Abstract
Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies.
Highlights
Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level
Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, a specialized microenvironment that is required for the steady development of new blood cells and the self-renewal of HSCs3–5
Technical advances in fluorescent microscopy led to the application of confocal microscopy and two-photon microscopy (TPM) in BM imaging
Summary
Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. An ideal method for studying the BM niche is intravital microscopy, which allows in vivo observation of the BM microenvironment at the single-cell level. Technical advances in fluorescent microscopy led to the application of confocal microscopy and two-photon microscopy (TPM) in BM imaging The use of both extrinsic labelling dyes and intrinsic contrasts, such as second-harmonic generation (SHG) from collagen and autofluorescence from cell cytoplasm[19,25], in TPM enables the visualization of cells, vasculature, bone, and other compartments in the BM niche. Have been conducted and HSCs have been tracked successfully the first few days after transplantation using intravital microscopy of mouse calvarial BM18,33. Dye staining of BM stem cells leads to dilution of the dye via cell division, limiting the imaging time period[25]
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