced to focus at a close distance
rather than at infinity.

5.0 Resolution
The resolution of the light microscope cannot be small than the half of the wavelength of the
visible light, which is 0.4 - 0.7 m. The best resolution for an optical microscope is about 0.2
m and equal to 200 nm. The actual power or magnification of a compound optical microscope
is the product of the powers of the ocular (eyepiece) and the objective lens. The maximum
normal magnifications of the ocular and objective are 10 and100 respectively, giving a final
magnification of 1,000.

6.0 Application
This type of microscopy is used in microelectronics, nano physics, biotechnology and
microbiology. It has allowed us to distinguish between different cells and their components,
and also paved the way for the other types of microscopes which have enhanced how we view
the human body and also in the fight against disease
7.0 Micrograph image

Figure 4: Micrograph image of optical microscopy.

Figure 4 show the optical microscope image of a water-in-oil-in-water (W/O/W) multiple

emulsion right after preparation.
SCANNING ELECTRON MICROSCOPE

1.0 Introduction

In scanning electron microscopy (SEM) an electron beam is focused into a small probe and is
rastered across the surface of a specimen. Several interactions with the sample that result in the
emission of electrons or photons occur as the electrons penetrate the surface. These emitted
particles can be collected with the appropriate detector to yield valuable information about the
material. The most immediate result of observation in the scanning electron microscope is that it
displays the shape of the sample. The resolution is determined by beam diameter.

2.0 Basic signal of SEM

The electrons interact with the atoms at or close to sample surface produces signals that contain
information about the sample's surface topography, composition, and other properties such as
electrical conductivity. The types of signals produced by an SEM include secondary electrons,
back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence), specimen
current and transmitted electrons.

Secondary electrons: they are electrons generated as ionization products. They are called
'secondary' because they are generated by other radiation (the primary radiation). This radiation
can be in the form of ions, electrons, or photons with sufficiently high energy, i.e. exceeding the
ionization potential. Secondary electron detectors are common in all SEMs. A SEM with
secondary electron imaging or SEI can produce very high-resolution images of a sample surface,
revealing details less than 1 nm in size.

Back-scattered electrons (BSE): they are beam electrons that are reflected from the sample by
elastic scattering. BSE are often used in analytical SEM along with the spectra made from the
characteristic X-rays. Because the intensity of the BSE signal is strongly related to the atomic
number (Z) of the specimen, BSE images can provide information about the distribution of
different elements in the sample.

Characteristic X-rays: they are emitted when the electron beam removes an inner shell electron
from the sample, causing a higher energy electron to fill the shell and release energy. These
characteristic X-rays are used to identify the composition and measure the abundance of elements
in the sample.

3.0 Components

Figure 5: Components of scanning electron microscope.

Electron Source: Electrons are produced at the source by thermionic heating. These electrons
are then accelerated to a voltage between 1-40 kV and condensed into a narrow beam which
is used for imaging and analysis.
Lenses: A series of condenser lenses focus the electron beam as it moves from the source
down the column. The narrower the beam the smaller the spot it will have when contacting
the surface, thus the term spot size.
Scanning Coil: After the beam is focused, scanning coils are used to deflect the beam in the
X and Y axes so that it scans in a raster fashion over the surface of the sample.
Sample Chamber: Samples are mounted and placed into a chamber that is evacuated. The
sample chamber can include a translation stage, tilt and rotation devices, feed-throughs to the
outside, temperature stages, optical cameras, and a variety of other devices to assist in imaging
the sample.
 SEM Detector: The detectors for SEM collect the electrons coming off of the sample. Two
types of electrons are typically used for imaging: secondary electrons (SE) and backscattered
electrons (BSE)

4.0 Operation
The basic principle is that a beam of electrons is generated by a suitable source, typically a
tungsten filament or a field emission gun. The electron beam is accelerated through a high
voltage (e.g.: 20 kV) and pass through a system of apertures and electromagnetic lenses to
produce a thin beam of electrons. Then the beam scans the surface of the specimen Electrons
are emitted from the specimen by the action of the scanning beam and collected by a suitably-
positioned detector.

5.0 Resolution
In a SEM, an electron beam scans rapidly over the surface of the sample specimen and yield
an image of the topography of the surface. The resolution of a SEM is about 10 nm. The
resolution is limited by width of the exciting electron beam and the interaction volume of
electron in a solid.

6.0 Application
The SEM is routinely used to generate high-resolution images of shapes of objects (SEI) and
to show spatial variations in chemical compositions such as acquiring elemental maps or spot
chemical analyses using EDS, discrimination of phases based on mean atomic number
(commonly related to relative density) using BSE. The SEM is also widely used to identify
phases based on qualitative chemical analysis and/or crystalline structure.
7.0 Micrograph image

Figure 6: Micrograph image of scanning electron microscopy.

SEM image of comb-like nanostructure of ZnO, which is the result of surface polarization
induced growth.
TRANSMISSION ELECTRON MICROSCOPE

1.0 Introduction

The transmission electron microscope is a very powerful tool for material science. A high energy
beam of electrons is shone through a very thin sample, and the interactions between the electrons
and the atoms can be used to observe features such as the crystal structure and features in the
structure like dislocations and grain boundaries. Chemical analysis can also be performed. TEM
can be used to study the growth of layers, their composition and defects in semiconductors. High
resolution can be used to analyze the quality, shape, size and density of quantum wells, wires and
dots.

The TEM operates on the same basic principles as the light microscope but uses electrons instead
of light. Because the wavelength of electrons is much smaller than that of light, the optimal
resolution attainable for TEM images is many orders of magnitude better than that from a light
microscope. Thus, TEMs can reveal the finest details of internal structure - in some cases as small
as individual atoms.

2.0 Specification of TEM

In transmission electron microscopy (TEM), a beam of highly focused electrons is directed toward
a thinned sample (<200 nm). Normally no scanning required helps the high resolution, compared
to SEM. These highly energetic incident electrons interact with the atoms in the sample producing
characteristic radiation and particles providing information for materials characterization.
Information is obtained from both deflected and non-deflected transmitted electrons, backscattered
and secondary electrons, and emitted photons.
3.0 Components

Figure 7: Components of transmission electron microscope.

Electron gun: The function of the electron gun is to provide an intense beam of high energy
electrons.
Lenses: Electron lenses are the magnetic equivalent of the glass lenses in an optical
microscope and to a large extent, we can draw comparisons between the two.
Vacuum Pump: Electron beam must be generated and used in a high vacuum of 10-4 mbars
or less.
Camera and display: The viewing screen is a screen coated with a layer of electro
phosphorescent material. The screen can be tilted and viewed through the binoculars. These
usually magnify the image by about x10 to help resolve fine detail. The camera and screen do
not have to be precisely positioned because the depth of focus of the final lens is very large.
Eucentric Gonimeter: A goniometer stage allows the specimen to be traversed and tilted
simultaneously so that every part of the specimen can be examined at a variety of angles.
A eucentric goniometer stage can be adjusted so that the illuminated region of the specimen
stays the same as the stage is tilted.
4.0 Operation
When a beam of electrons is passed through a specimen, a part of it is transmitted and this
part when projected on fluorescent screen, its image can be seen by the observer. It is a
microscopy technique whereby a beam of electron is transmitted through an ultra-thin
specimen, interacting with the specimen as it passes through. An image is formed from the
interaction of the electron transmitted through the specimen, the images magnified and
focused onto an imaging device, such as fluorescent screen, on a layer o0f photographic film,
or to be detected by a sensor such as CCD camera.

5.0 Resolution
In a TEM, a monochromatic beam of electrons is accelerated through a potential of 40 to 100
kilovolts (kV) and passed through a strong magnetic field that acts as a lens. The resolution
of a TEM is about 0.2 nanometers (nm). This is the typical separation between two atoms in
a solid.

6.0 Application
A Transmission Electron Microscope is ideal for a number of different fields such as life
sciences, nanotechnology, medical, biological and material research, forensic analysis,
gemmology and metallurgy as well as industry and education. TEM provide topographical,
morphological, compositional and crystalline information. The images allow researchers to
view samples on a molecular level, making it possible to analyze structure and texture. This
information is useful in the study of crystals and metals, but also has industrial applications.
TEMs can be used in semiconductor analysis and production and the manufacturing of
computer and silicon chips. Technology companies use TEMs to identify flaws, fractures and
damages to micro-sized objects; this data can help fix problems and/or help to make a more
durable, efficient product. Colleges and universities can utilize TEMs for research and studies.
 7.0 Micrograph image

Figure 8: Micrograph image of transmission electron microscopy.

Figure 8 show the transmission electron microscopy (TEM) image of the as-synthesized SnO2
nanobelts.
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