MuscleJ2: a rebuilding of MuscleJ with new features for high-content analysis of skeletal muscle immunofluorescence slides

Abstract

Histological analysis of skeletal muscle is of major interest for understanding its behavior in different pathophysiological conditions, such as the response to different environments or myopathies. In this context, many software programs have been developed to perform automated high-content analysis. We created MuscleJ, a macro that runs in ImageJ/Fiji on batches of images. MuscleJ is a multianalysis tool that initially allows the analysis of muscle fibers, capillaries, and satellite cells. Since its creation, it has been used in many studies, and we have further developed the software and added new features, which are presented in this article. We converted the macro into a Java-language plugin with an improved user interface. MuscleJ2 provides quantitative analysis of fibrosis, vascularization, and cell phenotype in whole muscle sections. It also performs analysis of the peri-myonuclei, the individual capillaries, and any staining in the muscle fibers, providing accurate quantification within regional sublocalizations of the fiber. A multicartography option allows users to visualize multiple results simultaneously. The plugin is freely available to the muscle science community.

Keywords: Centro- and perinuclei; Extracellular matrix; Fiber typing; Histology; Interstitial cells; Muscle fiber morphology; Phenotype cartography; Sarcolemmal staining; Vascularization.

Fig. 1

Interface of the MuscleJ2 plugin. Screenshots of the plugin dialog boxes. A The main dialog box MuscleJ2 is divided into five sections where the user must select from a drop-down menu or check boxes. The lowercase letters in red refer to dialog box 2, in which the channels and staining information must be indicated. B The Channel information dialog box is used to indicate the channel number for each requested analysis. Depending on the analyses selected in the MuscleJ2 dialog box, the design of this dialog box changes. In the upper panel, the lowercase letters in red refer to the section Data Analysis by Section (a, b, c); in the lower panel, they refer to the section Data Analysis by Fiber (d, e, f) and Data Cartographies (g)

Fig. 2

New functionalities of the plugin. A Immunostaining of skeletal muscle with WGA showing the extracellular matrix (ECM) in green (SB = 600 µm) and respective quantification with MuscleJ2 in the "GlobalResults" file. B Immunostaining of skeletal muscle with laminin (gray) and CD31 showing the endothelial cells in red (SB = 600 µm) and quantification of vessels and capillaries with MuscleJ2. Tables present the results obtained after selecting the option Vascularization (section Data Analysis by Section) and Capillaries (section Data Analysis by Fibers). The gray table presents the results obtained in the "GlobalResults" file, and the green table presents some of the results obtained in the "CapillaryDetails" file (SB = 600 µm). C Immunostaining of skeletal muscle with laminin (gray), DAPI (blue), and F4/80 showing the macrophages in red and quantification of specific cells with MuscleJ2 (SB = 600 µm). The gray table presents the results obtained in the "GlobalResults" file, and the green table presents some of the results obtained in the "SpecificCells" file. Nucleus GC X and Y correspond to the coordinates of identified specific cells colabeled with DAPI. All areas are indicated in µm²

Fig. 3

Measurement of Fiber Intensity by ROI. A Representation of the different ROIs in MuscleJ2. ROI^F, ROI Fiber; ROI^CNF, ROI Centronucleated Fiber; ROI^SC, ROI Satellite Cell; ROI^Cap, ROI Capillary; ROI^MB, ROI Membrane. B Original image of skeletal muscle stained with dystrophin and corresponding cartographies representing the different ROIs obtained after MuscleJ2 analysis with the Fiber Intensity option. C For each fiber, the intensity of the staining and the percentage of positive pixels in each ROI are given. D Quantification of dystrophin staining in the different ROIs. The gray table presents the results obtained in the "GlobalResults" file, and the green table presents some of the results obtained in the "FiberDetails" file

Fig. 4

Novelty of the cartography section. A Representative images of the cartographies obtained for specific cells, ECM detection, and capillaries (SB = 600 µm). B Representation of the image obtained after selection of the multicartography option, in which different cartographies are assembled on the same image. In this example, the image was stained with dystrophin, and the results are represented in the different cartographies (SB = 300 µm)