The goal of this project is to characterize the shape, molecular weight distribution, and elemental composition of individual macromolecules and macromolecular assemblies. Such assemblies are critical to many cell functions, and their behavior in vitro reflects their function and regulation in intact cells. This project depends on an unique instrument - a low-temperature, high-resolution, field-emission scanning transmission electron microscope (STEM) - for molecular weight mapping and chemical analysis by parallel electron energy loss spectroscopy (PEELS). The development of this instrument, and new preparation techniques for directly-frozen thin films and high-resolution platinum replicas were described last year. Now, the capabilities of this STEM have been extended to ultrathin cryosections of directly frozen tissues. Mass mapping and calcium analysis of sections of cerebellar cortex have characterized two distinct classes of endoplasmic reticulum (ER) in the dendrites of Purkinje cells. In addition, spectrum imaging, a new technique for high-resolution elemental mapping by PEELS, has bee,) implemented and used to image the distribution of calcium-sequestering ER within these dendrites. To measure the distribution of water within Purkinje cell dendrites, a new method based on analyzing the low-loss region of low-dose PEELS map of frozen hydrated sections has been developed. The combination of low-dose mass mapping and high-dose PEELS spectrum imaging would appear to be a practical method for correlating ion and water fluxes that accompany neuronal activity.
