Lee and colleagues investigated the role of the intestinal microbiota in steady-state hematopoieisis, demonstrating that microbiota-derived DNA circulates to the bone marrow, where uptake by mononuclear cells leads to inflammatory cytokine production favoring myeloid-cell maturation of hematopoietic progenitors.

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

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.

Delineating brain tumor margin is critical for maximizing tumor removal while sparing adjacent normal tissue for better clinical outcome. We describe the use of moxifloxacin-based two-photon (TP)/coherent anti-Stokes Raman scattering (CARS) combined microscopy for differentiating normal mouse brain tissue from metastatic brain tumor tissue based on histoarchitectural and biochemical differences. Moxifloxacin, an FDA-approved compound, was used to label cells in the brain, and moxifloxacin-based two-photon microscopy (TPM) revealed tumor lesions with significantly high cellular density and invading edges in a metastatic brain tumor model. Besides, label-free CARS microscopy showed diminishing of lipid signal due to the destruction of myelin at the tumor site compared to a normal brain tissue site resulting in a complementary contrast for tumor detection. This study demonstrates that moxifloxacin-based TP/CARS combined microscopy might be advantageous for tumor margin identification in the brain that has been a long-standing challenge in the operating room.

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