Measurements of absolute values in biochemical fluorescence spectroscopy

Abstract

Fluoresce nce spectroscopy is an important tool of the bioc he mist studyin g th e structure and interac tion s of prote in s and nu cle ic acid s. The four bas ic quantities to measure accurately are: 1. s pec tra (co rrected excitation and e mi ss ion). 2. quantum yields (0), 3. fluoresce nce decay c haracte ri stics, and 4 _ poLarization_ Co mmercially avail ab le instruments, with little modification, can be used to obtain these meas ure me nts, but th e bioc he mi st in this fi eld is ve ry de pende nt on the accuracy of measure me nts of substan ces he uses as standard s. Co nfusion arises from di sagree ment be twee n re ported values for standard s whi c h may be used to ca ubrate a de tector sys te m to obtain quantum yields, or to set up Ijfe time and polarization photometers. For in stan ce, the prot ein c he mi st is fond of usin g tryptophan and quinine as quantum yield standards, but Q for tryptophan has been variou s ly reported as 0.13 and 0.20, and Q values for quinine bi sulfate range from 0.4 to 0.7. The bioc he mist should also be aware of th e probl e ms inhe re nt in the use of co mme ri cally avai lable instrume nts in a bsolute measurements, as we ll as the s pecial co mpli cation s arisin g in co mplex bioc he mi cal systems where the flu oresce nce is hete rogeneous.Key words : Ab so lute fluorometry; a bso lute flu o rometry in bioche mistry; standard refe re nce materials in fluorom etry.

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Microfluidic discharge-based optical sources for detection of biochemicals

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How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

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