I am performing cellular uptake studies with peptides labelled with fluorochromes. I did flow cytometry experiments and now I want to validate it by fluorimetry by measuring the emission spectrum of fluorochromes after excitation. Unfortunately none of the labs in the institute has a plate reader with possibility for excitation of fluorochromes (Cy5 and FITC). The traditional ELISA plate readers have the option to measure absorbance at a particular wave length. Is it a good idea to measure the absorption at the excitation wave length of the fluorochromes? I am not sure about the difference between absorbance and excitation peaks. In my opinion its not an appropriate method to quantify the fluorescent molecules.
The quantitative value of a fluorescent molecule is the emission of light at a different wavelength from the one of excitation. The excitation power is too high to be quantitative in absorbance. And at a lower power, the background will mask your signal.
The quantitative value of a fluorescent molecule is the emission of light at a different wavelength from the one of excitation. The excitation power is too high to be quantitative in absorbance. And at a lower power, the background will mask your signal.
A fluorchrome's excitation spectrum is the wavelength range and absorbance at each wavelength, as a function of wavelength. This energy is then re-emited as a photon of lower energy (longer wavelength) with a spectrum of its wavelength range and intensity at each wavelength. Fluorescence is a sensitive tool because the emission is high relative to background and therefore easily detectable and quantifiable. The absorbance of the fluorochrome is much less specific given other molecular absorbances in a cell, so it is not equivalent to a spectrophotometer's absorbance measurements (e.g., for DNA and RNA).
I think Flow quantitation is good enough and more reliable that doing flurometry because individual event is measured in FACS while a gross fluroscence is measured in fluorometry. Also the detectors in FACS are very efficient in collecting the minute signals.
Best luck
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