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HSS 2305Oct 28 2013Chapter 18Techniques in Cell and Molecular Biology181The Light MicroscopeCondenser lensgathers diffused rays from light source and illuminates the specimienObjective lenscollects the light rays focused on the specimen by the condenser lensOcular lensuses the image formed by the objective lens to make an enlarged and virtual imageResolutionVisibilityoFactors that allow an object to actually be observedBrightfield microscopeuses a bright background against which the image of the specimen must be contrastedPreparation of specimens for BF microscopesoWhole mountan intact object living or deadoSectiona thin slice of tissueTo prep need to kill the cells in a fixative which immobilizes the tissue in a phasespotPhasecontrast microscopyoPH microscope makes highly transparent objects more visibleoCauses light rays from two sources to interfere with one anotheroIs a type of interference microscopeoDICdifferential interference contrast OR Nomarski interference gives 3D qualityFluorescence microscopyrelated techniquesoAllows viewers to see the location of certain molecs called fluorophores or fluorochromesoFluorophores absorb invisible UV radiation and release a portion of the energy in the more visible spectrumoEarliest applicationimmunofluorescencea small fluorophore is covalently linked to an antibodyoGFPgreen fluorescent protein doesnt need an additional cofactor to absorb and emit Does it by selfmodificationUsed in FRET fluorescence resonance energy transfer to measure distances btw fluorophores Ex measure changes in distance btw two parts of a proteinoDsReddistantly related red fluorescent tetrameric proteinLaser scanning confocal microscopy Marvin Minskyoproduces an image of a thin plane situated within a much thicker specimen182Transmission Electron Microscopy TEMTEMs form images using electrons that are transmitted through a specimenoMuch better resolution than light microscopeo1000x250000x resolutionSscanningEms utilize electrons that have bounced off the surface of the specimenSpecimen prepoNeed to be fixed embedded and sectioned but more critical to get rightoUsed osmium tetroxide as a fixative Is a heavy metal
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