Microscopes

Micrographia Robert HookeRobert Hooke First report of cell structureFirst report of cell structure corkcork First illustrated book on microscopy

Binocular Microscope

Early Microscope Early 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 BrightfieldPhase contrast NomarskiDarkfield

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 ConventionalConfocal

3D Reconstruction Via Confocal 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

TEM SEM DIC Comparison of EM methods