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The well-known red shift of fluorescence spectra in thick tissue causes significant cross-talk and inaccurate quantification of fluorophores using multispectral imaging (MSI). However, fluorescence lifetimes (FLT) are largely unaffected by tissue light propagation and results in superior quantitation accuracy. Here we present a systematic experimental comparison of quantitative MSI and FLT of multiple fluorescent molecules in tissue. In experiments using one or two fluorescence species in mixture with tissue autofluorescence, MSI showed significantly higher error rates (>20%) than FLT (<5%). These results show promise for in vivo FLT multiplexing based imaging of receptor expression in cancer and other diseases.
Rahul Pal andAnand T. N. Kumar
"Quantitative multiplexing in biological tissue using multispectral and fluorescence lifetime imaging", Proc. SPIE 11639, Optical Tomography and Spectroscopy of Tissue XIV, 116391V (5 March 2021); https://doi.org/10.1117/12.2578437
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Rahul Pal, Anand T. N. Kumar, "Quantitative multiplexing in biological tissue using multispectral and fluorescence lifetime imaging," Proc. SPIE 11639, Optical Tomography and Spectroscopy of Tissue XIV, 116391V (5 March 2021); https://doi.org/10.1117/12.2578437