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

An experimental set-up is described that allows the combination of rapid tr ansmembrane 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 mic roscope photometer, The pools of the vaseline gap chamber that contain the solutions surrounding the cut ends and the voltage-clamped segment of the m uscle fibre are closed and have volumes of 20-50 mu l. Thin tubes allow rap id solution change or continuous perfusion in the chamber compartments. Acc essory 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 in tensity with increasing numbers of flashes were quantified with an instrume nt that integrates the photo-current of a UV-sensitive silicon diode. The f unction of the set-up was demonstrated by measuring the efficiency of Ca2release from DM-nitrophen in quartz capillaries using the Ca2+-sensitive dy e antipyrylazo III and by recording the flash-induced recovery of L-type ca lcium currents in muscle fibres blocked by the light-sensitive dihydropyrid ine drug nifedipine.