Light Microscopy and Electronic Imaging for the Biomedical

SciencesE. D. Salmon and Kerry Bloom

Biology 188

History of the Microscope, Thomas E. Jones

http://www.utmem.edu/~thjones/hist/hist_mic.htm

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

See also Molecular Expressions, a Microscope Primer at:http://micro.magnet.fsu.edu/primer/index.html

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 m

“Cells” Discovered

Leeuwenhoek Microscope Had Resolution of About 1 m

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 ofLeeuwenhoek's microscopes, and it appears that someof 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 ofthickened 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 mechanicalimprovements 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, andJohn 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 = nsin) 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 StructuralDetail 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 Epi-

Fluorescence 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 ofDifferentMolecules RelativeTo 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 inMid-1980’s and After

30 - 60 um/min

+-

Kinesin

60 - 120 um/min

Dynein

Microtubule Motor Driven Organelle Motility

Coverslip

Optical Trap

8 nm Step5 pN Stall Force100 Steps/sec at No Load1 ATP Hydrolized Per Step

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• 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

• 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

Orca ER CCD

Laser Input (fiber optic)

Filter Wheel

Focus motor

PC with MetaMorph

software

High Resolution,High Signal-Noise,1Kx1K Pixel ImagesRecorded in 200ms

ImmunofluorescenceMicroscopy 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 Imaginge.g. GFP-Fusion Proteins

Alexa-488-Eb1

Bound to the Growing Ends(10 m/min)of Microtubulesin Early PrometaphaseSpindle in Xenopus EggExtracts(Jen Ternauer)

Green:GFP-Cdc20At Kinetochores

Red:Phase ContrastImages of PtK1Tissue Cells

Cdc20 PersistsAt KinetochoresThroughout Mitosis and Exhibits FastKinetics:FRAP t1/2 =[4 sec (attached)25 sec (unattached]

Biological System: Budding Yeast

• 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 OogemaAnd

Paul Maddox