return to ground state results in emission of radiation (fluorochrome). absorption of photon...
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Return to ground state results in emission of radiation (fluorochrome).
Absorption of photon elevates chromophore to excited state.
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Absorbance (solid) and Fluorescence (dashed) Spectra of Tryptophan
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• enzyme assays• nucleic acids
• measurement• detection (gels)
• microscopy• flow cytometry
Applications of Fluorescence
substrate product*
Detection Limits for Nucleic Acids
• UV absorbance 1 g/ml• ethidium bromide 10 ng/ml
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Fluorescent Microscopy• label cells with fluorescent probe• illuminate with UV light• examine epifluorescence
ethidium bromide (DNA/RNA)
DAPI (only DNA) rhodamine 123 (mitochondria)
C5-CMB-ceramide (Golgi) free Ca2+ indicatorspH indicatorsmembrane potential indicators
antibodies
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acridine orange• fluoresces when bound to
DNA and RNA• fluorescence exhibits
different wavelengths max DNA ~ 530 nm max RNA ~ 640 nm
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• filters can distinguish fluorochromes• dual-labeling experiments
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Confocal Laser Scanning Microscopy
• minimizes the light from outside the plane of focus• apertures (or ‘pinholes’)• objective lens = condenser
• wide field vs. scanning (x, y dimensions)• excitation with lasers
• movable stage allows ‘optical’ sections (z dimension)• generate 3-D images
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8 consecutive 0.5 m optical sections
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Fluorometry vs Microscopy
quantitative data average of mixture
qualitative data quantitative data
more difficult individual cells
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Flow Cytometer
• qualitative and quantitative data on individual cells• rapidly analyze > 10,000 cells
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DNA synthesis
nuclear division
cytokinesis
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Flow Cytometer
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Fluorescence Activated Cell Sorter (FACS)