Laser Irradiation and Raman Spectroscopy o f Single L iving Cells Chromosomes : Sample Degradation Occurs

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradat ion of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot 0.5 pm in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. W ith 5 m W of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heat ing due to absorpt ion of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradat ion of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanat ion therefore seems to be that the sample degradat ion is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentif ied sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples. IG 1991 Academic PWS, IN.