Many assays, including Cell-based assays, QPCR, some ELISA test, rely on the presence/absence of fluorescence. Diagnosis applications about cancer or antiobiotic resistance uses fluorescence ratio to draw a conclusion. All of these applications found a way around measuring the amount of fluorescence as reliable indicator of target potency. My question is exploratory, if we could reliably measure the value of the fluorescence of a cell, what application would be dramatically improved ?
Fluorescence, as it is usually measured, does not have an absolute value, which is why it is usually expressed as relative fluorescence units (RFU). It depends on many parameters, some of which are intrinsic to the sample and others are related to the apparatus and its settings. I think you would have to base any attempt at such a measurement on some internal standard, making it in effect ratiometric (like chemical shift in NMR).
Hello Adam, thank you for your answer. I agree with you and you are describing the current situation very well. My question rely on your first sentence "as it is usually measured". In my opinion it is both the lack of standard (either internal but reproducible or legal) and the difficulty of getting accurate measurement in Cell Imaging that has oriented the applications toward relative or ratiometric measurement. So My question is : if you could have a perfectly reproducible way to measure fluorescence in assays and a consequences could compare fluorescence intensity measurement accross any assay in the world, hence gettting reliable quantitative fluorescence measurement, what application would be greatly improved ? Adam B Shapiro
For fluorescence measurements you can use facs on which you can take fluorescence measurements at different wavelengths, can provide quantitative and qualitative data from cell surface receptors or intracellular molecules
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