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biases, which will be discussed further. This chapter elaborates
on key stereology methods in analyses of OI in neuroscience. The
discussion and protocol of stereology herein focus on the optical
fractionator stereological technique using Stereo Investigator soft-
ware, the predominant method used in determining signals or OI.
The Protocol, (Sects. 3-5), provides a practical guide for experimental
design and execution, with caveats and discussions. This chapter
applies established stereology protocols for quantitative analysis of
OI, which are useful for fi rst-time as well as novice stereology users.
Due to the numerous stereological software packages available,
some information and characteristics about each are provided.
The last section (Sect. 7) focuses on 3D volume analysis. Recent
technological advances in biomedical imaging, such as non-invasive
scanning techniques, have resulted in an explosive growth of research
and scientifi c activities into the analysis of high-quality 3D volumet-
ric images. This Section focuses on 3D analysis of neuroanatomical
images and will describe a set of typical protocols used to (1) quantify
morphological characteristics of the anatomy and (2) identify struc-
tural abnormalities that are associated with a particular condition to
aid diagnosis, treatment, and/or scientifi c investigation.
2. Stereology
2.1. Practical
Applications
for Stereology
One of the most basic applications of stereology is to count the
number of cells in a given region of interest (ROI) while identify-
ing cells by immunostaining or Nissl staining. Immunostaining
for nuclear antigens can provide an easily distinguished label for
counting, compared to background staining or unstained cells.
Establishing clear criteria for inclusion and exclusion is important
in order to avoid estimation errors.
There are a number of stereological methods. Each is slightly
different and more or less appropriate, based on the OI, the nature
of the experiment, and the parameter desired to quantify as detailed
in Table 1 . Many stereological protocols make use of the disector,
a 3D stereological probe that samples OI in histological sections as
they come into the focal plane. The most common of these stereo-
logical techniques is known as the optical disector, which is distinctly
different from the physical dissector from the respect that the user
does not need to worry about assessing whether two pieces of an
OI appear in two adjacent sections ( 1 ). The optical fractionator
combines the optical disector and the fractionator sampling (i.e.,
using systematic random sampling to select which sections to sam-
ple and where to sample on them) ( 1 ); how to set the sampling
fraction will be discussed in subsequent sections.
Counting neurons is one of the most common uses for stereology.
For example, this study used stereology to assess the difference in
2.1.1. Counting Neurons
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