Visualizing Cells

Learning Objectives ! History of Microscopy (Hooke, Zeiss, Perkin) ! Anatomy of Compound Microscope ! Capabilities & Limitations of Various Technologies ! Fluorescence Microscopy ! Laser Scanning Microscopy ! Fluorescent Proteins

Early Cell Biologists

Robert Hooke (1635 - 1703)

Built one of the first useful compound microscopes Observed structure of cork Coined the term Cell. Published Micrographia (1665)

A Brief History of Optical Microscopy 1665 1678 1838-9 Hooke publishes Micrographia van Leeuwenhoek observes protozoa (little animals) Schleiden & Schwann proposed Cell Theory

1857 1865

Carl Zeiss produces the Stand 1 microscope Perkins invents synthetic (aniline) dyes

Microscopy for the Rest of Us

Carl Zeiss (1816 1888)

Stand 1 Microscope

stem section

mouse fibroblast

A Brief History of Optical Microscopy 1665 1678 1838-9 Hooke publishes Micrographia van Leeuwenhoek observes protozoa (little animals) Schleiden & Schwann proposed Cell Theory

1857 1865

Carl Zeiss produces the Stand 1 microscope Perkins invents synthetic (aniline) dyes

Pioneers of Organic Synthesis

August Wilhelm von Hoffman (1818 1892)

William Henry Perkin (1838 1907)

Early Anti-malarial Drugs

Chichona calisaya

Quinine quina-quina

Mauveine (Perkins Mauve)

3-amino-2,9-dimethyl-5-phenyl-7(p-tolylamino)phenazinium acetate

Imperial (Tyrian) Purple

Justinian I (482 - 565)

Bolinus brandaris (spiny dye-murex snail)

Figure 1-5 Essential Cell Biology ( Garland Science 2010)

Discovery of Chromosomes (Colored Bodies) & Mitosis

Walther Flemming (1843 1905)

Zellsubstanz, Kern und Zelltheilung, 1882

A Brief History of Optical Microscopy 1931 Ruska invents electron microscope

1932 1955

Zerniki develops phase contrast microscopy Minsky invents the laser scanning microscope (LSM)

1989 1995

Webb, Denk & Strickler invent multiphoton LSM Stefan Hell invents Super Resolution Microscopy

Compound Microscope

Detection & Analysis

Magnification

Light Gathering Illumination

Olympus IX71 Inverted Microscope

Olympus BX51 Upright Microscope

Issac Newton

Alhazen

George B. Airy

James C. Maxwell

Albert Einstein

Richard Feynman

Electromagnetic Theory of Light Propagation

James C. Maxwell

Wave-like Properties of Light

refraction

diffraction

Refraction at the Air - Water Interface

Optical Convergence using a Thin Lens

Snells Law For n2 > n1 air water

#

n1sin! = n2sin"# n2 n1 = sin! sin"

nair = 1.0 sin!# n2 = sin"#

nwater = 1.33 nglass = 1.51

Ripple Tank

Diffraction is a characteristic of wave dynamics

barrier

barrier slit Slit ~ wavelength ($)#

Slit ~ 4 $#

Interference

Positive Interference

Negative Interference

Phase Contrast Microscopy

Images of a Fibroblast Bright Field Phase Contrast

Differential Interference Contrast (DIC) Nomarski

Dark Field

Objective Lens captures only a small portion of the light rays that are diffracted and refracted by the specimen.

The optical image is always incomplete

Limit of Spatial Resolution of Optical Microscopy Limit of resolution (D) D= (0.61) $ n sin (!) $!= wavelength of light n = refractive index ! = angular aperture
NOT A FUNCTION OF MAGNIFYING POWER OF LENS

Limit of Spatial Resolution of Optical Microscopy Limit of resolution (D) D= (0.61) $ n sin (!) Numerical Aperture (light gathering ability) N.A. ~ 0.3 - 1.65

Limit of Spatial Resolution of Optical Microscopy Limit of resolution (D) D= (0.61) $ n sin (!) Numerical Aperture (light gathering ability) N.A. ~ 0.3 - 1.65 Higher is Better

Objective Lens

Limit of Spatial Resolution of Optical Microscopy Limit of resolution (D) D= (0.61) $ n sin (!) Wavelength of Light Shorter is Better

Visible Spectrum

Resolution is ultimately limited by the wavelength of the illuminating light

D=
70o

(0.61) $ n sin (!) (0.61) (450 nm) (1.5) sin (70o)

D=

~ 200 nm glass & oil ~ 0.2 m (~ $ /2)

Limit of Resolution approaches ~ ! of the wavelength

Electron Microscopy Wavelength ($) of an (accelerated) electron = 0.004 nm Resolution = $ / 2 ~ 0.002 nm This is 100,000 times better than optical microscopy ~ 200 nm

Array of Au atoms

Light Microscopy visible light glass glass

TEM electrons magnet

magnet

glass retina

magnet

phosphor

Transmission electron microscopy (TEM)

Actin Filaments

Figure 9-54 Molecular Biology of the Cell ( Garland Science 2008)

Scanning electron microscopy (SEM)

Figure 9-52 Molecular Biology of the Cell ( Garland Science 2008)

SEM of a metal cast of a wheat flower. Its not so much about size -Its about resolution

Stereocillia from bullfrog auditory hair cell

DIC

SEM

TEM

Fluorescence Microscopy

mouse fibroblast

mouse fibroblast

Stuff Fluoresces

Fluorescence Spectrum of FITC

Stokes Shift

{
absorbance emission

blue

red

Fluorescence Spectrum of FITC

Stokes Shift

{
absorbance emission

fluorescein isothiocyanate (FITC)

blue

red

Sigma (%) vs Pi (&) Bonding

%#

%#

Filter Cube Enables Fluorescence Microscopy

Filter Cube

Filter Cube Enables Fluorescence Microscopy

Filter Cube

Synthetic Fluorescent Tags (fluorochromes) have been produced that span the entire visible spectrum These are attached to affinity reagents (e.g., antibodies) to label intracellular molecules of interest for examination by fluorescence microscopy

Indirect Immunofluorescence

Figure 9-18 Molecular Biology of the Cell ( Garland Science 2008)

All organic fluors undergo photobleaching

Quantum Dots

Quantum Dots Highly resistant to photobleaching All are excited by UV light Emitted color is a function of size

Conventional Fluorescence Microscopy

slit

Conventional Fluorescence Microscopy

Epifluorescence Image More photons

Confocal Image Better photons

All emitted light under the objective lens

Only light emitted from within the focal plane

Confocal Imaging by laser scanning microscopy (LSM)

Laser
Mirror

Image plane

Pinhole
Galvanometer y Scanner

Galvanometer x Scanner

Tubus Lens
Objective Lens

laser

Light Source

Object Plane y

Objective Lens

Object plane

Confocal Microscope

Line Scan

Confocal Imaging
z-Stack
Image plane

Pinhole

Tubus Lens
z

Light Source

Objective Lens

Object plane

Confocal Microscope

Reconstructed Neuron

50 m

3D Reconstruction from Consecutive Optical Sections

Metaphase Spindle Complex

Aequorea victoria

Green Fluorescent Protein

Green Fluorescent Protein

GFP Fluorophor - Ser65 - Tyr66 - Gly67 -

GFP transcriptional reporter

GFP transcriptional reporter

GFP variants produced by artificial selection in E coli.

Roger Tsien Lab

Tsien, Shimomura, Chalfie 2008 Nobel Prize in Chemistry

Roger Tsien

Deconvolution Microscopy

Point Spread Function for 2 Fluorescent Dyes

Deconvolution Microscopy

Acquired Image

Deconvolved Image