MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool

Background Skeletal muscle has the capacity to adapt to environmental changes and regenerate upon injury. To study these processes, most experimental methods use quantification of parameters obtained from images of immunostained skeletal muscle. Muscle cross-sectional area, fiber typing, localization of nuclei within the muscle fiber, the number of vessels, and fiber-associated stem cells are used to assess muscle physiology. Manual quantification of these parameters is time consuming and only poorly reproducible. While current state-of-the-art software tools are unable to analyze all these parameters simultaneously, we have developed MuscleJ, a new bioinformatics tool to do so. Methods Running on the popular open source Fiji software platform, MuscleJ simultaneously analyzes parameters from immunofluorescent staining, imaged by different acquisition systems in a completely automated manner. Results After segmentation of muscle fibers, up to three other channels can be analyzed simultaneously. Dialog boxes make MuscleJ easy-to-use for biologists. In addition, we have implemented color in situ cartographies of results, allowing the user to directly visualize results on reconstituted muscle sections. Conclusion We report here that MuscleJ results were comparable to manual observations made by five experts. MuscleJ markedly enhances statistical analysis by allowing reliable comparison of skeletal muscle physiology-pathology results obtained from different laboratories using different acquisition systems. Providing fast robust multi-parameter analyses of skeletal muscle physiology-pathology, MuscleJ is available as a free tool for the skeletal muscle community. Electronic supplementary material The online version of this article (10.1186/s13395-018-0171-0) contains supplementary material, which is available to authorized users.


3/ Tutorial
The tutorial will be posted on the website where MuscleJ will be deposited. Both will be updated regularly depending on new features or major bug corrections.

MuscleJ Tutorial
MuscleJ: A high content analysis method to study skeletal muscles.
The MuscleJ macro is a compilation of tools allowing for the analysis of fiber phenotypes.

Installation of MuscleJ in the FiJi environment
The automated image analysis workflow was implemented in the FiJi (NIH, Bethesda, MD, USA) environment as a macro and will be upgrade to a plugin with further functionalities.

Description: MUSCLEJ Fiber Phenotype Dialog Boxes
The first step : The user provides the requested information

Data Acquisition
Microscopy selection Select the type of microscopy used when generating the data. Based on the selection a specific pretreatment will be performed.

Volume choice
The Z stack option will apply a maximum projection along z axis. Scanned muscle area choice Entire: the whole section or a large part of the section (min. 50%) will be been scanned.
Crop: if only a small area is to be scanned the analysis will be less efficient despite a series of additional filters that track the maximum intensity of the fiber contours in Laminin channel.

Data Format
The Original File Format is for files readable by the Bioformat Importer Plugin such as czi, lsm, lif, etc. formats. Additionally, tiff files containing all channels (a stack file from FiJi or ImageJ) are readable.
The TIFF (16bits) by channel format is for cases when your images have been exported from your acquisition system by channel.

Data Analysis
Analysis check boxes: multiple optionsyou can select the series of analysis to be performed. However, the Fiber Morphology analysis is mandatory whenever you start a new batch run. Artefact Detection option: sets the minimum threshold for the fiber area detection. This is used for the initial quality check of the fiber shape signal (Laminin). Example: %area min=xx, if less than xx% of the total area of the section does not contain segmentable fibers, the section will be automatically placed into the "Artefact" directory and the analysis will not be performed for this section.

Data Cartography
Cartography Automatically backs up the in situ cartography according to the analysis performed (single choice): "Fiber Area Classes", "Centro Nuclei Classes", "Sat Cell", "Fiber Types" or "Vessels". Note: If "Fiber Morphology" analysis has been performed during a previous run, the corresponding cartography ("Fiber Area Classes") can be chosen independently from the current analysis. Legend If chosen, for the Centro Nuclei Fibers, Sat Cells, Types, and Vessels a legend will be automatically drawn onto the cartography in situ on the bottom left. For the distribution by fiber surface the legend is put in a column at the top left of the cartography with the range of surface distribution displayed in μm 2 .

Channel Information
To identify the channel order, please open the first image from the input folder with the Bioformat Importer.
Depending on the analysis requested by the user, the order of channels will be need to be set.
In the right dialog box, the analysis of fiber typing has been demanded. You must enter the corresponding fiber channel number to the track of the fiber shape, before the analysis of intensity by fiber typing channel can be performed.
For the same analysis request, if the tiff format by channel option has been checked the information about the number of channels, the x pixel size in microns and the y pixel size in microns must be entered before the analysis can be performed.

Note: all fields in the two previous dialog boxes are mandatory
The second step: Setting the directory path for the Input and the Output data • Select the Image File Folder by Batch run • Select an empty folder to save the ROIs, the Results by file and the Cartographies During the first run, a series of directories will be automatically created in the root of the selected folder:

III. Recommendations / Limitations
Images obtained from any microscope magnification (10x, 20x, 25x, 40x, 63x) can be used. The limitations are relative to the quality of image staining and a minimum of 1.5x1.5mm scanned surface area.
For an optimal utilization of MUSCLEJ we recommend using the tool under the following conditions: When gather the data use an Apotome / Widefield microscope when imagining a single Z slice or use a confocal/Spinning Disk microscope when imaging a Z-stack. When analyzing a whole section (set the minimum detection threshold of 40 % to reject section artefacts).
Use the Original File Format to keep the metadata associated.
Images folder: all images of the batch folder should have the same parameters (number of channels, channel orders, stainings and formats). The input and output folders have to be named without using spaces and symbols.
File format that are not supported are: Jpeg, Png, Tiff-8bits, a time series.
For the option « Tiff 16 bits by channel»: Each file must contain one channel and have the following nomenclature FileName1_C# where # is the number of channel (1,2,3 or 4). Do not use spaces and symbols when naming the files.
Note: All the files have to be gather into the image folder