The ever-increasing speed and resolution of modern microscopes make the storage and post-processing of images challenging and prevent thorough statistical analyses in developmental biology. Here, instead of deploying massive storage and computing power, we exploit the spherical geometry of zebrafish embryos by computing a radial maximum intensity projection in real time with a 240-fold reduction in data rate. In our four-lens selective plane illumination microscope (SPIM) setup the development of multiple embryos is recorded in parallel and a map of all labelled cells is obtained for each embryo in <10 s. In these panoramic projections, cell segmentation and flow analysis reveal characteristic migration patterns and global tissue remodelling in the early endoderm. Merging data from many samples uncover stereotypic patterns that are fundamental to endoderm development in every embryo. We demonstrate that processing and compressing raw image data in real time is not only efficient but indispensable for image-based systems biology.

Key Points• Normally, engrafting HSCs reside and oscillate within confined bone marrow niches.• HSCs harvested from mice bearing acute infection are migratory and interact with larger niches.Hematopoietic stem cells (HSCs) maintain the turnover of mature blood cells during steady state and in response to systemic perturbations such as infections. Their function critically depends on complex signal exchanges with the bone marrow (BM) microenvironment in which they reside, but the cellular mechanisms involved in HSC-niche interactions and regulating HSC function in vivo remain elusive. We used a natural mouse parasite, Trichinella spiralis, and multipoint intravital time-lapse confocal microscopy of mouse calvarium BM to test whether HSC-niche interactions may change when hematopoiesis is perturbed. We find that steady-state HSCs stably engage confined niches in the BM whereas HSCs harvested during acute infection are motile and therefore interact with larger niches. These changes are accompanied by increased long-term repopulation ability and expression of CD44 and CXCR4. Administration of a CXCR4 antagonist affects the duration of HSC-niche interactions. These findings suggest that HSC-niche interactions may be modulated during infection. IntroductionThe function of hematopoietic stem cells (HSCs) depends on their interaction with complex niches within the bone marrow (BM) 1,2 and must readily adjust to meet the changing needs for differentiated cells during situations of stress, such as the immune responses generated by infectious agents.3-5 The current working hypothesis is that changes in HSC-niche interactions may cause changes in HSC function and therefore a major challenge in the field is to understand the specific cellular behaviors (eg, duration of cell-cell/cell-matrix interactions) resulting in different HSC fates (quiescence vs proliferation, self-renewal vs differentiation). Direct visualization of HSCs in vivo and over time is the ideal approach to monitor the nature of the cellular interactions between individual stem cells and their niches and can be achieved by intravital microscopy of mouse calvarium BM. 6,7 We studied a naturally occurring, nonlethal mouse model of infection initiated by ingestion of the parasitic nematode Trichinella spiralis, the infectious agent causing trichinosis. Ingested T spiralis reside for several days in the intestinal epithelium, where they mature, proliferate, and release larvae, which migrate into the circulatory system and invade the skeletal muscle, where they form cysts and survive for the life of the host.8 Migration of parasites causes extensive tissue damage and intense inflammation over the early phase of infection (weeks 1-3), which is ameliorated by the induction of regulatory cytokines during the chronic phase of the disease. 9,10Here we show that acute T spiralis infection affects hematopoietic dynamics and boosts HSC function. Using time-lapse intravital microscopy of transplanted HSCs, we show that acute infection is accompanied by changes in HSC-nic...

Recent breakthroughs in magnetic resonance imaging (MRI) enabled quantitative relaxometry and diffusion-weighted imaging with sub-millimeter resolution. Combined with biophysical models of MR contrast the emerging methods promise in vivo mapping of cyto- and myelo-architectonics, i.e., in vivo histology using MRI (hMRI) in humans. The hMRI methods require histological reference data for model building and validation. This is currently provided by MRI on post mortem human brain tissue in combination with classical histology on sections. However, this well established approach is limited to qualitative 2D information, while a systematic validation of hMRI requires quantitative 3D information on macroscopic voxels.We present a promising histological method based on optical 3D imaging combined with a tissue clearing method, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel (CLARITY), adapted for hMRI validation. Adapting CLARITY to the needs of hMRI is challenging due to poor antibody penetration into large sample volumes and high opacity of aged post mortem human brain tissue. In a pilot experiment we achieved transparency of up to 8 mm-thick and immunohistochemical staining of up to 5 mm-thick post mortem brain tissue by a combination of active and passive clearing, prolonged clearing and staining times. We combined 3D optical imaging of the cleared samples with tailored image processing methods. We demonstrated the feasibility for quantification of neuron density, fiber orientation distribution and cell type classification within a volume with size similar to a typical MRI voxel. The presented combination of MRI, 3D optical microscopy and image processing is a promising tool for validation of MRI-based microstructure estimates.

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