Figure 1.

Illustrating the importance of fully three-dimensional (3-D) registration. Adjacent image stacks with an equal number of optical slices may still be offset axially relative to the objects in the tissue. (A) Two adjacent confocal stacks represented by horizontal rectangles, and hypothetical objects within the confocal image stacks represented by colored ovals. Arbitrarily registering the stacks using the top or bottom optical slice of the image stack does not yield an accurate montage. (B) One stack must be shifted along the z axis relative to the other. In practice, confocal image stacks often contain varying numbers of optical slices. Panels (C) and (D) are slice 30 from adjacent confocal stacks of rat brain tissue stained with a fluorescent antibody against the microglial-specific protein Iba-1. However, they are not the matching slices. For correct alignment, the stack in (D) should be shifted about 5 slices in the z-direction. Panel (E) shows slice 30 of the correctly aligned montage produced by our 3-D registration algorithm. Panels (C), (D), and (E) are taken from data set #2.