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Microscopes Micrographia • Robert Hooke • 1665 • First report of cell structure • cork First illustrated book on microscopy Binocular Microscope compound EarlyEarly Microscope 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 • Light reflected • Objects bright against dark background • Slightly improved resolution Click on image to start video 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 Nomarski Phase contrast 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 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 Scanning EM Comparison of EM methods DIC SEM TEM
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