Research Papers: Imaging

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

[+] Author Affiliations
Lorena Sigaut, Mariano Barella, Rocío Espada, María Laura Ponce, Silvina Ponce Dawson

Ciudad Universitaria, Departamento de Física, FCEN-UBA, and IFIBA, CONICET, Pabellón I, (1428) Buenos Aires, Argentina

J. Biomed. Opt. 16(6), 066013 (June 22, 2011). doi:10.1117/1.3592497
History: Received January 26, 2011; Revised April 25, 2011; Accepted April 28, 2011; Published June 22, 2011; Online June 22, 2011
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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.

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© 2011 Society of Photo-Optical Instrumentation Engineers (SPIE)

Citation

Lorena Sigaut ; Mariano Barella ; Rocío Espada ; María Laura Ponce and Silvina Ponce Dawson
"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", J. Biomed. Opt. 16(6), 066013 (June 22, 2011). ; http://dx.doi.org/10.1117/1.3592497


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