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Unformatted text preview: ? Microscopy Biochemistry Molecular Biology Genetics A sense of scale between tissues, living cells and atoms If you want to look at the shape of a cell… use light microscopy Stained cells
fibroblasts myoblasts Retinal ganglion Bright field Phase contrast Nomarsky Dark field Tobacco cells If you want to look at a fluorescent macromolecule inside a live cell use… GFP-based fluorescence microscopy Green fluorescent protein (GFP)
Useful when you want to find out the location of a particular protein in cells, to a radius of ~200 nm of its locale You need to make a gene fusion between the genes encoding GFP and your protein of interest Cells are not fixed prior to visualization under the microscope; therefore, the technique is used when you want to visualize a protein (a fusion protein) in ‘real time’ Detecting a nucleoporin in live yeast Nup2p-GFP DAPI wild-type nup60Δ 20 neurons in live fly embryo The confocal fluorescence microscope yields greater resolutionDrosophila embryo actin staining conventional confocal Pollen grains in three Z-axis optical slices & three dimensional reconstruction 30 z-slices If you want to look at a macromolecule inside a cell, but it is not fluorescent… use indirect immuno-fluorescence microscopy Useful when you need to find out the location of a particular protein in cells, to a radius of ~200 nm of its locale. You must have antibodies against the protein of interest; if your protein is tagged with MYC or FLAG peptides then you need antibodies against the tag (commercially available). Cells must be fixed with formaldehyde prior to incubation with fluorescence-labeled antidody; therefore, the technique is not used when you want to visualize a protein in ‘real time’ Cell in mitosis: (spindles, centrosomes & DNA) TIRF (total internal reflection fluorescence) microscopy is used to detect single-fluorescent molecules GFP myosin binding individual (invisible) actin cables If you want to look at the surface of a cell or subcellular compartment… Use Scanning Electron Microscopy (SEM) or TEM with etching & shadowing
TEM in conjunction with etching & metal shadowing Protein filaments in muscle cells SEM
Light microscopy TEM The surface topology of cilia in a audi...
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