Tools for flash-photolysis experiments on voltage-clamped muscle fibre segments

Abstract

An experimental set-up is described that allows the combination of rapid transmembrane voltage changes and photometric calcium recording with the fast photochemical turnover of substances applied externally or intracellularly to cut skeletal muscle fibres. It consists of a double-vaseline-gap system, designed for use with a xenon-flash-lamp device and a dual-wavelength microscope photometer. The pools of the vaseline gap chamber that contain the solutions surrounding the cut ends and the voltage-clamped segment of the muscle fibre are closed and have volumes of 20–50 µl. Thin tubes allow rapid solution change or continuous perfusion in the chamber compartments. Accessory tools were constructed to simplify focussing and measuring the flash-light intensity. A pilot light delivered from a red laser diode is used as a guide beam to target the ultraviolet (UV) flash to the preparation. The light distribution in the focal region and the relative changes in flash intensity with increasing numbers of flashes were quantified with an instrument that integrates the photo-current of a UV-sensitive silicon diode. The function of the set-up was demonstrated by measuring the efficiency of Ca²⁺ release from DM-nitrophen in quartz capillaries using the Ca²⁺-sensitive dye antipyrylazo III and by recording the flash-induced recovery of L-type calcium currents in muscle fibres blocked by the light-sensitive dihydropyridine drug nifedipine.