Control of sarcomere length in skinned muscle fibres of Rana temporaria during mechanical transients

A new technique is described for high-speed measurement of striation spacing of single skinned muscle fibres. A galvanometer mirror directs helium-neon laser light on to the muscle fibre at a variable angle. Light diffracted by the cross-striations is collected by a position-sensitive photodetector. The incident angle necessary to centre the diffracted light beam on to the photodetector is related to sarcomere length. The instrument was tested by comparison with measurements obtained with a compound microscope. Discrepancies of several nanometers per half sarcomere were observed between these two methods. When the incident angle of the laser beam was varied sinusoidally about its mean position the magnitude of the discrepancy was reduced. During steady passive shortening of the muscle fibres the output of the diffraction instrument often displayed pauses and brief periods of rapid shortening. These irregularities were eliminated by averaging the sarcomere length output over a range of illumination angles by oscillating the incident angle of the laser beam. The results suggest that at the spatial resolution of several nanometers per half sarcomere, volume diffraction effects can cause the apparent sarcomere length measured from the angle of coherent light diffraction to differ from the mean striation spacing. With incident-angle oscillation the time and spatial resolution of the equipment were satisfactory for the sarcomere length signal to be fed back to a length controller for a 'sarcomere length clamp'. In active contractions, stiffness was closely related to steady developed tension at sub-saturating calcium concentrations. Skinned fibres are less stiff than intact fibres at a given level of developed tension.