Abstract
A simple hemolytic assay of the photodamage suffered by individual intact erythrocytes upon localized exposure to high laser intensities such as those encountered in fluorescence photobleaching recovery (FPR) experiments has been characterized. At incident beam powers over 100,000 W/cm2 at 514 or 568 nm, hemoglobin absorption induces thermal-shock lysis. Below the thermal-shock threshold, other intracellular chromophores appear to catalyze membrane contraction and lysis with linear dose kinetics in anaerobic preparations. A variety of chemical agents that inhibit photodamage to red cell ghost membranes at low intensities (less than 450 W/cm2) do not benefit intact cells at realistic FPR bleaching doses of 10,000 to 1,000,000 W/cm2. However, we find that sample deoxygenation dramatically photostabilizes erythrocytes, thus we have developed a physiologically compatible in situ deoxygenation method utilizing submitochondrial particles. The extent to which this and other deoxygenation procedures modify fluorophore photolability has been determined. FPR measurements become practical on intact erythrocytes when a combination of low bleach power, thorough deoxygenation, and long wavelength excitable (approximately 650 nm) fluorescence probes is employed; the equivalent of 60,000 photobleaches per cell can then be tolerated without detectable damage.