CELL PHOTODAMAGE, A POTENTIAL HAZARD WHEN MEASURING CYTOPLASMIC CA-2+ WITH FURA-2

Photodamage of insulin-releasing pancreatic beta-cells during measurements of the cytoplasmic Ca2+ concentration ([Ca2+]i) with fura-2 was studied. Keeping the fluorescence intensities at low levels, regular oscillations of [Ca2+]i with a frequency of 0.2-0.5 min-1 could be recorded for more than 60 min in glucose-stimulated cells. However, after a tenfold increase of the excitation energies, oscillations disappeared in most cells, the glucose response being transformed into an elevated concentration of [Ca2+]i with irregular fluctuations. Under corresponding conditions, the loss of fluorescence due to photobleaching during the initial 10 min increased from 7 to 16% in cell-sized fura-2-containing droplets. In further attempts to investigate the effects of photodamage, cells were irradiated by pulses (< 1 ms) of intense UV light (300-400 nm). A single flash of 4-7 mJ/mm2 perturbed the oscillatory pattern with maintenance of the glucose response in terms of raised [Ca2+]i. More severe lesions obtained by repeated pulses of 13-15 mJ/mm2 involved an excessive and uncontrolled rise of [Ca2+]i. It is concluded that there is a significant potential risk of photodamage when using the fura-2 technique. In the pancreatic beta-cells, exposure to intense excitation light may even result in a selective suppression of the oscillatory response to glucose.