title: 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.
abstract: The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 - 400 nm) enters through an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows to perform the photolysis of caged compounds over wide areas (∼ 200 μm) and obtain confocal fluorescence images simultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulate the amount of photolyzed compounds. In the paper we characterize the properties of the system and show its capabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrate its applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals.
authors: Ciudad Universitaria, Departamento de Física, FCEN-UBA, and IFIBA, CONICET, Pabellón I, (1428) Buenos Aires, Argentina. lorena@df.uba.ar
authors: Lorena
authors: L
authors: Sigaut
authors: Mariano
authors: M
authors: Barella
authors: Rocío
authors: R
authors: Espada
authors: María Laura
authors: ML
authors: Ponce
authors: Silvina Ponce
authors: SP
authors: Dawson
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004867:Equipment Design; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry* / Q000378:metabolism*; D016014:Linear Models; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D009865:Oocytes / Q000378:metabolism; D010782:Photolysis; D014466:Ultraviolet Rays; D014982:Xenopus laevis
chemical_list: C085112:2-nitrophenyl-EGTA; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium