UC Irvine

Cell damage in UV and NIR laser microscopes by highly focused micromanipulation and fluorescence excitation microbeams has been studied. Damage in erythrocytes, spermatozoa and Chinese hamster ovary cells was detected by monitoring morphology changes, autofluorescence detection, cloning assay, and viability screening. It was found that 364 nm/365 nm UVA radiation induced irreversible cell damage at radiant exposures as low as <10 J/cm2. NIR CW microradiation used in laser tweezers was also able to damage cells via a two-photon excitation process, in particular, when using <800 nm trapping beams. Non- destructive two-photon excitation in femtosecond NIR microscopes is possible within a narrow intensity window. The lower limit is determined by two-photon absorption coefficients and detector efficiency, the higher by intracellular optical breakdown in the extranuclear region. Above certain wavelength-dependent intensity thresholds in femtosecond microscopy, cells were completely destroyed by fragmentation concomitant with plasma generation. The influence of excitation and micromanipulation microbeams should be considered when studying physiology and metabolism of vital cells. ©2004 Copyright SPIE - The International Society for Optical Engineering.