Custom-made modification of a commercial confocal microscope to photolyze caged compounds using the conventional illumination module and its application to the observation of Inositol 1,4,5-trisphosphate-mediated calcium signals

LORENA SIGAUT; MARIANO BARELLA; ROCÍO ESPADA; MARÍA LAURA PONCE; SILVINA PONCE DAWSON

JOURNAL OF BIOMEDICAL OPTICS

SPIE-SOC PHOTOPTICAL INSTRUMENTATION ENGINEERS

Año: 2011 p. 660131 - 660137

1083-3668

Abstract. The flash photolysis of ?caged? compounds is a powerful experimental technique for producing rapidchanges in concentrations of bioactive signaling molecules. These caged compounds are inactive and becomeactive when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympusconfocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscopefor conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 − 400 nm) entersthrough an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows toperform the photolysis of caged compounds over wide areas (∼200 μm) and obtain confocal fluorescence imagessimultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulatethe amount of photolyzed compounds. In the paper we characterize the properties of the system and show itscapabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrateits applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals. C 2011 Society ofPhoto-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3592497]