Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells

Abstract

We report a novel analytical approach to identify individual components of a cell’s endogenous fluorescence, recorded by spectrally-resolved time-correlated single photon counting (TCSPC). Time-resolved area-normalized emission spectroscopy (TRANES) and principal component analysis (PCA) were applied to estimate the number of spectral components after metabolic modulation of cardiac cells following excitation with a 375 nm picosecond laser. Linear unmixing of TCSPC data spectrally decomposed individual components in living cells, while using characteristics of endogenously fluorescing molecules in solvents as a reference spectral database. Our data demonstrate the presence of three individual components, corresponding to the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in organic and inorganic solvents and to the residual flavoprotein fluorescence. The presented analytical approach offers a new alternative for the spectral separation of multi-wavelength fluorescence lifetime spectroscopy data to the conventional analysis, and opens a new possibility for the use of pattern recognition for fast resolution of components in 2D fluorescence lifetime microscopy images.

Correlation of intracellular oxygen and cell metabolism by simultaneous PLIM of phosphorescent TLD1433 and FLIM of NAD(P)H

“…Free NAD(P)H typically possesses a short fluorescence lifetime around 400 picoseconds, whereas if it is bound to proteins, the lifetime is much longer, 1.5 to 4 nanoseconds depending on the binding target . Heterogeneous surrounding of the coenzyme and different protein binding possibilities lead to a complex fluorescence lifetime distribution in the cells . Nevertheless, in most cases, the relative amount of free and protein‐bound NAD(P)H can be analyzed by a double‐exponential fitting procedure .…”

Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions

“…An essential disadvantage of this method is, however, that the cells need to be stained—meaning that they are not in their native state anymore. Intrinsic NAD(P)H fluorescence has been used to create confocal images by two photon excitation in cardiac myocytes [ 14 ]. This method, although promising for studies of metabolic states in living cells, only allows for the study of one biomarker.…”

Correlative NAD(P)H‐FLIM and oxygen sensing‐PLIM for metabolic mapping

“…If it is bound to proteins the lifetime is much longer (1–6.5 ns, depending on the target to which the cofactor binds) . Due to conformational heterogeneity of the different enzymes, bound NAD(P)H can have complex lifetime distributions with more than one exponential component . Also due to the existence of the two redox couples NAD + /NADH and NADP + /NADPH in the cells the lifetime of bound NAD(P)H varies .…”