Transcript Light Microscopy and Electronic Imaging for the Biomedical

Light Microscopy and Electronic
Imaging for the Biomedical
Sciences
E. D. Salmon and Kerry Bloom
Biology 188
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/index.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
Janssen Microscope Was One of the First
“Microscope” Named and a 2-lens "Huygens
Eyepiece Introduced in Early 1600’s
Italian microscope Galileo might have used
Hooke Microscope Had a
Resolution of About 5 mm
“Cells” Discovered
Leeuwenhoek Microscope Had
Resolution of About 1 mm
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.
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.
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.
Spherical Aberration Not Solved Until
1830 by Joseph Jackson Lester
Tulley/Lister Corrected
Lens Microscope, 1830's
Adjustable Objective
by Ross, circa 1840
Abbe Discovers in 1877 The Importance of
Numerical Aperture (NA = nsinq) for
Resolution
Developed Apochromatic Optics
Microscopes in the Mid-Late 1800’s
Zeiss
Köhler
illumination was
first introduced
in 1893 by
August Köhler of
the Carl Zeiss
corporation as a
method of
providing the
optimum
specimen
illumination
Objective Turrets Developed and
Modern Condenser Design
Parfocal Objectives
Abbe condensers with
Cond. Diaphragm and Turret
Fritz Zernike Invented Phase Contrast in 1930’s
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
Differential Interference Microscopy
(DIC) Invented by Nomarski and Smith
in 1960’s
Live Cell Imaging By Phase, DIC and Pol Microsocopy
Cellular Histology Developed Over Last
150 Years
Ploem Invented
EpiFluorescence
Illuminator in
Early 1970’s
Mono-Clonal and Affnitiy Purified Antibody
Methods and Beginning of Molecular Probe
Development Began in 1970’s
Multi-Wavelength
Fluorescence
Microscopy:
Co-Localization of
Different
Molecules Relative
To Cellular
Structures
Video-Enhanced Contrast Methods
Developed in Early 1980’s by Inoue and
Allen Revealed Cellular Structures and
Macromolecular Complexes Invisible by
Eye or Film
Video-Enhanced
DIC Microscope
System from
1985
VE-DIC Motility Assays
Lead to Discovery of
Microtubule Motor
Proteins Like Kinesin in
Mid-1980’s and After
Microtubule Motor Driven Organelle Motility
8 nm Step
5 pN Stall Force
30 - 60 um/min
100 Steps/sec at
No Load
1 ATP Hydrolized
Per Step
Optical Trap
Kinesin
-
+
Dynein
Coverslip
60 - 120 um/min
Simulation from Ron Milligan and Ron Vale of
Kinesin Mechanochemical cycle
Fluorescence microscopy pushed forward in
early 1980’s by new fluorophores (start of
Molecular Probes) and intensified video
cameras
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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)
In early 1980’s video cameras
with image intensifiers:
Today: e.g. Hamamatsu Orca ER
Cooled CCD Camera
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Low readout noise (~8 electrons)
High Quantum Efficiency
Broad spectral response
Fast readout: ~8MHz
No distortion
1024x1024 pixels
>20,000 e deep wells
FRAP Scope with Cooled CCD Camera
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. 150:1-17.
1987: John White and Brad Amos
Invented Modern Laser Scanning
Confocal Fluorescence Microscope
In Mid 1990’s Went from Single
Photon to Multiphoton Imaging
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
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)
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
In early 1990’s, went to semi-automated,
multimode,wide-field microscopes with
cooled CCD cameras, shutters, filter wheels
and computer control
Multi-Wavelength Immunofluorescence
Microscopy
Confocal Scanning
Head
Nikon TE300
inverted microscope
Filter Wheel
Orca ER
CCD
PC with
MetaMorph
software
Laser Input
(fiber optic)
Focus motor
High Resolution,
High Signal-Noise,
1Kx1K Pixel Images
Recorded in 200ms
Immunofluorescence
Microscopy of
Microtubules (Green)
And
Chromosomes (Red)
In Mitotic PtK1 Cell
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
Aequorea victoria
Green Fluorescent Protein (GFP)
GFP Vectors from Clontech
Cellular Imaging is Key to
Understanding Protein Function in Cells
Genomics
Proteomics
Cellular Imaging
e.g. GFP-Fusion
Proteins
Alexa-488-Eb1
Bound to the
Growing Ends
(10 mm/min)
of
Microtubules
in Early
Prometaphase
Spindle in
Xenopus Egg
Extracts
(Jen Ternauer)
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
Biological System: Budding
Yeast
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Saccharomyces cerevisiae
Short cell cycle.
Genetics.
Ease of Gfp constructs.
Conserved mitotic
processes.
Budding Yeast Anaphase and
Cytokinesis: GFP-Tubulin and
CFP-Myo1(Myosin)
Paul Maddox
GFP-Microtubule Dynamics in A First
Division C. elegans Embryo
Karen Oogema
And
Paul Maddox