Spectrally resolved time-correlated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes

Abstract

A new setup for time-resolved fluorescence micro-spectroscopy of cells, based on multi-dimensional time-correlated single photon counting, was designed and tested. Here we demonstrate that the spectrometer allows fast and reproducible measurements of endogenous flavin fluorescence measured directly in living cardiac cells after excitation with visible picosecond laser diodes. Two complementary approaches for the analysis of spectrally- and time-resolved autofluorescence data are presented, comprising the fluorescence decay fitting by exponential series and the time-resolved emission spectroscopy analysis. In isolated cardiac myocytes, we observed three distinct lifetime pools with characteristic lifetime values spanning from picosecond to nanosecond range and the time-dependent red shift of the autofluorescence emission spectra. We compared obtained results to in vitro recordings of free flavin adenine dinucleotide (FAD) and FAD in lipoamide dehydrogenase (LipDH). The developed setup combines the strength of both spectral and fluorescence lifetime analysis and provides a solid base for the study of complex systems with intrinsic fluorescence, such as identification of the individual flavinoprotein components in living cardiac cells. This approach therefore constitutes an important instrumental advancement towards redox fluorimetry of living cardiomyocytes, with the perspective of its applications in the investigation of oxidative metabolic state under pathophysiological conditions, such as ischemia and/or metabolic disorders.

Rejection of transplanted hearts in patients evaluated by the component analysis of multi‐wavelength NAD(P)H fluorescence lifetime spectroscopy

“…Data were analyzed using SPCImage software (Becker&Hickl, Boston Electronics, U.S.A), as described previously [8,12]. Data are shown as mean AE standard error (SEM).…”

“…Our previously obtained data in rat cardiomyocytes [6][7][8] clearly showed that metabolic state can be determined directly in living cardiac cells by monitoring their naturally occurring endogenous AF. Such AF, generated after excitation with UV or visible light is localized in mitochondria and is mainly resulting from mitochondrial oxidized flavins (FAD * ) and reduced NAD(P)H [9], principal endogenous indicators of cellular oxidative metabolism.…”

“…Adenosine triphosphate (ATP) is produced by mitochondrial oxidative phosphorylation, a process coupled to oxidation of reduced NADH, the principal electron donor for electrochemical gradient. We have now applied spectrally resolved TCSPC [8] method in cardiomyocytes from rat, as well as human hearts and studied spectral and lifetime properties of both the endogenous flavins [7,8], as well as the NAD(P)H [11,12], and thus gathered an additional information on functioning of enzymes responsible for generation of electrochemical gradient in the respiratory chain. Furthermore, we have used an original approach of multispectral imaging followed by spectral decomposition and linear unmixing [13,14] and developed analytical methods for the separation of individual components in spectrally-resolved confocal images [7] and time-resolved signals from both the cell AF [15] and fluorescence probes [16].…”

“…NAD(P)H fluorescence was measured in isolated cardiac cells using Spectrally-Resolved Time-Correlated Single Photon Counting (TCSPC) setup on Axiovert 200 inverted microscope, as previously described [8]. Picosecond laser diode with emission of 375 nm (BDL-375, Becker&Hickl, Boston Electronics, U.S.A.) was employed at 20 MHz repetition rate as an excitation source (output power $1 mW).…”

Effect of ouabain on metabolic oxidative state in living cardiomyocytes evaluated by time-resolved spectroscopy of endogenous NAD(P)H fluorescence

“…7,12 Picosecond laser diode BDL-375 (Becker&Hickl, Boston Electronics, USA) with emission at 375 nm was deployed at the 20 MHz repetition rate as an excitation source. All experiments were performed at room temperature.…”