Photodegradation of indo-1 and its effect on apparent Ca2+ concentrations

Abstract

Photodegradation processes like that observed for indo-1 are likely to be possible for all cation indicators that contain bis(carboxymethyl)anilino moieties, which include essentially all fluorescent indicators for Ca2+ and Mg2+ currently in biological use. If unrecognized, this photochemical dealkylation leads to an underestimation of the analyte concentrations depending on the intensity and duration of illumination. The problem can be avoided by including cell-permeant antioxidants such as Trolox in the bathing solution. The ultimate solution would be to redesign the indicators to minimize photodegradation in the absence of antioxidants.

Stable Interactions between Mitochondria and Endoplasmic Reticulum Allow Rapid Accumulation of Calcium in a Subpopulation of Mitochondria

“…The reason for this discrepancy is currently unknown, but may lie in the illumination protocol. Indeed, it is known that dyes closely related of rhod-2 (such as fura-2 and indo-1) undergo a photoisomerization process upon laser illumination, with the formation of fluorescent, but Ca 2ϩ -insensitive compounds (28,29). To our knowledge there is only one other report, in pancreatic ␤-cells, suggesting a possible desensitization of the mitochondrial Ca 2ϩ uniporter (27).…”

On the Role of Voltage-Dependent Calcium Channels in Calcium Signaling of AstrocytesIn Situ

“…Occasionally, a slight decrease was observed in R405: 485 basal levels. Indeed, prolonged UV irradiation of Indo-1 can cause overall photobleaching and conversion to a fluorescent, but C a 2ϩ -insensitive, species (Scheenen et al, 1996). In several experiments, we used 100 M Trolox, a vitamin E analog that inhibits formation of Indo-1 photodegradation products (Scheenen et al, 1996).…”

Measurement of Intracellular Calcium

“…However, rapid photobleaching has the potential to decrease signal-to-noise ratio (416). In addition, it has been reported that UV illumination induces photodegradation of indo 1 (342). Ultraviolet illumination generates a Ca 2ϩ -insensitive fluorescent compound from indo 1 whose emission spectrum is quite close to Ca 2ϩ -free indo 1, resulting in underestimation of [Ca 2ϩ ] measurements (342).…”

“…In addition, it has been reported that UV illumination induces photodegradation of indo 1 (342). Ultraviolet illumination generates a Ca 2ϩ -insensitive fluorescent compound from indo 1 whose emission spectrum is quite close to Ca 2ϩ -free indo 1, resulting in underestimation of [Ca 2ϩ ] measurements (342). Moreover, the intracellular milieu can change indo 1's emission spectrum (161,289).…”