Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type - International Review of Cell and Molecular Biology - page 289

(see below) could be explained by several factors: (1) Image J and Image-Pro
Plus are well known and are widely accepted methods for accurate measure-
ments of CSAs; (2) knowledge about availability of different software for
automated CSA measurements is limited, and most of these software are
not validated/compared to manual measurements with Image J or Image-
Pro Plus; (3) software for automated CSA measurements is more expensive
and may not be accessible to small laboratories with limited resources, while
Image J is a free software.
5.3. Automated analysis of skeletal muscle structure
With recent advances in computers and software, new approaches to CSA
measurements were introduced to reduce time needed for the analysis. For
example, Klemencic et al. (1998) developed a segmentation method for
CSAmeasurements; the method required selection of the point in the center
of each muscle fiber after which the program would automatically perform
outlining and CSAmeasurements with fast and accurate outcome. Kim et al.
(2007) further developed this method by describing fully automated seg-
mentation of the H&E-stained images of muscle fibers.
For fully automated analyses of skeletal muscle fibers, images are loaded
into the appropriate software and specially developed algorithms develop a
binary “mask” that correspond to the sarcolemmal/basal lamina staining
used for visualization of the fiber outlines; in cases where the fiber outline
is incomplete, the algorithms “fill in the gaps” of the outline to estimate the
fiber boundaries. The mask is then used to uniquely identify all fibers that
are present within the image, and CSA data, as well as other parameters such
as characterizations of fiber shape (e.g., the degree of circularity) are output
as a data table, on a fiber-by-fiber basis. The data table can then be accessed
for graphing or statistical analyses, either by the software package, itself, or
the data table exported to spreadsheet programs, such as Microsoft Excel or
GraphPad. This approach significantly shortens the time involved in CSA
measurements. Instead of minutes/hours required for semiautomated
CSA measurements, the automated image-analysis algorithms require only
a few seconds to accurately analyze the same image. The important require-
ments for increased use of software for fully automated quantification of
CSA in skeletal muscle research are accessibility (price, availability for com-
puter download) and verification of the accuracy of measurements.
Software programs that are widely being used for automated measure-
ments of CSA include MetaMorph (Molecular Devices, Sunnyvale, CA),