2-PHOTON-EXCITED LIFETIME IMAGING OF AUTOFLUORESCENCE IN CELLS DURINGUVA AND NIR PHOTOSTRESS

By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (N IR) radiation (two-photon absorption) was compared with exposure to lo w-power UVA radiation (one-photon absorption), Excitation radiation fr om a tunable Ti-sapphire laser, focused through high-numerical-apertur e microscope optics, provided diffraction-limited microbeams of an adj ustable peak power, Laser scanning NIR microscopy was used to detect s patially the intracellular distribution of fluorescent coenzymes by fl uorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster o vary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure t o 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm(- 2)) resulted in oxidative stress correlated with increased autofluores cence intensity, onset of nuclear fluorescence, and a fluorescence lif etime decrease, The cellular response to femtosecond NIR microbeams de pended significantly on peak power, Peak powers above a threshold valu e 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 (100x) than the int racellular NAD(P)H fluorescence. This luminescence, accompanied by des truction of cellular morphology, was localized and occurred in the mit ochondrial region. In contrast, beams at a power of less than 0.5 kW a llowed nondestructive fluorophore detection with high spatial and temp oral resolution without modification of cellular redox state or cell m orphology.