Abstract
Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection.
Highlights
Fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide hydrogen (NADH) has proven to be a valuable tool for the characterization of cell metabolism and mitochondrial function with a broad range of bioenergetics applications (Schaefer et al 2019)
As compared to other fluorescence microscopic approaches, FLIM of NADH stands out because it makes no use of fluorescent dyes but rather uses the intrinsic fluorescence of NADH in living cells of a defined physiological state
FLIM is extremely sensitive to slight adaptations that are convenient for subsequent electron microscopy (EM) preparation
Summary
Fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide hydrogen (NADH) has proven to be a valuable tool for the characterization of cell metabolism and mitochondrial function with a broad range of bioenergetics applications (Schaefer et al 2019). Being limited to the spatial resolution of light microscopes, FLIM can not be used to investigate the cellular ultrastructure. This study aims to combine FLIM with electron microscopy. The underlying assumption was that FLIM and EM would suit each other well, since FLIM measurement is carried out in unstained living cells in the native state. In contrast to fluorescence microscopy of stained cells, no ultrastructural changes are introduced that could hamper EM imaging. A correlative FLIM and EM (FLEM) protocol was established and evaluated using imaging of
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