Light diffractometry for determining the sarcomere length of striated muscle: an evaluation

Single semitendinosus muscle fibres of frog were illuminated at normal incidence with an argon-ion laser lasing at 514.5, 496.5, 488.0 or 476.5 nm. The meridional diffraction was projected directly on to a photographic film and recorded. A scanning densitometer plotted the diffraction recorded on film. The densitometer scans yielded the centroid positions of the diffraction columns. The shift of the centroid position upon a change of the wavelength of the laser beam obeyed the grating equation. Relative to the undiffracted beam, the positions of the fine structure within the first- and second-order diffractions were measured with a spectroscopic plate reader to a precision of 1 micrometer. The shifts of the fine structures also followed the prediction of the grating equation when the wavelength of the laser beam varied. The fine structures of the left and right diffraction columns were different. The difference in position and intensity of the corresponding fine structures of the left and right diffraction columns was explained by assuming that the fibre acted as a quasi-homogeneous optical medium and that the myofibrils were tilted at most by 5 degrees against the fibre axis. Each diffraction fine structure was interpreted as the superposition of the light scattered from a group of sarcomeres of equal length. Its position allowed an accurate determination of the sarcomere length according to the grating equation.