Naturally occurring endogenous fluorescence of flavins, arising in response to excitation by visible light, offers broad opportunity to investigate mitochondrial metabolic state directly in living cells and tissues, including in clinical settings. However, photobleaching, the loss of the autofluorescence intensity following prolonged exposure to light is an inherent phenomenon occurring during the fluorescence acquisition, which can have a negative impact on the recorded data, particularly in the context of measurement of metabolic modulations in pathophysiological conditions. In the presented study, we present a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally-resolved confocal imaging. We demonstrate significant nonuniform photobleaching related to different bleaching rates of individual flavin components, resolved by linear spectral unmixing of the recorded signals. Induced photodamage was without effect on the cell morphology, but lead to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. These findings point to the necessity of inducing limited photobleaching during metabolic screening in all studies involving visible light excitation and fluorescence acquisition in living cells. © 2018 International Society for Advancement of Cytometry.
Keywords: autofluorescence; cardiac myocytes; confocal imaging; flavin; photobleaching; spectral unmixing.
© 2018 International Society for Advancement of Cytometry.