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.

We report on cell damage of single cells confined in continuous-wave (cw), near-infrared (NIR) multimode optical traps as a result of multiphoton absorption phenomena. Trapping beams at NIR wavelengths less than 800 nm are capable of damaging cells through a two-photon absorption process. Cell damage is more pronounced in multimode cw traps compared with single-frequency true cw NIR traps because of transient power enhancement by longitudinal mode beating. Partial mode locking in tunable cw Ti:sapphire lasers used as trapping beam sources can produce unstable subnanosecond pulses at certain wavelengths that amplify multiphoton absorption effects significantly. We recommend the use of single-frequency long-wavelength NIR trapping beams for optical micromanipulation of vital cells.

We report the observation of two-photon f luorescence excitation in a continuous-wave (cw) single-beam gradient force optical trap and demonstrate its use as an in situ probe to study the physiological state of an optically confined sample. In particular, a cw Nd:YAG (1064-nm) laser is used simultaneously to confine, and excite visible f luorescence from submicrometer regions of, cell specimens. Two-photon f luorescence emission spectra are presented for motile human sperm cells and immotile Chinese hamster ovary cells that have been labeled with nucleic acid (Propidium Iodide) and pH-sensitive (Snarf) f luorescent probes. The resulting spectra are correlated to light-induced changes in the physiological state experienced by the trapped cells. This spectral technique should prove extremely useful for monitoring cellular activity and the effects of confinement by optical tweezers. © 1995, Optical Society of America.

The influence of femtosecond near-infrared (NIR) microirradiation on cell vitality and cellular reproduction has been studied. Chinese hamster ovary cells exposed to a highly focused 150-fs scanning beam at 730, 760, and 800 nm (80 MHz, 80-mus pixel dwell time) of /=6 -mW mean power, cells were unable to form clones. They died or became giant cells. Complete cell destruction, including cell fragmentation, occurred at mean powers >10 mW. Cell death was accompanied by intense luminescence in the mitochondrial region. When we consider the diffraction-limited spot size in the submicrometer region, intensities and photon flux densities of 0.8-kW pulses (10-mW mean power) are of the order of terawatts per square centimeter (10(12)W/cm (2)) and 10(32) photons cm(-2) s(-1) , respectively. Extremely high fields may induce destructive intracellular plasma formation. The power limitations should be considered during NIR femtosecond microscopy of vital cells and in the design of compact NIR femtosecond solid-state lasers for two-photon microscopes.

We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ∼0.035 μmoles/106 cells/h. © 2013 Biophysical Society.