Lau et al.

Skeletal Muscle (2018) 8:32

https://doi.org/10.1186/s13395-018-0178-6

SOFTWARE Open Access

Automated muscle histopathology analysis

using CellProfiler
Yeh Siang Lau, Li Xu, Yandi Gao and Renzhi Han*

Abstract
Background: Histological assessment of skeletal muscle sections is important for the research of muscle physiology
and diseases. Quantifiable measures of skeletal muscle often include mean fiber diameter, fiber size distribution, and
centrally nucleated muscle fibers. These parameters offer insights into the dynamic adaptation of skeletal muscle
cells during repeated cycles of degeneration and regeneration associated with many muscle diseases and injuries.
Computational programs designed to obtain these parameters would greatly facilitate such efforts and offer significant
advantage over manual image analysis, which is very labor-intensive and often subjective. Here, we describe a customized
pipeline termed MuscleAnalyzer for muscle histology analysis based upon CellProfiler, a free, open-source software for
measuring and analyzing cell images.
Results: The MuscleAnalyzer pipeline consists of loading, adjusting, and running a series of image-processing modules
provided by CellProfiler. This pipeline was evaluated using wild-type and mdx muscle sections co-stained with laminin
(to demarcate the muscle fiber boundaries) and 4′,6-diamidino-2-phenylindole (DAPI, to label the nuclei). The
immunofluorescence images analyzed using the MuscleAnalyzer pipeline or manually yielded similar results in
the number of muscle fibers per image (p = 0.42) and central nucleated fiber (CNF) percentage (p = 0.29) in
mdx mice. However, for a total of 67 images, CellProfiler completed the analysis in ~ 10 min on a regular PC
while it took an investigator ~ 3 h using the manual approach in order to quantify the number of muscle
fibers and CNF. Moreover, the MuscleAnalyzer pipeline also provided the measurement of the cross-sectional
area (CSA) and minimal Feret’s diameter (MFD) of muscle fibers, and thus fiber size distribution can be plotted.
Conclusions: Our data indicate that the MuscleAnalyzer pipeline can efficiently and accurately analyze laminin and
DAPI co-stained muscle images in a batch format and provide quantitative measurements for muscle histological
properties such as muscle fiber diameters, fiber size distribution, and CNF percentage.
Keywords: Histology, Muscle, Image segmentation, Quantitative analysis, mdx mouse

Background result in increased variation of fiber size and muscle fibers

Skeletal muscle is an exceptionally adaptive tissue. During with central nuclei [9, 10]. Examination of muscle
endurance exercise, skeletal muscle undergoes extensive cross-sections is therefore often carried out to assess such
adaptation by changing their fiber type composition and changes in the fields of myopathy and rehabilitation sci-
fiber size [1–3]. Upon injuries, satellite cells associated ence. However, the methods to quantify these changes
with skeletal muscle are activated to proliferate, fuse to remain challenging among investigators and often require
form myotubes, and eventually regenerate new muscle fi- painstaking manual procedures [11, 12]. Traditionally,
bers [4–7]. In genetic myopathies such as Duchenne mus- visually identifying muscle nuclei and manually measuring
cular dystrophy (DMD), a fatal X-linked recessive muscle the muscle fiber size manual tracing of individual fibers
disease caused by genetic mutations leading to the loss of are relatively subjective and time consuming. These tasks
dystrophin [8], repeated cycles of muscle injury, and repair are highly susceptible to both inter-individual and
inter-laboratory variability, often resulting in discrep-
* Correspondence: [email protected] ancies within the literature, despite the use of similar
Department of Surgery, Davis Heart and Lung Research Institute, Biomedical animal models under similar experimental settings.
Sciences Graduate Program, Biophysics Graduate Program, The Ohio State
University Wexner Medical Center, Columbus, OH 43210, USA Several semi-automatic approaches to analyze muscle

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Lau et al. Skeletal Muscle (2018) 8:32 Page 2 of 9

histopathology currently exist [12–15]. However, their Both ND2 and TIFF formats can be used as input im-
usage has not been widely adopted, likely due to the ages for CellProfiler.
cost or the difficulty to implement them with some
requiring basic programming skills. CellProfiler-facilitated automation of image processing
Recently, a free, open-source software called CellProfiler The stable version (2.2.0) of CellProfiler downloaded from
has increasingly gained popularity and visibility in the field the CellProfiler website (www.cellprofiler.org) and installed
of automated image analysis, which provides a platform on a PC (Intel Xeon CPU E5–1620 v2 @3.70 GHz, 32.0 GB
for the user to create customized pipelines for image ana- RAM, and 64-bit Windows 7 operating system) was used
lysis. CellProfiler is developed by the Carpenter Labora- for the data processing in this manuscript. The current
tory at the Broad Institute of Harvard and MIT that stable version is 3.0.0 at the time of this manuscript submis-
allows investigators with little prior bioinformatics know- sion. CellProfiler is available for Windows, Mac and Linux.
ledge to automate image analysis and collect large Java installation is required prior to installing CellProfiler.
amounts of phenotypic data relatively easily [16, 17]. The Users are encouraged to read the CellProfiler manuals
goal of this work is to provide a free, easy-use, fast, and re- (http://cellprofiler.org/manuals/) before testing the pipeline.
liable pipeline for CellProfiler to analyze and quantify The MuscleAnalyzer pipeline (ND2) for CellProfiler version
muscle histological properties using immunofluorescence 2.2.0 (Additional file 1) and 3.0.0 (Additional file 2), as well
images of muscle cross-sections. as the MuscleAnalyzer pipeline (TIFF) for CellProfiler ver-
sion 3.0.0 (Additional file 3) are available online.
Implementation
Manual muscle fiber counting and CNF determination
Mice
To further validate the data generated by CellProfiler,
Mice (C57BL/10ScSn and C57BL/10ScSn-Dmdmdx/J) were
muscle fiber counting, CNF percentage, CSA, and MFD
maintained at The Ohio State University Laboratory Ani-
were determined manually using Nikon NIS Elements
mal Resources in accordance with animal use guidelines.
software (version 4.3, Nikon). For CNF counting, two
All animal studies were authorized by the Animal Care,
different classes were assigned for CNF and total fibers
Use, and Review Committee of the Ohio State University.
under count and taxonomy from manual measurement
control window. To determine CSA and MFD, each in-
Immunofluorescence staining of muscle cross-sections dividual muscle fibers were detected as object manually.
and imaging The results were exported to Excel files. Total of seven
Quadriceps muscles were collected from five male non-overlapping images per section of each mouse were
wild-type (WT) and five male mdx mice at the 8 weeks captured, and the percentage of CNF was determined.
of age. Skeletal muscle tissues were mounted in Optimal
Cutting Temperature (OCT) and frozen in liquid nitro- Results
gen cooled isopentane. Muscle cryosections were pre- Muscle fiber and nuclei identification
pared using Leica CM3050S cryostat (Leica Biosystems, The laminin α2 (green) and DAPI (blue) co-stained muscle
Buffalo Grove, IL, USA) at a thickness of 7 μm. The sec- sections of WT and mdx mice were imaged and saved to
tions were fixed with 4% paraformaldehyde for 15 min ND2 files using the NIS-Elements Advanced Research soft-
at room temperature followed by two washes with PBS ware provided by Nikon (Fig. 1). CellProfiler supports a
and 1 h incubation with blocking solution (5% bovine wide variety of image formats, including most of those
serum albumin) prior to overnight incubation at 4 °C used in imaging, by using a library called Bio-Formats
with primary antibody against laminin α2 (ALX-804-190, (http://docs.openmicroscopy.org/bio-formats/5.7.0/suppor-
1:100, Alexis). The slides were then extensively washed ted-formats.html). In our initial test, we found that Cell-
with PBS and incubated with secondary antibodies Profiler can directly analyze both ND2 files and TIFF files,
(Alexa Fluor 488 goat anti-rat IgG, 1:500, Invitrogen) for and thus for all our following studies, we used ND2 files
1 h at room temperature. Finally, the slides were without prior conversion into TIFF files. Upon startup, the
mounted using VECTASHIELD® Mounting Medium user is provided with an empty pipeline, which consists of
with DAPI (Vector Laboratories, Inc.) and imaged with Input modules, analysis modules, and output settings
a × 20 lens in an inverted Nikon microscope (Nikon). A (Fig. 2). A typical CellProfiler workflow is summarized in
total of 70 non-overlapping images (approximately 250 the flowchart (Additional file 4). Basically, the images are
muscle fibers per image for WT and 200 for mdx) were loaded and then processed (e.g., cropping, illumination cor-
captured from 5 WT and 5 mdx mice (7 images per rection, object identification, object classification, measure-
mouse) and saved in the ND2 file format with green ments, and data output). A step-by-step video tutorial is
channel for laminin and blue channel for DAPI. These provided to guide the implementation of MuscleAnalyzer
images were also exported into the TIFF file format. pipeline (Additional file 5).
Lau et al. Skeletal Muscle (2018) 8:32 Page 3 of 9

Fig. 1 Representative immunofluorescence images of WT and mdx skeletal muscle sections stained with laminin α2 (green) and DAPI (blue)

To demonstrate the process of automated imaging image. Under the “Analysis modules,” individual ana-
analysis using CellProfiler, we first loaded an mdx lysis modules can be added.
image in ND2 file format by dragging the file into the For analyzing ND2 color images (Fig. 3a), the first step
“File list” of the “Images modules” under “Input mod- is to split the color images into grayscale images for each
ules” (Fig. 2). The metadata of the image can be ex- channel using the “ColorToGray” module (Fig. 1). The
tracted using the “Metadata module.” You may also green channel of laminin staining (Fig. 3b) will be used
need to set up the correct image type using the to identify the muscle fiber and blue channel of DAPI
“NamesAndTypes module” and assign a name to the staining (Fig. 3c) will be used to identify the nuclei. The

Fig. 2 CellProfiler (version 2.2.0) interface. a The pipeline panel consists of input modules for data entry (image file or folder name, image type,
and grouping of images). The analysis modules is the platform to build up the analysis pipeline from the module ‘ColorToGray’ to ExportToSpreadsheet,’
which can be inserted by clicking the ‘+’ sign below the pipeline panel
Lau et al. Skeletal Muscle (2018) 8:32 Page 4 of 9

Fig. 3 Sample image processing by CellProfiler. a Original RBG image of mdx muscle section stained with laminin α2 (green) and DAPI
(blue). b, c Converted grayscale images of each channel after running the “ColorToGray” module. d Inverted green channel image. e Pseudo-colored
image to show individual muscle fibers identified by using ‘IdentifyPrimaryObjects’ module. f The identified muscle objects were shrunk by seven
pixels in order to determine if they contain central nuclei. g Outlines of identified muscle fibers were overlaid with original green channel image to
illustrate the accuracy of muscle fiber identification. h Pseudo-colored image to show individual nuclei identified by using ‘IdentifyPrimaryObjects’
module. i The outlines of identified muscle fibers and nuclei were overlaid with original green channel image to illustrate the accuracy of classification
with either normal (blue) or central nucleated muscles (red)

grayscale image of the green channel was then inverted are located on the muscle edge or in the center. An over-
using the “ImageMath” module (Fig. 3d). Next, the lay of the identified muscle fibers (red) with the original
“IdentifyPrimaryObjects” module was used on the green channel image (gray) in Fig. 3g showed that the ma-
inverted image of the green channel to identify the muscle jority of the muscle fibers were correctly identified.
fiber. By setting the minimal and maximal diameter of To identify the nuclei on the DAPI-stained nuclei
muscle fibers, we can filter out any objects outside the image in the blue channel, we applied the “IdentifyPri-
diameter range. We used the “RobustBackground” maryObjects” module again by using the “Automatic”
method to threshold the image, and the key parameters threshold strategy. The smoothing filter size and maxima
such as threshold correction factor and size of smoothing suppression distance were again important for faithfully
filter for declumping are important to determine the reli- detecting the nuclei. As shown in Fig. 3h, we were able
ability of muscle fiber identification. By adjusting these pa- to detect the nuclei on the sample image. For central nu-
rameters, we were able to achieve a satisfactory result in clei classification, we again used the “ExpandOrShrin-
identifying muscle fibers on our sample image (Fig. 3e). By kObjects” module to shrink the nuclei object to a point
applying the “MeasureObjectSizeShape” module here, we to minimize the chance whereas the nuclei objects
can obtain total muscle fiber numbers and the muscle touching the border of muscle objects were incorrectly
fiber area, which can be exported into an Excel file later classified as central nuclei.
using the “ExportToSpreadsheet” module. The muscle
fiber objects were shrunk by several pixels (in our sample Count CNF and normal muscle fibers
image, we set the number of pixels to be 7) using the After having successfully identified the muscle fibers and
“ExpandOrShrinkObjects” module (Fig. 3f) in order to the nuclei, our next step is to relate these two objects so
classify them as CNF or normal later. Using shrunk that we can count the muscle fibers with or without cen-
muscle fibers would ensure that these associated nuclei tral nuclei. For this purpose, we used the “RelateObjects”
Lau et al. Skeletal Muscle (2018) 8:32 Page 5 of 9

module to relate these two objects. The shrunk nuclei It took about 11 min for CellProfiler on a PC (Intel
were used as the “child objects” while the shrunk muscle Xeon CPU E5-1620 v2 @3.70 GHz, 32.0 GB RAM,
fibers as the “parent objects.” This allows us to count and 64-bit Windows 7 operating system) to complete
the number of child objects (shrunk nuclei) associated all these 67 images. One image from each of the 5
with each parent object (shrunk muscle fibers). After WT and 5 mdx muscles were shown in Fig. 4a.
this step, the “ClassifyObjects” module can be applied to Clearly, the majority of the WT muscle fibers were
classify the shrunk muscle fibers with 0 (normal) or at identified as normal (blue) while the majority of the
least 1 nuclei (CNF). As shown in Fig. 3i, the CNF (red) mdx muscle fibers were identified as CNF (red). Care-
and normal muscle fibers (blue) were correctly differen- ful examination of the analyzed images found three were
tiated. At the end of the pipeline, “SaveImages” and not correctly processed with a large black area (Fig. 4b).
“ExportToSpreadsheet” modules can be used to save This was due to the “threshold correction factor” being set
images and export measurements to Excel files, at too high. By lowering down this value from 0.985 to
respectively. 0.975, we can recover the missing muscle fibers on this
particular image (Fig. 4b). Careful comparison of the
Comparison between manual and semi-automated pseudo-colored muscle fiber image (the right image) with
approaches the original laminin-stained image shown on the left
To compare the performance of the MuscleAnalyzer (Fig. 4b) showed that several mistakes were made by Cell-
pipeline versus manual fiber counting, we captured 7 Profiler. Some inter-muscle fiber regions were identified
images of randomly chosen non-overlapping regions as muscle fibers (Fig. 4b, blue star). In addition, some
per muscle section from WT and mdx mice (5 each), muscle fibers were split into two (Fig. 4b, blue boxes).
resulting in a total of 70 images to be analyzed. A However, most muscle fibers were correctly identified.
quick examination of these 70 images found that We also analyzed these 67 images by the manual ap-
three images had large area of artifact staining in blue proach. It took an experienced investigator roughly 3 h
channel and were excluded from the analysis. We to complete the task. The average number of muscle fi-
thus analyzed the 67 images (Additional file 6) by ei- bers identified per image with CellProfiler was similar to
ther CellProfiler or the traditional manual approach. that counted by the manual approach in mdx samples;

Fig. 4 Examples of correctly and incorrectly processed muscle images. a Representative pseudo-colored images from all five WT and mdx mice
after processed by CellProfiler. Red, centrally nucleated fibers; blue, normal fiber. b An incorrectly processed image with a large black area, which
was due to a high threshold setting and can be corrected by lowering the threshold from 0.985 to 0.955. The blue stars showing the inter-fiber
spaces that were mistakenly identified as muscle fibers; the blue boxes indicating individual muscle fibers that were mistakenly split into two
Lau et al. Skeletal Muscle (2018) 8:32 Page 6 of 9

however, there was about 6% less of muscle fibers identi- for the measurement of CSA and MFD using three
fied by CellProfiler than the manual approach for the independent images per genotype. As shown in
WT samples (Fig. 5a). The calculated CNF percentage Fig. 5c, d, CellProfiler obtained very similar measure-
was again fairly comparable between the two approaches ments as the manual approach; however, the time
for mdx samples (56.7 by CellProfiler vs 61.8 manually, used by CellProfiler to complete the same task was
p = 0.29) (Fig. 5b), indicating that the CellProfiler can be only a small portion of that used by the latter.
used to automate the process for muscle immunofluor- Finally, we used CellProfiler to automate the meas-
escence image analysis. It is of note that CellProfiler ob- urement of CSA for all 67 images to derive the fiber
tained significantly more CNFs than the manual size distribution. Consistent with the muscular dys-
approach did in WT samples (1.9 by CellProfiler vs 0.1 trophy phenotype, mdx muscle showed an increase
manually, p = 0.001) (Fig. 5b), however, these numbers in both very small (regenerative) and very large
are still within the normal range of healthy sample varia- (hyper-contracted) muscle fibers, while WT muscles
tions. Moreover, we also compared these two approaches showed a more even distribution (Fig. 5e).

Fig. 5 Quantitative measurements of the 67 images processed manually or by CellProfiler. a The average number of muscle fibers per
image. b The percentage of CNF. c CSA measurements. d MFD measurements determined by CellProfiler or the manual approach. e Size
distribution of WT and mdx muscle fibers generated from the CSA data produced by CellProfiler
Lau et al. Skeletal Muscle (2018) 8:32 Page 7 of 9

Discussion the remaining pixels, and the threshold as the mean

In this study, we compiled a pipeline termed MuscleA- + 2 times the standard deviation [16, 19]. The thresh-
nalyzer for CellProfiler to automatically process im- old can be further adjusted either upwards or down-
munofluorescence images of muscle cross-sections wards through multiplying it by the “threshold
stained with laminin α2 and DAPI. Laminin α2 staining correction factor.” The strategy that we used to clas-
was used to facilitate the determination of individual sify the CNF is to relate the nuclei and muscle fibers
muscle fibers while DAPI staining was used to label cell using the “RelateObjects” module after shrinking
nuclei. The parallel comparison with MuscleAnalyzer muscle fibers by several pixels and nuclei to a point.
and manual approach showed that the MuscleAnalyzer This strategy appears to be robust; however, the num-
pipeline can provide relatively accurate measurements of ber of pixels to be shrunk for muscle fibers need to
muscle features such as CNF percentage, fiber diame- be empirically determined.
ters, and fiber size distribution with minimal efforts and Three common errors associated with identification of
time. This should aid the pathophysiological studies of muscle fibers include (1) some inter-muscle fiber spaces
muscle diseases and evaluation of therapeutic impact. and blood vessels were mistakenly counted as muscle
The most critical steps involve the identification of fibers due to the fact that the laminin staining on the
muscle fibers and nuclei, and then classification of surrounding muscle fibers formed closed compartments;
muscle fibers into CNF or normal through correlation of (2) some muscle fibers were merged due to the difficulty
nuclei with muscle fibers. CellProfiler uses the term “ob- in correctly finding the borders of the touching muscle
ject” as a generic term to refer to an identified feature fibers; and (3) some muscle fibers were split into smaller
(for example, nuclei and muscle fibers) in an image. ones due to high background intracellular staining.
Nuclei are more easily identifiable due to their more Carefully adjusting the parameters for thresholding can
uniform morphology, high contrast relative to the back- greatly minimize the rate of these errors but does not
ground with DAPI staining, and good separation be- seem to completely get rid of them. It is worthy to test if
tween adjacent nuclei. However, muscle cells often have adding a third staining of muscle fibers such as collagen
irregular morphology, varying sizes, uneven and more (to label inter-muscle fiber regions) and muscle-specific
diffused staining patterns, making them much more cytoplasmic proteins (i.e., desmin and α-actinin, to label
challenging to identify than nuclei [12, 18]. Moreover, muscle fibers) could help to further reduce the errors in
muscle cells often touch their neighbors making it the future.
harder to delineate the cell borders. To use the “Identify- Several other semi-automatic analysis tools have
PrimaryObjects” module to identify either nuclei or been reported [12–15]. We have attempted to test
muscle fibers, the ND2 images should be converted to and compare these tools with CellProfiler and found
grayscale images for the corresponding channels. The CellProfiler is relatively easy to implement. We did
laminin-stained channel needs to be inverted using the not test all these tools because they are either not
“ImageMath” module. Object identification (segmenta- available on internet or purchase is required. From
tion) is performed through image thresholding, recogni- the original reference, it appears that MuscleQNT can
tion and division of clumped objects, and removal or quantify only CSA and fiber distribution, while the
merging of objects on the basis of size or shape [16]. MuscleAnalyzer pipeline can obtain CNF, MFD, CSA,
Therefore, it is important to test the thresholding pa- and potentially other more parameters with some
rameters for correct segmentation. The “Test Mode” modifications. We were able to download, install, and
provided by CellProfiler makes it convenient to test indi- test the standalone SMASH program, unfortunately
vidual modules of the pipeline before final batch analysis we were unable to export the data to Excel files at
of a large set of images. the end. Moreover, SMASH can only analyze the im-
For laminin-stained muscle sections, we found that ages one-by-one, while CellProfiler can analyze the
the “Global” strategy of thresholding, which calculates data in a batch format, enabling full automation. Fi-
a single threshold value based on the unmasked pixels nally, CellProfiler provides a free and flexible platform
of the input image and use that value to classify to a wide range of users in performing image analysis,
pixels above the threshold as foreground and below which has been cited more than 6000 times. The
as background, and the “RobustBackground” method established pipelines are easy to share among the re-
for finding thresholds automatically, provided the best search community allowing fast improvement and in-
results in muscle fiber identification. The “Robust- creased scientific reproducibility.
Background” method assumes that the background There are several limitations for the current version of
distribution approximates a Gaussian by trimming the our MuscleAnalyzer pipeline. First, it does not incorpor-
brightest and dimmest 5% of pixel intensities, and ate the function to manually correct the wrongly identi-
then calculates the mean and standard deviation of fied muscle fibers. However, our initial study with the 67
Lau et al. Skeletal Muscle (2018) 8:32 Page 8 of 9

images showed that the error for muscle fiber identifica- Acknowledgements
tion was less than 10%. In a fully automation setting to We would like to acknowledge members of the Han Lab for beta testing the
MuscleAnalyzer.
analyze a large number of images, such an error rate
does not appear to affect the conclusion of CNF, CSA, Funding
and MFD, particularly in diseased muscles. Second, we R.H. is supported by US National Institutes of Health grants (R01HL116546
and R01 AR064241).
have not incorporated the function to analyze other use-
ful parameters for muscle biology, such as satellite cells, Availability of data and materials
muscle fiber types, and muscle fibrosis. Future improve- The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
ments can be made to incorporate these functions. Last
but not least, it is important to acquire high quality im- Authors’ contributions
ages in order for the MuscleAnalyzer pipeline to accur- RH conceived the study, compiled the MuscleAnalyzer pipeline, and wrote
ately identify muscle fibers and nuclei. Tissue tearing/ up the manuscript. YSL carried the experimental procedures and helped in
data analysis. LX and YG contributed to mouse maintenance and experimental
folding and freezing artifacts during tissue section prep- analysis. All authors have read and approved the manuscript.
aration should be minimized.
Ethics approval and consent to participate
Animal care and experimental procedures were approved by the Animal
Conclusions Care, Use, and Review Committee of the Ohio State University and complied
Taken together, the MuscleAnalyzer pipeline for CellPro- with the Guide for the Care and Use of Laboratory Animals, Institute of
filer allows rapid and accurate batch analysis of skeletal Laboratory Animal Resources, Commission on Life Sciences, National Research
Council (Washington: National Academy Press, 1996).
muscle cross-sectional immunofluorescence images. Al-
though we only test it for CNF, CSA, and MFD, one can Consent for publication
envision it would allow for quantification of other traits Not applicable.
of skeletal muscle such as characterization of muscle sat-
Competing interests
ellite cell, muscle fiber type, necrosis and fibrosis with The authors declare that they have no competing interests.
minor modifications of the pipeline. This should aid the
unbiased pathophysiological studies of muscle diseases Publisher’s Note
and evaluation of therapeutic impact. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Availability and requirements Received: 20 April 2018 Accepted: 4 October 2018
Project name: MuscleAnalyzer pipeline for CellProfiler
version 2.2.0 and 3.0.0.
Project homepage: N/A. References
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