Electron micrographs obtained from biological specimens generally have very low signal-to-noise ratios. To determine the structure of macromolecular assemblies and cellular organelles by electron microscopy, it is therefore necessary to employ image processing methods that must often be adapted for particular applications. To address such problems in our laboratory, we are developing and implementing a series of computational tools. Structures of filamentous macromolecules that exhibit tight and variable curvature can be obtained by first straightening them, computationally, prior to image averaging, classification, and three-dimensional reconstruction. We have developed a computer program that straightens filaments, having any curvature profile, by fitting non-uniform cubic-splines to their center lines. The program was implemented as a plug in of the ImageJ program package, and can be downloaded from http://rsb.info.nih.gov/ij/plugins/straighten.html. Circular macromolecules may have rotational symmetries that are hidden by noise. To be able to perform image averaging one needs to know if such symmetry exists in the given structure, and what is the order of symmetry. We are developing a program that detects rotational symmetries of macromolecules, and determines the statistical significance of the detected symmetry(ies). The program will be executable on any computer (operating system). Electron tomography of subcellular components on the nanometer scale in the transmission electron microscope provides a three-dimensional visualization of supramolecular arrangements and associations. We are calculating the three-dimensional structures of several biological specimens provided by other NIH investigators, and visualizing them with different rendering techniques. We are also studying the extent to which energy-filtered imaging can enhance collection of such information. Energy filtering completely removes the inelastic scattering from the image and can thus significantly improve tomographic reconstructions of lightly stained specimens where inelastic scattering predominates over elastic scattering.
