Components of intrinsic fluorescence revealed by Metabolic Modulation Matrix in isolated rat cardiac myocytes

Abstract

Aim: Recent developments in multi-wavelength fluorescence lifetime spectroscopy brought simultaneous measurement of fluorescence spectra and lifetimes in complex samples, namely cell and tissue intrinsic fluorescence. However, emission spectra of endogenous fluorophores are often superimposed in broad bands over the 400-600 nm wavelength region, making their separation particularly difficult. We therefore present a newly developed method of separation. Based on Metabolic Modulation Matrix approach, this method is built on evaluation of spectral modulation of endogenous fluorescence following changes in the cell metabolic state. Methods: Spectral fingerprints of time-resolved fluorescence are determined in isolated cardiomyocytes after excitation by 375-nm pulsed picosecond laser diode using SPC-830 TCSPC measurement system (Becker-Hickl on Zeiss Axiovert 200). Metabolic modulation was induced by respiratory chain and/or oxidative stress regulators. The number and profiles of the most significant spectral components were identified by time-resolved area-normalized emission spectroscopy and principal component analysis. Results: The Metabolic Modulation Matrix approach applied to time-resolved spectroscopy data in living cardiac cells demonstrated the presence of at least 3 significant spectrally-distinct components of NAD(P)H fluorescence corresponding to: i) NAD(P)H in water-like environment, ii) NAD(P)H in restricted-motion environment and iii) a flavin-type component. Lifetimes, revealed by fluorescence decay analysis showed values of τ1∼0.4 ± 0.1 ns and τ 2∼1.0 ± 0.2 ns for component i), τ ∼3.2 ± 0.8 ns, for component ii), and τ∼2.5-5 ns for component iii). Conclusions: Presented Metabolic Modulation Matrix concept, in conjunction with spectrally-resolved fluorescence lifetime detection of the cell intrinsic fluorescence, is a promising, highly versatile tool for quantitative assessment of oxidative metabolism in living cells.