THE USE OF EXOGENOUS FLUORESCENT PROBES FOR TEMPERATURE MEASUREMENTS IN SINGLE LIVING CELLS

The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazo1-4-y1 (NBD) and 6-dodeca-noy1-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2°C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1°C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.     

AUTOFLUORESCENCE SPECTROSCOPY OF OPTICALLY TRAPPED CELLS

Abstract— 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 β-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.