Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress

By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UVA radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser, focused through high-numerical-aperture microscope optics, provided diffraction-limited microbeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster ovary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm-2) resulted in oxidative stress correlated with increased autofluorescence intensity, onset of nuclear fluorescence, and a fluorescence lifetime decrease. The cellular response to femtosecond NIR microbeams depended significantly on peak power. Peak powers above a threshold value of about 0.5 kW (average power: 6 mW). 0.55 kW (7 mW) and 0.8 kW (10 mW) at 730 nm, 760 nm and 800 nm, respectively, resulted in the onset of short-lived luminescence with higher intensity (100 x) than the intracellular NAD(P)H fluorescence. This luminescence, accompanied by destruction of cellular morphology, was localized and occurred in the mitochondrial region. In contrast, beams at a power of less than 0.5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.