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Unformatted text preview: Correction: mu=10-6 , n= 10-9 Fluorescence Fluorescence • A variety of Techniques that use fluorescence • Fluorescent dye= fluorochrome o Can be excited with a particular wavelength of light and emit light at a longer wavelength Flourceine • Light in is 485 nm, out is 535 nm. Vital Fluorescence microscopy and spectroscopy • Use of fluorochromes to monitor some physiological aspect of physiological parameter in living cells. • Problems o Dyes were not membrane permeable o Solution Add Acetoxymethyl to get in, but once it gets in, it must come off. Causes cleaving by an enzyme (esterases) • Examples o Mitochondrial activity PMF/ Proton motive force • I.e. Rhodamine 123 • JC-1 o Stains living mitochondria o orange (very high motive force) or green (not so active) Membrane integrity • Live/ Dead assays o Calcien AM/ Propidium Iodide Propidium Iodide stains red o Invitro toxicology Intracellular Calcium • Fluoro AM Intracellular PH • BCECF AM How to measure? • Microspectrofluorometry o Qualitative information/ picture o Not Quantatative • Plate reading spectrophotometer o Cytofluor Not qualitative, but is quantitative o Highthroughput screening...
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This note was uploaded on 04/13/2009 for the course BIOL 311 taught by Professor Vanbuskirk during the Spring '08 term at Binghamton University.
- Spring '08
- cell biology