Spectral phasor analysis of autofluorescence responses from cells embedded in turbid media containing collagen

Max Kreider; Andrew I. Rodriguez; Karthik Vishwanath; Paul Urayama

doi:10.1117/12.2546398

20 February 2020 Spectral phasor analysis of autofluorescence responses from cells embedded in turbid media containing collagen

Max Kreider, Andrew I. Rodriguez, Karthik Vishwanath, Paul Urayama

Author Affiliations +

Proceedings Volume 11251, Label-free Biomedical Imaging and Sensing (LBIS) 2020; 112510B (2020) https://doi.org/10.1117/12.2546398
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

Autofluorescence spectroscopy can provide information on the metabolic status of cellular systems, but extensions of these techniques to turbid media such as tissues is complicated by the presence of multiple scattering, background fluorescence, and intrinsic absorption. Phasor analysis is a class of analytical approaches for the real-time assessment of emission signals that could be used to decipher cellular-level metabolic status of tissues. Spectral phasor analysis was originally developed for the rapid segmentation of hyperspectral images and has since been used for monitoring cellular NAD(P)H conformation from UV-excited cellular autofluorescence. Specifically, we showed previously that chemically induced autofluorescence responses in Saccharomyces cerevisiae (baker’s yeast) suspensions could not be accounted for using the two-component free vs. protein-bound model for conformation. Rather, by considering a series of physically similar and dissimilar chemicals acting on multiple metabolic pathways, we showed that responses affecting different pathways, e.g., involving cellular respiration versus oxidative stress, could be distinguished. Here, we seek to extend this pathway-level interpretation to the sensing of cellular metabolism in tissues by monitoring the cyanide-induced metabolic response of yeast cells embedded in media containing 9-cyanoanthracene or collagen as sources of background emission. Despite the similarity between autofluorescence and background spectra, we observe spectral behavior consistent with the discrimination of the metabolic response from the background emission. Performance over specifically selected noncontinuous spectral bands to reject chromophore absorption is also assessed.

Citation Download Citation

Max Kreider, Andrew I. Rodriguez, Karthik Vishwanath, and Paul Urayama "Spectral phasor analysis of autofluorescence responses from cells embedded in turbid media containing collagen", Proc. SPIE 11251, Label-free Biomedical Imaging and Sensing (LBIS) 2020, 112510B (20 February 2020); https://doi.org/10.1117/12.2546398

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