FIGURE 4.6. The space balls method for estimating the total length of a linear biological structure within a given tissue.


The space balls method for estimating the total length of a linear biological structure within a given tissue. The procedure is shown for collagen IV-immunoreactive capillaries in the mediodorsal nucleus of the thalamus from a human brain. The postmortem brain was fixed by immersion in 10% formalin prior to histologic processing (for details, see [132]). The right hemisphere was embedded in gelatin, deeply frozen at −60° C, and cut into serial 600–700-μm-thick coronal sections. Approximately 4 cm × 4 cm small pieces containing the thalamus were cut out from these thick sections, frozen, and cut into 50-μm-thick coronal sections. To prevent shrinkage in section thickness, the sections were not dehydrated but mounted on gelatin-coated slides and coverslipped with 80% glycerol in TRIS-buffered saline. Panel (a) provides a low-power overview of capillaries in the mediodorsal nucleus. The square indicates the region at which the high-power photomicrographs (b–l) were taken. These show the upper surface of the section as well as the same microscopic field at 10 consecutive focal planes below the upper surface, with a distance of 4 μm between the focal planes. Between −4 μm and −32 μm, the intersections of a hemisphere (a [semi-]“space ball”) and the focal plane at the investigated focal depth are shown as circles. This (semi-)space ball was centered at a depth of −4 μm and had a radius of 30 μm. At −8 μm, −12 μm, −16 μm, and −24 μm, intersections between the hemisphere and the capillaries in focus at the point of intersection were found (arrows). From the total number of intersections and the size of the hemisphere, the capillary length density of the investigated microscopic field can be calculated [135]. By placing (semi-)space balls in a systematic-random manner throughout the entire mediodorsal nucleus, an estimate of the average capillary length density within this region is obtained. Finally, by combining length density with an estimate of the volume of the region (as shown in Figure 4.2), one can obtain an unbiased estimate of the total capillary length in the structure. [Scale bar = 150 μm (a); 30 μm (high-power photomicrographs).]

From: Chapter 4, Design-Based Stereology in Brain Aging Research

Cover of Brain Aging
Brain Aging: Models, Methods, and Mechanisms.
Riddle DR, editor.
Boca Raton (FL): CRC Press/Taylor & Francis; 2007.
Copyright © 2007, Taylor & Francis Group, LLC.

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