Biophysical Journal 68: 2429-2443 (1995)
© 1995 the Biophysical Society

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Identification of source of oscillations in apparent sarcomere length measured by laser diffraction.

MRC Muscle and Cell Motility Unit, King's College London, United Kingdom.

ABSTRACT

The most widely used technique for dynamic estimates of sarcomere length in muscle is laser light diffraction. We have identified conditions under which artifactual oscillations can arise in apparent sarcomere length measured by this technique and report methods to reduce the effect. Altringham et al. (1984) first reported that the diffraction angle can exhibit one cycle of oscillation for each sarcomere length displacement of the illuminated portion of the fiber. We find that the amplitude of similar oscillations is strongly dependent on the intensity of light scattered from objects near the fiber and on the spacing between fiber and scatterer. The oscillations can be eliminated by minimizing scattered light and positioning the fiber a few millimeters from sources of scattering. A theoretical description shows that oscillations of this kind are expected from interference of scattered and diffracted light. Interference fringes were observed along the meridian of the pattern, and these moved during translation of either a fiber or a grating. The movement of fringes across the diffraction order shifts the centroid back and forth and, when associated with steady shortening, can give rise to "steps" and "pauses" in apparent striation spacing.

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