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

摘要

Autofluorescence spectroscopy can provide information on the metabolic status of cellular systems, but extensions of thesetechniques 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 signalsthat could be used to decipher cellular-level metabolic status of tissues. Spectral phasor analysis was originally developedfor the rapid segmentation of hyperspectral images and has since been used for monitoring cellular NAD(P)H conformationfrom UV-excited cellular autofluorescence. Specifically, we showed previously that chemically induced autofluorescenceresponses in Saccharomyces cerevisiae (baker’s yeast) suspensions could not be accounted for using the two-componentfree vs. protein-bound model for conformation. Rather, by considering a series of physically similar and dissimilarchemicals acting on multiple metabolic pathways, we showed that responses affecting different pathways, e.g., involvingcellular respiration versus oxidative stress, could be distinguished. Here, we seek to extend this pathway-levelinterpretation to the sensing of cellular metabolism in tissues by monitoring the cyanide-induced metabolic response ofyeast cells embedded in media containing 9-cyanoanthracene or collagen as sources of background emission. Despite thesimilarity between autofluorescence and background spectra, we observe spectral behavior consistent with thediscrimination of the metabolic response from the background emission. Performance over specifically selected noncontinuousspectral bands to reject chromophore absorption is also assessed.