Achieving uniform optical resolution for a large tissue sample is a major challenge for deep imaging. For conventional tissue clearing methods, loss of resolution and quality in deep regions is inevitable due to limited transparency. Here we describe the Transparent Embedding Solvent System (TESOS) method, which combines tissue clearing, transparent embedding, sectioning and block-face imaging. We used TESOS to acquire volumetric images of uniform resolution for an adult mouse whole-body sample. The TESOS method is highly versatile and can be combined with different microscopy systems to achieve uniformly high resolution. With a light sheet microscope, we imaged the whole body of an adult mouse, including skin, at a uniform 0.8 × 0.8 × 3.5 μm³ voxel resolution within 120 h. With a confocal microscope and a 40×/1.3 numerical aperture objective, we achieved a uniform sub-micron resolution in the whole sample to reveal a complete projection of individual nerve axons within the central or peripheral nervous system. Furthermore, TESOS allowed the first mesoscale connectome mapping of individual sensory neuron axons spanning 5 cm from adult mouse digits to the spinal cord at a uniform sub-micron resolution.

Type II collagen-positive (Col2⁺) cells have been reported as skeletal stem cells (SSCs), but the contribution of Col2⁺ progenitors to skeletal development both prenatally and postnatally during aging remains unclear. To address this question, we generated new mouse models with ablation of Col2⁺ cells at either the embryonic or postnatal stages. The embryonic ablation of Col2⁺ progenitors resulted in the death of newborn mice due to a decrease in skeletal blood vessels, loss of all vertebral bones and absence of most other bones except part of the craniofacial bone, the clavicle bone and a small piece of the long bone and ribs, which suggested that intramembranous ossification is involved in long bone development but does not participate in spine development. The postnatal ablation of Col2⁺ cells resulted in mouse growth retardation and a collagenopathy phenotype. Lineage tracing experiments with embryonic or postnatal mice revealed that Col2⁺ progenitors occurred predominantly in the growth plate (GP) and articular cartilage, but a limited number of Col2⁺ cells were detected in the bone marrow. Moreover, the number and differentiation ability of Col2⁺ progenitors in the long bone and knee joints decreased with increasing age. The fate-mapping study further revealed Col2⁺ lineage cells contributed to, in addition to osteoblasts and chondrocytes, CD31⁺ blood vessels in both the calvarial bone and long bone. Specifically, almost all blood vessels in calvarial bone and 25.4% of blood vessels in long bone were Col2⁺ lineage cells. However, during fracture healing, 95.5% of CD31⁺ blood vessels in long bone were Col2⁺ lineage cells. In vitro studies further confirmed that Col2⁺ progenitors from calvarial bone and GP could form CD31⁺ vascular lumens. Thus, this study provides the first demonstration that intramembranous ossification is involved in long bone and rib development but not spine development. Col2⁺ progenitors contribute to CD31⁺ skeletal blood vessel formation, but the percentage differs between long bone and skull bone. The number and differentiation ability of Col2⁺ progenitors decreases with increasing age.

Axially swept light sheet microscopy (ASLM) is an emerging technique that enables isotropic, subcellular resolution imaging with high optical sectioning capability over a large field-of-view (FOV). Due to its versatility across a broad range of immersion media, it has been utilized to image specimens that may range from live cells to intact chemically cleared organs. However, because of its design, the performance of ASLM-based microscopes is impeded by a low detection signal and the maximum achievable frame-rate for full FOV imaging. Here we present a new optical concept that pushes the limits of ASLM further by scanning two staggered light sheets and simultaneously synchronizing the rolling shutter of a scientific camera. For a particular peak-illumination-intensity, this idea can make ASLMs image twice as fast without compromising the detection signal. Alternately, for a particular frame rate our method doubles the detection signal without requiring to double the peak-illumination-power, thereby offering a gentler illumination scheme compared to tradition single-focus ASLM. We demonstrate the performance of our instrument by imaging fluorescent beads and a PEGASOS cleared-tissue mouse brain.

Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional (3D) architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions. Deciphering the spatial interaction of leptin receptor-expressing (LepR⁺) stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue. In this study, we introduce an optimized protocol for the 3D analysis of skeletal tissues, enabling the visualization of hematopoietic and stromal cells, especially LepR⁺ stromal cells, within optically cleared bone hemisections. Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae. The protocol entails tissue fixation, decalcification, and cryosectioning to reveal the marrow cavity. Completed within approximately 12 days, this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals. The bone hemisections are compatible with diverse antibody labeling strategies. Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software, assessing a spectrum of parameters. With proper storage, the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months. This protocol is robust, straightforward to implement, and highly reproducible, offering a valuable tool for tissue architecture and cellular interaction studies.