Estimation of Myonuclear Domains from the Assisted Segmentation of Muscle Fiber Nuclei
Collaboration
C. Høgsbjerg and A. Mackey, Bispebjerg Hospital, University of Copenhagen
Research Background
The tips of human muscle fibres were probed with RNAscope and imaged with 3D microscopy, to characterise the arrangement of cells and myonuclei at the myotendinous junction. In this collaboration, we developed a way to estimate the myonucleus density along human muscle fibers and to characterize the myonuclei domains (MND).
Method
Segmentation (Semi-automatic)
For analysis of the nuclei 3D spatial organisation, the spinning disc confocal images of single myofibres were imported into Imaris. The myofibre volumes were generated from the phalloidin staining channel using the Imaris surface segmentation function, and manually evaluated to ensure close overlap with the phalloidin and dystrophin staining. The 3D spatial coordinates of the nuclei were detected using the Imaris spot segmentation function. Excess spots were generated to ensure capture of all nuclei and were removed manually, preferentially leaving the centremost spot. Nuclei spots were then classified as either inside (myonuclei) or outside (nuclei of mononuclear cells) the myofibres, with a spot being classified as inside if located within the dystrophin staining. Furthermore, we used vimentin as an additional marker to distinguish between inside and outside nuclei, if determination by dystrophin was ambiguous. If the Hoechst signal showed a major decrease in quality through the Z-stack, deconvolution of the Hoechst channel was performed in Imaris to improve nuclei segmentation.
Automatic measurements and results visualization
The resutling 3D nuclei and surface objects were analysed using our Imaris XT MATLAB module. In brief, the software imports the Imaris objects to MATLAB and calculates the distance of nuclei spots to the myofibre tip, the distance of outside nuclei spots to the myofibre surface, the NN of inside nuclei spots, the MND of inside nuclei spots, and the outward-facing surface of MNDs (MNDS). All data are exported in an Excel sheet together with accompanying MATLAB files.
For more details, see the full publication and code linked below .
Publication
Christian Hoegsbjerg, Ask Møbjerg, Ching-Yan Chloé Yeung, Peter Schjerling, Michael R. Krogsgaard, Manuel Koch, Michael Kjaer, Arvind G. von Keudell, Abigail L. Mackey; Fibre type differences in the organisation of mononuclear cells and myonuclei at the tips of human myofibres. J Cell Sci 15 January 2025; 138 (2): JCS263660. doi: https://doi.org/10.1242/jcs.263660
Code Repository
https://github.com/DBI-INFRA/Automated-muscle-fiber-analysis