21 February 2013 Predicting errors from spectral overlap in multi-probe and multi-laser flow cytometry
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Abstract
In flow cytometry a group of cells labeled with a fluorescent probe molecule or dye is focused into a single cell stream passing through a laser light source. The fluorescent light is filtered and sampled by an array of detectors. In many cases a single light source and one probe/dye molecule have been used. But additional information can be obtained if several different laser wavelengths and multiple probes fluorescing at other wavelengths are used. We consider four lasers at 488nm, 532nm, 640nm and 785nm which occur near the peak absorptions of common fluorescent probes, Alexa488, Alexa532, Alexa647 and Alexa750, respectively. In some cases overlapping of the various fluorescent spectra occur. This effect can be mitigated by checking the emitted signals in individual wavelength channels and subtracting them, a practice known as compensation. But residual amounts of fluorescence as well as phosphorescence may not be completely taken into account because of the photodetector sensitivity. Using our unique numerical we calculated both the fluorescence and phosphorescence intensities in a multi-laser and multi-probe configuration. The total intensities of the fluorescent state and phosphorescent state are calculated for a range of laser powers from 5mW to 100mW. We found that there can be significant overlap between fluorescence and phosphorescence emission from multiple dyes.
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M. Potasek, M. Potasek, E. Parilov, E. Parilov, K. Beeson, K. Beeson, } "Predicting errors from spectral overlap in multi-probe and multi-laser flow cytometry ", Proc. SPIE 8596, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications V, 85960T (21 February 2013); doi: 10.1117/12.2003179; https://doi.org/10.1117/12.2003179
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