Lecture 1--microscopy

Mercury light or led cells can be labeled with

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Unformatted text preview: ed against a dark, unstained background. Fluorescence Microscopy Janet Iwasa Frequently used fluorophores GFP gets exited most at 488nm, but it will also get excited at other wavelengths most samples have some s tructures that are already fluorescent Emission Excitation binds to DNA likes nucleus Two major ways to introduce fluorophores fluorophore (e.g. fluoroscein, rhodamine)coupled antibodies are used to image fixed cells Fluorescent proteins (GFP, RFP) are used to image live cells (or animals!) Principles of immunofluorescence Immunofluorescence is a technique making use of antibodies to visualize specific structures in fixed cells In its simplest form, an antibody that recognizes a specific cellular protein is conjugated to a fluorophore. Cells, tissues or animals are fixed and exposed to the antibody, which sticks only where its antigen is found. Fluorescence microscopy can then be used to visualize the location of the antigen. molecules made nby immune syste to recognize s pecific antigens Making primary antibodies A secondary antibody can by used to amplify the fluorescence signal -Add primary antibodies -Add fluorophore-conjugated secondary antibodies -image with fluorescence microscopy Secondary antibodies recognize nonvariable regions of the primary antibodies and can be conjugated to a fluorophore A secondary antibody can by used to amplify the fluorescence signal -Add primary antibodies -Add fluorophore-conjugated secondary antibodies -image with fluorescence microscopy Two major ways to introduce fluorophores fluorophore (fluoroscein, rhodamine)-coupled antibodies are used to image fixed cells Fluorescent proteins (GFP, RFP) are used to image live cells (or animals!) Naturally fluorescent proteins have been isolated from marine organisms variants of fluorescent proteins have been made by mutation mutations in GFP and it would absorb different wavelengths of light and emit others pick the one whos properties are ideal Fluorescent proteins can be genetically encoded 1. Use molecular biology methods to create a transgene with a tissue-specific promoter driving a fluorescent protein some times the trangene becomes incorporated into genome 2. Inject into an animal egg to make a transgenic animal 3. Grow to adulthood have to fuse the protein and GFP...
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