Autofluorescence spectroscopy of optically trapped cells.

Cellular autofluorescence spectra were monitored in a single-beam gradient force optical trap ("optical tweezers") in order to probe the physiological effects of near infrared and UVA (320-400 nm) microirradiation. Prior to trapping, Chinese hamster ovary cells exhibited weak UVA-excited autofluorescence with maxima at 455 nm characteristic of beta-nicotinamide adenine dinucleotide (phosphate) emission. No strong effect of a 1064 nm NIR microbeam on fluorescence intensity and spectral characteristics was found during trapping, even for power densities up to 70 MW/cm2 and radiant exposures of 100 GJ/cm2. In contrast to the 1064 nm trap, a 760 nm trapping beam caused a two-fold autofluorescence increase within 5 min (about 20 GJ/cm2). Exposure to 365 nm UVA (1 W/cm2) during 1064 nm trapping significantly altered cellular autofluorescence, causing, within 10 min, a five-fold increase and a 6 nm red shift versus initial levels. We conclude that 1064 nm microbeams can be applied for an extended period without producing autofluorescence changes characteristic of alterations in the cellular redox state. However, 760 nm effects may occur via a two-photon absorption mechanism, which, in a manner similar to UVA exposure, alters the redox balance and places the cell in a state of oxidative stress.