Ds qimc camera

For high-throughput cell phenotyping, fluorescence images of immuno-stained cells were collected with a DS-QiMc camera (Nikon) mounted on a Nikon TE300 fluorescence microscope with a 10× Plan Fluor lens (Nikon). Morphometric analysis and fluorescence intensity was assessed with a customized Matlab code[23 ]. More than 800 cells were tested per independent biological repeat (n = 3).

Cross sections (10 μm thick) from the midbelly of the muscles frozen in optimal cutting temperature compound were obtained with a cryostat and fixed in acetone for 10 min at −30°C. The sections were warmed to room temperature (RT) for 5 min and rehydrated with PBS for 15 min. The sections were then incubated in solution A (PBS containing 0.5% BSA and 0.5% Triton X-100) for 20 min and probed with the indicated primary antibodies (dissolved in solution A) for 1 hour at RT. After washing with PBS, the sections were incubated with the appropriate fluorophore-conjugated secondary antibodies (dissolved in solution A) for 1 hour at RT. When nuclei staining was needed, the sections were subsequently incubated with propidium iodine (20 μg/ml) for 5 min. After a final wash with PBS, fluorescent signals from each secondary antibody or propidium iodine were captured with a DS-QiMc camera on an 80i epifluorescence microscope (both from Nikon) at RT, and the resulting monochrome images were merged with NIS-Elements D image analysis software (Nikon). Investigators that were blinded to the sample identification then used NIS-Elements D to measure the total fiber number per muscle cross section, average fiber CSA, myonuclei per fiber, macrophage density, and myonuclear localization.

Certainly when observing a collection of CMs, but even within an individual CM, the deformation taking place is not uniform across the field of view. The details of the full-field behavior are often critical to developing a deeper understanding of the biomechanics at play. To measure full-field displacements, we used digital image correlation (DIC) [77 ]. DIC requires an image having a random high-contrast pattern, which in this case was a phase contrast image of the cells obtained with a Nikon Eclipse Ti microscope with a Plan Fluor 10× NA 0.3 objective and Nikon DS-QiMc camera. In our preliminary analyses, we found the results to be the same with two different free software packages, Ncorr (https://github.com/justinblaber/ncorr_2D_matlab) [78 (link)] and Fast Iterative Digital Image Correlation (FIDIC, https://github.com/FranckLab/FIDIC) [79 (link)]. Results presented here used FIDIC.