FIGURE 45.

Finding the top focal plane of a section. The three images of Giemsa stained methacrylate embedded rat cortex were acquired with 0.6 μm steps along the z‐axis. Image (a) is clearly out of focus. Some cellular features appear sharp and the background becomes finely structured in Image (b), which may represent the early recognition of the top focal plane in Figure 44. While sharpness further increases in the Image (c), a fine grain of dust resting on the surface of the section (arrowheads) is now out of focus. The depth of field should be able to contain both dust and tissue at the very top of the section. Image (c) may represent the late recognition of the top focal plane in Figure 44. Scale bar: 10 μm [Color figure can be viewed at wileyonlinelibrary.com]