Microscopic Mapping of Subnanometric Motion with Multiple-Beam Differential Holographic Technique

The measurement of ultrasmall displacement is usually performed by laser interferometry. In most cases, this method is specified for the surface measurement and requires a relatively smooth surface capable of reflecting light. In this research, a newly developed method, mutiple -beam microdifferential holography, is introduced to measure a small configuration change. This configuration change can happen on the surface of an object or inside a semitransparent object. In the experiment, two reference beams are used to record a pair of phase biased holographic images simultaneously. During the image reconstruction, the CCD image acquisition system is employed to record the pair of images one at a time and then process them digitally. The subtraction image intuitively shows that the deformation of tested object occurs between the double exposures applied during the holographic recording. A second object beam, usually a plane wave, is added to the imaging system for the purpose of image registration, which is required for the image processing. Several developments upgraded the system performance. The calibration was done with an extremely consistent moving object, a small air bubble drifting in a glycerine-filled capillary. Displacements as small as 0.4 nanometer are reported. In application, a living cell, a single frog muscle fiber, was under examination. This part of the research focused mainly on the crossbridge mechanism of striated muscle contraction. The images made at the plateau of tetanus suggest either that the cycling time constant is much longer than 10 msec, that the displacement for a power stroke is substantially less than 12 nanometer, or that the crossbridge is not cycling during the isometric force generation. The images made at the initial state of force development suggest that a large number of crossbridges shift toward the actin filament at the onset of the force development and stay there (at least without large scale rotation) even when the force has started to develop.