The purpose of this project is to examine the subcellular and extracellular structure of nerve and muscle and relate such structure to function. Electron microscopy in TEM, STEM and analytical electron beam probe modes, such as EELS and EDAX, determination of proteins contributing to these structures and structural modeling are methods used in this study. The following structures are probed: 1) Neuroplasmic lattice, 2) neurofilaments, 3) microtubules, 4) axolemma, 5) glial cell membranes, and 6) myofilaments. Methods developed and used in this study are: 1) Stereoscopic imaging, 2) optical autocorrelation, 3) fast Fourier transformation (FFT) of STEM video images, and 4) STEM video image filtering and image enhancement using reverse Fourier transformation. Video imaged light microscopy is used to study living neurons in differential interference contrast. A new method was developed for direct visualization of particle velocity distribution. By viewing image pairs separated by an appropriate time interval in sequential recording of the subject, the positive or negative parallax arising from particle motion results in the binocular image of a particle being perceived as raised or lowered relative to an immobile background plane depending on its direction of movement. The degree of perceived elevation is proportional to particle speed. Using this method, measured particle movement during axoplasmic transport in squid axons ranged from 0.05 to 0.75 mum/sec. The dynamic behavior of filopodia in variety of cultured neuronal cell types while attaching to substrate have been analyzed. The configurational changes, particularly branching and sliding of branch points, have been and continue to be a focus of this study with particular emphasis on specific localization of fibrous proteins such as f-actin using fluorescently-labelled phalloidin. The reversible effects of hypotonicity on the structure of filopodia as well as filopodial tension have been studied and remain under investigation.
