Light Microscopy and Electronic Imaging for the Biomedical Sciences

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Light Microscopy and Electronic Imaging for the
Biomedical Sciences

• E. D. Salmon and Kerry Bloom
• Biology 188

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History of the Microscope, Thomas E. Jones
http//www.utmem.edu/thjones/hist/hist_mic.htm
See also Molecular Expressions, a Microscope
Primer at http//micro.magnet.fsu.edu/primer/inde
x.html
There is one very early description of an
isolated use of spectacles. Pliny the Elder
wrote the following in 23-79 A.D. "Emeralds are
usually concave so that they may concentrate the
visual rays. The Emperor Nero used to watch in an
Emerald the gladatorial combats."
The modern reinvention of spectacles occurred
around 1280-1285 in Florence, Italy
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Janssen Microscope Was One of the First
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Microscope Named and a 2-lens "Huygens Eyepiece
Introduced in Early 1600s
Italian microscope Galileo might have used
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Hooke Microscope Had a Resolution of About 5 mm
Cells Discovered
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Leeuwenhoek Microscope Had Resolution of About 1
mm
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Leeuwenhoek's Secret Lenses Leeuwenhoek's method
of making the tiny, high-quality and high power
lenses was kept secret. A study has recently
been done on the few remaining copies
of Leeuwenhoek's microscopes, and it appears that
some of the lenses may have been made by
grinding, while the best ones were blown.
Leeuwenhoek learned that when a glass bulb is
blown, a small drop of thickened glass forms at
the bottom of the bulb (much like a drop sits in
the bottom of a blown soap bubble.) By carefully
breaking away the excess glass, this tiny drop
can be used as a lens.
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Chromatic and Spherical Aberration Limited
Resolution
While the 18th century produced some great
mechanical improvements for the microscope,
making it much more sturdy and easy to use, the
images obtainable remained rather blurry with
colorful halos around objects. This was largely
due to the problems of "Chromatic and Aspheric
Aberration." The reason the single lens "simple"
microscopes remained important throughout the
century was that a single lens system has much
less aberration because the distortion becomes
synergistic with multiple lenses. This allowed
simple microscopes to attain around 2 micron
resolution, while the best compound microscopes
were limited to around 5 microns.
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Chromatic Aberration Corrected by the Achromatic
Doublet
Chester More Hall Makes the Discovery in 1730,
diddles, and John Dolland Learns the Secret, and
Patents it in about 1759.
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Spherical Aberration Not Solved Until 1830 by
Joseph Jackson Lester
Tulley/Lister Corrected Lens Microscope, 1830's
Adjustable Objective by Ross, circa 1840
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Abbe Discovers in 1877 The Importance of
Numerical Aperture (NA nsinq) for Resolution
Developed Apochromatic Optics
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Microscopes in the Mid-Late 1800s
Zeiss
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Köhler illumination was first introduced in 1893
by August Köhler of the Carl Zeiss corporation as
a method of providing the optimum specimen
illumination
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Objective Turrets Developed and Modern Condenser
Design
Parfocal Objectives Abbe condensers
with Cond. Diaphragm and Turret
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Fritz Zernike Invented Phase Contrast in 1930s
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Phase Contrast Gives Contrast to
Structural Detail in Transparent Specimens
In focus Image Get phase contrast by slight
out-of-focus, but loss of resolution
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Differential Interference Microscopy (DIC)
Invented by Nomarski and Smith in 1960s
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Live Cell Imaging By Phase, DIC and Pol
Microsocopy
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Cellular Histology Developed Over Last 150 Years
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Ploem Invented Epi-Fluorescence Illuminator in
Early 1970s
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Mono-Clonal and Affnitiy Purified Antibody
Methods and Beginning of Molecular Probe
Development Began in 1970s
Multi-Wavelength Fluorescence Microscopy
Co-Localization of Different Molecules
Relative To Cellular Structures
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Video-Enhanced Contrast Methods Developed in
Early 1980s by Inoue and Allen Revealed Cellular
Structures and Macromolecular Complexes Invisible
by Eye or Film
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Video-Enhanced DIC Microscope System from 1985
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VE-DIC Motility Assays Lead to Discovery of
Microtubule Motor Proteins Like Kinesin
in Mid-1980s and After
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8 nm Step 5 pN Stall Force 100 Steps/sec at
No Load 1 ATP Hydrolized Per Step
Optical Trap
Coverslip
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Simulation from Ron Milligan and Ron Vale of
Kinesin Mechanochemical cycle
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Fluorescence microscopy pushed forward in early
1980s by new fluorophores (start of Molecular
Probes) and intensified video cameras

• Detect fluorescence invisible to eye or film
• Quantitative fluorescence measurements
• Fluorescent protein analogs of live cells
• Ratio measurements for ion dynamics (e.g. Fura 2
  for calcium ion)
• Molecular dynamics from Measurements of
  fluorescence recovery after photobleaching (FRAP)

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In early 1980s video cameras with image
intensifiers
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Today e.g. Hamamatsu Orca ER Cooled CCD Camera

• Low readout noise (8 electrons)
• High Quantum Efficiency
• Broad spectral response
• Fast readout 8MHz
• No distortion
• 1024x1024 pixels
• gt20,000 e deep wells

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FRAP Scope with Cooled CCD Camera
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Measurements of Fluorescence Recovery After
Photobleaching (FRAP) Shows that Alexa488- or
GFP-Mad2 Turns-Over Rapidly at Unattached
Kinetochores ( a 20-25 sec half-life)
Howell et al., 2000, J. Cell Biol. 1501-17.
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1987 John White and Brad Amos Invented Modern
Laser Scanning Confocal Fluorescence Microscope
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In Mid 1990s Went from Single Photon to
Multiphoton Imaging
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The Modern Era of Light Microscopy

• New microscope optics generate brilliant images
  over wide wavelengths
• Computers control x-y-z specimen position,
  wavelength selection, illumination and image
  acquisition
• Electronic cameras quantitatively record light
  intensity of specimens invisible or undetectable
  by eye or film
• Confocal and deconvolution methods give 3-D views
  of cellular architectural dynamics
• New fluorescent molecular probes and biophysical
  methods report on the temporal and spatial
  activities of the molecular machinery of living
  cells and single molecule imaging
• Micromanipulation, ablation, force measurement

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Modern Upright Research Light Microscope (1995)
Bright, High Contrast Optics Epi-Fluorescence
Phase-Contrast Polarization DIC Diffraction
Limited Resolution Multiple Ports Auto.
Photography Electronic Imaging- (Video---CCD)
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The Modern Era of Light Microscopy

• New microscope optics generate brilliant images
  over wide wavelengths
• Computers control x-y-z specimen position,
  wavelength selection, illumination and image
  acquisition
• Electronic cameras quantitatively record light
  intensity of specimens invisible or undetectable
  by eye or film
• Confocal and deconvolution methods give 3-D views
  of cellular architectural dynamics
• New fluorescent molecular probes and biophysical
  methods report on the temporal and spatial
  activities of the molecular machinery of living
  cells and single molecule imaging
• Micromanipulation, ablation, force measurement

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In early 1990s, went to semi-automated,
multimode,wide-field microscopes with cooled CCD
cameras, shutters, filter wheels and computer
control
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Multi-Wavelength Immunofluorescence Microscopy
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Confocal Scanning Head
Nikon TE300 inverted microscope
Filter Wheel
Orca ER CCD
PC with MetaMorph software
Laser Input (fiber optic)
Focus motor
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High Resolution, High Signal-Noise, 1Kx1K Pixel
Images Recorded in 200ms
Immunofluorescence Microscopy of Microtubules
(Green) And Chromosomes (Red) In Mitotic PtK1
Cell
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Molecular Fluorescent Probes

• Specific Fluorescent Dyes (e.g. DAPI)
• Covalently bind fluorescent dye to purified
  protein
• Fluorescent Antibodies (e.g immunofluorescent
  labeling with primary and fluorescent secondary
  antibodies)
• Express in cells Green Fluorescent Protein (GFP)
  fused to protein of interest

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Aequorea victoria
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Green Fluorescent Protein (GFP)
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GFP Vectors from Clontech
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Cellular Imaging is Key to Understanding Protein
Function in Cells
Genomics
Cellular Imaging e.g. GFP-Fusion Proteins
Proteomics
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Alexa-488-Eb1 Bound to the Growing Ends(10
mm/min)of Microtubulesin Early
PrometaphaseSpindle in Xenopus
EggExtracts(Jen Ternauer)
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Cdc20 Persists At Kinetochores Throughout
Mitosis and Exhibits Fast Kinetics FRAP t1/2
4 sec (attached) 25 sec (unattached
Green GFP-Cdc20 At Kinetochores
Red Phase Contrast Images of PtK1 Tissue Cells
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Biological System Budding Yeast

• Saccharomyces cerevisiae
• Short cell cycle.
• Genetics.
• Ease of Gfp constructs.
• Conserved mitotic processes.

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Budding Yeast Anaphase and Cytokinesis
GFP-Tubulin and CFP-Myo1(Myosin)
Paul Maddox
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GFP-Microtubule Dynamics in A First Division C.
elegans Embryo
Karen Oogema And Paul Maddox
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