Inverted double-gap isolation chamber for high-resolution calcium fluorimetry in skeletal muscle fibers.

Here we describe an improved inverted double-grease-gap isolation chamber that allows the formation of grease seals of high mechanical stability and high electrical resistance. We also provide a detailed description of the procedure used to mount the muscle fibers onto the apparatus. The new chamber permits the electrophysiological study of muscle fibers using an inverted microscope and high-resolution objectives, thus complying with the requirements of modern fluorescence confocal detection methods. The simplicity and reliability of the mounting procedure make this chamber preferable over other gap isolation chambers currently used for simultaneous electrophysiological and optical studies of calcium release. Experimental results obtained from amphibian muscle fibers are presented to illustrate the performance of the chamber when using global fluorescence detection, confocal spot detection, and laser confocal scanning imaging.