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