The freeze-fracture thaw-fix (FfTF) technique described in earlier papers is applied in the present work to more detailed study of the chicken erythrocyte, by transmission replicas and high resolution scanning electron microscopy (3 nm scan beam size). The three-dimensional structure of the chromatin, and possibly the non-histone protein matrix, of fractured nuclei is to a large extent retained in this method of preparation and seen in stereomicrographs. In these micrographs the helical sub-structure of the 25 nm chromatin strands can be seen at about the same resolution as that of previously published micrographs in which extracted chromatin is viewed by negative contrast or after metal shadowing. The useful resolution of the secondary electron micrographs, for a suitably mounted specimen, is shown to be as good as that of transmission micrographs of platinum-carbon replicas of the same material.

Methods are described for the use of a sensitive photomultiplier, improved photomultiplier voltage supply circuitry, quartz lens and a half-parabolic mirror to increase the amount of light collected from a luminescing specimen examined in the scanning electron microscope. The combined effect of these improvements increases cathodoluminescence detection twenty times. Sources of background light are investigated and recommendations are made for decreasing this noise component, which seriously limits sensitivity of cathodoluminescence signal detection.An appendix describes the natural luminescence (autoluminescence) of plant tissues under the impact of the electron beam. The biological applications of this mode of scanning microscopy have been little explored, but autocathodoluminescence is found to be of wide occurrence in plant tissues.

This paper continues work reported in an earlier paper on modification of a Cambridge Stereoscan Mk IIA to improve the quality of cathodoluminescent micrographs of biological material. In the work presently described the microscope gun has been offset laterally by 2 mm, to prevent light from the filament passing down through the column to the specimen chamber. The electron beam is brought onto the column axis by deflection coils. This modification effectively eliminates background light in the chamber, and a full parabolic mirror is fitted to maximize light collection. Results for yeasts and wheat seed sections are described.

E. coli were examined by the freeze-fracture thaw-fix technique, embedded in thin fibrin gels. After glutaraldehyde fixation the bacterial nucleoid was found spread out over the surrounding fibrin. Addition of calcium and uranyl acetate to the fixative preserved the nucleoid in compact form. The spread nucleoid was then examined against a smooth mica background after freeze-thaw and osmotic lysis. These spreads were critical-point dried, rotary shadowed with platinum-carbon and viewed as stereo-pair micrographs. Structures seen are tentatively interpreted as clusters of polyribosomes, extended DNA, and supercoiled DNA complexed with proteins or polyamines. After osmotic lysis, glutaraldehyde alone preserves the nucleoid in compact form. Only where strands are broken, in freeze-fracture or freeze-thaw lysis, must uranyl acetate be added to the fixative to preserve a compact structure.