J. H. McAlear
1961 Journal of Cell Biology  
One of the limitations of conventional techniques in electron microscopy comes from the bars of the grids which periodically interrupt the field. This was pointed out by Gasser (1), for example, who attempted to get total fiber counts in cross-sections of cat nerves. Techniques have been developed to mount serial sections for electron microscopy (2), but it is often desirable to have uninterrupted fields for the examination of sections with appreciable dimensions in both axes. A more recent
more » ... . A more recent report gives a method for the preparation of unsupported thick films for the examination of large areas with the electron microscope (3). A similar method has been applied successfully in the investigation of total numbers of nerve fibers found in the cross-sections of various nerves, including submicroscopic ones. Grids for unsupported films were prepared by cutting out about 40 grid squares from standard (2 mm.) grids with a sharp razor blade and flattening the cut edges of the copper against glass (see insert in Fig. 1 ). Formvar films were prepared on glass slides and floated upon a water bath in the usual manner. A number of Formvar solutions were tried, but it was found that the most successful films were prepared from two layers of 0.1 per cent solution. The special grids were placed upon the floating film and picked up together by moving a wire mesh down upon them and through the water. The films were then covered with a 100 to 150 A thick carbon layer. It was possible to prepare a reasonably large number of grids successfully in this manner. The sections were cut with a diamond knife on a Porter-Blum ultramicrotome and picked up squarely over the unsupported portion of the film. The sections were examined and photographed in a Siemens Elmiskop I electron microscope at magnifications of less than 1000 using the inter-mediate projector lens connected to a rheostat for continuous magnification. The magnification was selected in a manner such that the image of the 50 # objective aperture was just outside of the field of view. Focusing was achieved by removing the objective aperture and bringing the objective lens control to the point where the minimum contrast in the image was seen, and then replacing the aperture before taking photographs. Calibration of the magnification was accomplished by taking an area in the image at a higher fixed magnification and comparing the corresponding distances in the same region in the low magnification map. The accelerating voltage was 60 kv. and the condenser II lens was completely defocused. Nerves and nerve tracts up to 0.4 mm. in diameter were mapped by this technique. These included the circumesophageal connective, the nerves in the large chelated leg, and the ventral nerve cord in the crayfish, the circumesophageal connective in the roach, and the optic nerve of the mouse. Allowing for proper overlap of the fields, from 12 to 36 plates were required for each map. Fig. 1 shows a montage of the circumesophageal connective of the crayfish. The negatives were developed for 15 minutes in Finex-L 1 developer and, following routine processing, were printed at a X 3 enlargement. The prints were fitted together by matching each print with its neighbor, taping them loosely together with cellophane tape, and then cutting through the median line of overlap with a razor blade. After removing the cut-off overlap areas, the adjacent prints were applied together on the reverse side with cellophane tape. Because the magnification at the edge of a negative is slightly higher than at the center, it was not possible to achieve a perfect 1 Ansco, Binghamton, New York. 133 on
doi:10.1083/jcb.10.1.133 fatcat:rt3zagatrfbn3hogcixql5ruie