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feb5 notes - Correction mu=10-6 n= 10-9 Fluorescence...

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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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