Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type - International Review of Cell and Molecular Biology - page 287

fluorescent staining for ConA lectin in the green fluorescent channel, with
imaging of MF-20, a monoclonal antibody which specifically binds myosin,
in the red channel; the multichannel images that were produced featured
muscle fibers with a green outline (from ConA lectin), outlining fibers with
a red interior (MF-20 antibody-labeled myosin). Thus, the study by
Mozdziak et al. foreshadowed the use of multichannel fluorescence micros-
copy to calculate CSAs and to quantify biomarkers, visualized in a separate
optical channel, on a fiber-by-fiber basis.
5.2. Digital camera revolution and image-analysis programs
Starting in the mid 1990s, the digital cameras interfaced to microscopes
allowed digital images to be directly captured to a computer's hard drive,
eliminating the need for an initial film-based image capture. In two studies
illustrating this approach, CSAs were measured from ATPase-stained rat
muscle using an Olympus microscope with attached camera and personal
computer ( Delp and Duan, 1996 ), whereas Mannion et al. (1997) evaluated
fibers in human erector spinae muscle utilizing an Olympus microscope/
camera/Apple Macintosh Quadra 950 workstation. These authors also uti-
lized NIH Image, a public domain, general purpose, image-analysis program
for outlining the fibers and calculating CSAs (NIH Image was the forerunner
of Image J, which is currently one of the most widely used general image-
analysis program ( Schneider et al., 2012 ), available for free download from
the NIH at http://imagej.nih.gov/ij/index.html ) . Several other imaging
programs for CSA measurements were used at that time as well. Examples
include Abrams et al. (2000) who used the Image 1 analysis program
(Universal Imaging Corp.) for CSA analysis in rabbit EDL muscle after
tenotomy, Miller et al. (2001) who used the Bioquant image-processing sys-
tem for analysis of rat soleus muscle after hind-limb unloading, Smith et al.
(2002) who used the Scion Image Program (derived from NIH Image) for
analysis of overload-induced hypertrophy of rat skeletal muscle, Linderman
and Blough (2002) who used Java Jandel Video Analysis Software for mea-
surements of the CSAs in ATPase-stained rat muscles, and Gilson et al.
(2007) who used a Kontron Image-analysis system to evaluate muscle fibers
in myostatin knockout mice. Unfortunately, the programs mentioned above
may not be currently supported, as Google searches did not identify Web
sites for these programs.
Examples of studies published from this time period, utilizing software
programs that are still supported, include the analysis of denervated rat