Figure 1.

Thickness estimation of ultrathin sections for 3D reconstruction. (A) Minimal folds of an ultrathin section cut by a microtome set to cut 95 nm sections. Width of the minimal folds, twice the section thickness, was 130 nm, much less than expected. Note the black vertical line at the center of the fold, which is the adhered, folded membrane perpendicular to the plane of a section. (B) Correlation between section thicknesses set by the microtome and those measured by a color laser 3D microscope or the minimal folds method. The thicknesses measured by the optical method were more similar to those set by the microtome than those measured by the minimal folds method, which were 50–80% of the set section thickness. (C, D) Three-dimensional view of ultrathin section (90 nm thickness, pink) on glass slide (beige) obtained by the laser scanning microscope. The border of the section and slide surface is clearly identified in larger magnification (C). (E) The pseudocolor image representing height of the ultrathin section (orange) and glass slide (blue). Note that the colors are uniformly distributed on the surface of the section and glass slide, indicating the surface flatness. Thickness of the ultrathin section was estimated from the difference between average heights of the two areas. Rectangles (about 50 μm²), indicate areas used for evaluating the average height. (F) Light microscopic photograph of red blood cells. (G, H) The 3D images of the red blood cells reconstructed assuming 50 nm (G) and 90 nm (H) in section thickness. The view of the upper images is the same as in the light microscopic photograph in (F); the view of the lower images is “side-on”. The red blood cells reconstructed assuming a 90 nm section thickness were more similar in shape and size to those in the light microscopic photograph than were cells reconstructed assuming a 50 nm section thickness.