A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections

Abstract

To observe certain quantitative features of neuronal geometry and microcircuitry, it is necessary to reconstruct neurons from electron micrographs of serial, ultra-thin sections. We describe here an approach to preparing, photographing, and analyzing moderately long series (100-500 sections). A series is prepared using an assembly line approach: one operator cuts while a second mounts ribbons of sections using various mechanical aids. Photographs are taken in the electron microscope at low magnification and high accelerating voltage. Sequential negatives are aligned using an image combiner and copied, using quasi-coherent illumination, onto 35 mm film. The resulting "movie' is mounted on a precision film transport mounted on an X-Y stage controlled by stepping motors. The movie is viewed through a high resolution video system while a video storage device and switching system permit rapid alternation between frames for comparisons. The profiles of a process in successive frames are "microaligned' by small adjustments of the transport's X-Y position. The absolute X-Y biological coordinates for each frame and the correction necessary to bring it into alignment are stored in a Z80 microprocessor as a process vector. When the movie is re-examined with the stepping motors under control of the computer, the microaligned process shows almost no frame-to-frame jitter. The process vector may be used to generate a "branch schematic' of the neuron. The microaligned profiles can also be digitized and displayed as a reconstruction using a PDP 11/34 computer. Uses of the approach are presented with examples from the cat retina and visual cortex.