Cell photodamage, a potential hazard when measuring cytoplasmic Ca2+ 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.
Grapengiesser
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Department of Medical Cell Biology, University of Uppsala, Sweden.
D000818:Animals; D002118:Calcium / Q000032:analysis*; D003593:Cytoplasm / Q000737:chemistry / Q000648:ultrastructure; D005453:Fluorescence; D016257:Fura-2; D005947:Glucose / Q000494:pharmacology; D007515:Islets of Langerhans / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D008027:Light / Q000009:adverse effects*; D051379:Mice; D008820:Mice, Obese; D010782:Photolysis
D005947:Glucose; D002118:Calcium; D016257:Fura-2