Morphometric study of glomerular slit diaphragms fixed by rapid-freezing and freeze-substitution. The widths of slit diaphragms in rat glomeruli were quantitatively measured on electron micrographs obtained by the rapid-freezing and freeze-substitution (FS) fixation method and compared to those prepared by conventional chemical fixation procedures, such as tannic acid, glutaraldehyde and osmium tetroxide (TGO) or glutaraldehyde and osmium tetroxide (GO) fixation. When fixed by FS using 2% osmium tetroxide, glomerular slit diaphragms had widths of 33.8 ± 3.6 nm, but when fixed by TGO or GO they had widths of 47.0 ± 4.3 and 48.7 ± 5.5 nm, respectively. The width of slit diaphragms was significantly narrower after cryofixation than after conventional TGO or GO fixation (P < 0.001). Similarly, the distance between neighboring foot processes at 50 nm from slit diaphragms was significantly narrower after cryofixation (P < 0.001). The widths were 98.6 ± 17.1 nm after TGO fixation, 122.0 ± 29.3 nm after GO fixation and only 54.8 ± 18.7 nm after FS fixation. These foot processes were significantly broader after FS fixation. The foot processes were 179.2 ± 59.4 nm in breadth after TGO fixation (P < 0.001), 188.8 ± 78.2 nm after GO fixation < 0.01) and 235.1 ± 87.2 nm after FS fixation. These results suggest that the cytoplasm of foot processes shrinks when the tissue is prepared by conventional chemical fixation methods, and that the broadening of slit diaphragms is an artifact separation of the margins of adjacent foot processes due to shrinkage. We propose that TGO fixation alters the structure of slit diaphragms by causing them to shrink.