Five-Channel In Vivo Imaging of Mouse Muscle During Regeneration (IMAGE)
Caption
a Schematic diagram illustrating the process of generating a targeting mouse hybrid model and the procedure of introducing mouse muscle injury model. H11-CAG-tdTomato mice were crossed with Pax7CreERT2/+ mice to generate mice where MuSCs were labeled with tdTomato. Subsequently these mice were crossed with CX3CR1-GFP mice to generate double-fluorescence labeled mice, where macrophages expressed GFP and MuSCs were labeled with tdTomato. Details of mouse muscle injury procedure were described in “Methods” part. b Three-channel three-photon fluorescence microscopic images of macrophages, MuSCs and muscle blood vessels before and after denoised by the MSAD-Net. The left panels displayed 3D structure images (volume size: 190 μm × 190 μm × 400 μm), while the right panels showed Y-Z plane images. Green represents macrophages, yellow represents MuSCs, red represents muscular blood vessels. Scale bar: 50 μm. c Intensity profiles along the dotted lines in the three-channel three-photon fluorescence microscopic images in (b). The identification of macrophages, MuSCs, and muscle blood vessels were significantly enhanced in the images denoised by the MSAD-Net. d Extracted volume of macrophages and MuSCs based on noisy images and denoised images (by the MSAD-Net). The box center indicates the median, the box edges represent the standard deviation, and the whiskers indicate the 5th and 95th percentiles. e Five-channel microscopic muscle images, including three-photon fluorescence microscopic images of macrophages, MuSCs and muscular blood vessels, SHG images of fibers, and THG images of fiber membranes, in uninjured mice and mice at 1-4 day(s) post-injury (1dpi-4dpi). Blue represents fiber membranes, green represents macrophages, yellow represents MuSCs, red represents muscular blood vessels, and gray represents fibers (details described in “Methods” part). The lower panels displayed enlarged five-channel images. MuSCs and muscular blood vessels were the morphological extraction results using “Surface” functionalities in the Imaris. Macrophages were the location results using “Spots” functionalities in the Imaris. Volume size: 340 μm × 340 μm × 400 μm. Scale bar: 100 μm.
Credit
Yifei Li et al., Zhejiang University, PhotoniX 2025.
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CC BY