microscopes. micrographia robert hookerobert hooke 16651665 first report of cell structurefirst...
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Microscopes
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Micrographia
• Robert HookeRobert Hooke
• 16651665
• First report of cell First report of cell structurestructure
• corkcork
First illustrated First illustrated book on book on
microscopymicroscopy
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Binocular Microscope
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Early MicroscopeEarly compound microscopes were
beautifully crafted but had
severe chromatic distortion
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Outline
• Units of Measurement
• Microscopes: features & uses– Light– Electron
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Units of Measurement
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Light Microscopes
• Brightlight
• Darkfield
• Phase Contrast
• Differential Interference Contrast
• Fluorescence
• Confocal
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Parts of the Microscope
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Changes in light as it passes through a cell
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Bright Light Microscopy
• FEATURES• structures > 0.2 um• specimen dark against
bright background• cheap and easy
• USES• Stained samples• Simple morphology• Count cells
AKA Bright field microscopy
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Stained bacteria
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Shape is easier to see after staining
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Granules & Capsules
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Darkfield Microscopy
• FEATURES• Special light source
for indirect reflected light– condenser disk
• object bright against black background
• improved resolution
• USES• living organisms• fragile organism• small microbes
– Treponema pallidum
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Flagella can be seen by Dark field microscopy
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Dark field Microscopy
Click on image to start video
• Light reflected
• Objects bright against dark background
• Slightly improved resolution
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Phase Contrast Microscopy
• FEATURES• Special condenser • Special objective lens• Uses diffracted light• No stains
• USES• Living Structures• Motility• Internal cell Structures
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Phase Light
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Effect of edges on phase light
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Bacteria by Phase Contrast
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Phase Contrast Microscopy
• Live cells
• internal organelles visible
• Dynamic processes
Click on image to show Video
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Differential Interference Contrast Microscopy
• FEATURES• Two Light Beams• Sample appears
coloured
• USES• 3D images
First developed by Nomarski in 1952
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World’s Largest Bacterium
• Visible to naked eye
• In gut of Surgeon fish
• Eats alga
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Procloron under DIG Microscopy
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Procloron dividing
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Four Types of Light Microscopy
Brightfield Phase contrast
Nomarski Darkfield
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Fluorescence Microscopy
• FEATURES• UV light source• Special lenses• objects fluorscent
against black background
• USES• Immunofluorescent• labelled antibodies• rapid identification
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Light path in a flourescent Scope
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Fluorecent cells
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Fluorescent Molecules
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Double fluorescent labelling
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Confocal Microscopy
• FEATURES• Laser light• multiple images• computer generated
• USES• 2D and 3D of fine
structural detail
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Confocal Microscopy
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Fluorescence Microscopy
Conventional Confocal
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3D Reconstruction ViaConfocal Microscopy
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Electron Microscopy
• Transmission Electron Microscopy
• Scanning Electron Microscopy
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Lens in Microscopes
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Plasma Membrane: Glycocalyx
Glycocalyx of plasma membrane of a T lymphocyte stained with ruthenium red
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Scanning EM
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Bacteria on Skin
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Flagella & Pili
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Scanning EM
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Scanning EM
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TEM
SEM
DICComparison of EM methods