Intravital imaging of neovascularization by two-photon laser scanning microscopy in tibial bone defects

Intravital imaging of neovascularization by two-photon laser scanning microscopy in tibial bone defects | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Intravital imaging of neovascularization by two-photon laser scanning microscopy in tibial bone defects Jiongnan Xu, Liang Zhu, Tingxiao Zhao, Weiyi Wu, Keyi Chen, Wangjie Fu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6196928/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neovascularization plays a critical role in bone regeneration and skeletal development. Our understanding of weight-bearing bone healing has been hindered by the lack of a reliable method that allows tracking neovascularization at a high spatiotemporal resolution in living model. Thus, we employed two-photon laser scanning microscopy (TPLSM) for longitudinal analysis of angiogenesis of tibial bone defects in mice. In this study, we established an effective model for long-term visualization and longitudinal analyses of angiogenesis in tibial bone defect healing. The vessel structural can be imaged and analyzed in healthy and tibial bone defects mice for over 3 weeks. Blood flow could be tracked for 21 days post-surgery. During this tibial bone healing process imaging, we found the blood flow start at 12–14 days after surgery and the velocity reach 0.6205 mm/sed and 0.9784 mm/sed. After 21 days recovery, the vessel structural and functional recovered to normal with velocity of 3.7644 mm/s which corresponding to baseline. The establishment of a in vivo imaging platform provides a unique tool to better understand angiogenesis in tibial bone defects repair, enabling further investigation of structure and function of vascularization during weight-bearing bone healing. Biological sciences/Biological techniques/Microscopy/Multiphoton microscopy Biological sciences/Physiology/Bone Two-photon laser scanning microscopy in vivo Weight-bearing bone Neovascularization Blood flow velocity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The skeleton is one of the most commonly injured tissues, with more than 16 million fractures of long bones occurs in the United States annually. [ 1 ] Although bones have excellent regenerative potential, the rate of delayed or non-union healing of fractures ranges is up to 15%. [ 2 ] The healing of bone injury includes a dynamic process of tissue regeneration driven by progenitor cells, necessitating the coordinated processes of osteogenesis and angiogenesis at the site of injury. [3.4] During the healing of bone injury, the vasculature plays a crucial role by recruiting mesenchymal stem cells (MSCs) and regulating the differentiation of perivascular MSCs into bone-forming cells. [ 5 – 7 ] While the structure and function of blood vessels within bones has been somewhat mysterious until recently, largely due to the technical challenges associated with imaging on calcified tissues. [ 8 ] The technical breakthrough in imaging of murine bones now has provided insights into the heterogeneity of blood vessels in bone. [ 9 – 11 ] Two-photon laser scanning microscopy (TPLSM) has emerged as an invaluable and invasive imaging for in vivo surveillance of cell or organ function at a cellular or subcellular level temporally and on live-animals. [ 12 , 13 ] TPLSM enables the sophisticated morphological and functional investigation of new blood vessels. [ 14 ] It offers the exclusive advantages comparing with conventional imaging methods, such as the high spatiotemporal resolution, minimal invasiveness, and the ability to capture 3D images. [ 15 – 18 ] In addition to capturing nonlinear fluorescence by using transgenic mice or labeling cells/structures with fluorescent dyes, TPLSM is also capable of visualizing the bone matrix through detecting the autofluorescence of collagen in bone known as second harmonic generation (SHG). [ 19 , 20 ] Owing to its substantial penetrating capability and high microscopic resolution, TPLSM is capable of measuring hemodynamic parameters, such as the flow rate within blood vessels. [ 21 ] With those unique advantages, the intravital TPLSM allows us to simultaneously visualize cells, the extracellular matrix (ECM), and the surrounding vascular networks and dynamically analyze the osteogenesis and angiogenesis spatiotemporal on live-animals during the whole repair and regenerative process of bone injury. [ 12 ] TPLSM has been initially used in studies of osteoclasts and hematopoietic stem cells images. [ 22 – 24 ] Later, TPLSM was used to image the interaction of angiogenesis with osteoblasts and bone regeneration during the bone repair in cranial bone defect. [ 25 ] However, the non-weight-bearing bone differs from the weight-bearing bone, including the bone cell functions, blood vessel regeneration and bone mineral composition. [ 26 – 28 ] To overcome the disadvantages, investigators implanted a gradient refractive index (GRIN) endoscopic lenses and visualize the blood vessels and bone cells in bone marrow of long bones. [ 29 ] Recently, Dr. Phan’s group developed a minimally invasive SOP for longitudinal imaging of endosteal bone cells and hematopoiesis in the place of intact tibiae with acceptable bone thickness. [ 30 ] However, the longitudinal intravital imaging method for visualizing the angiogenesis and bone regeneration long bone during bone injury repair is still lacking. The present study aims are to develop a TPLSM-based longitudinal imaging platform that enables the non-invasive and high-resolution analysis of osteogenesis and angiogenesis, particularly the function of newly formed vessels throughout the repair and regeneration process of tibial bone defects in mice. By employing Tek-Cre;Ai-14 mice, we are presenting for the first time, to visualize and functionally analyze the angiogenesis and its interaction with bone regeneration at the site of tibial defect repair in vivo longitudinally and spatiotemporally. Our research represents a unique and innovative tool, ideally suited for examining the temporal and spatial dynamics and functional aspects of angiogenesis throughout the process of bone defect repair. Result Surgical procedure and TPLSM imaging protocol for tibial bone defects in mice Angiogenesis is an important integral part of the fracture healing process. Due to technical limitations, traditional research study the physiologic function of blood vessels by examining their morphology, number, or composition of different subtypes of endothelial cells in vitro. However, there is a significant gap in testing blood vessels' function to transport blood in vivo. Therefore, there is urgently needed to conduct both qualitative and quantitative research on vessel function during the fracture healing process in live animals. To achieve repeated long-term observations of neovascularization in healing bone defects in live mice, we devised a detailed surgical protocol for creating tibial bone defects, shown in Figure 1a. Initially, consistent with standard protocols for murine surgical interventions, the mice were anesthetized via intraperitoneal injection. Subsequently, hair was removed from the right lower limb, and the area was sterilized to ensure a clean and intact surgical field. A scalpel blade was then employed to create a 1-cm vertical incision along the anterior border of the tibia. Ophthalmic forceps were utilized to carefully detach the surface-attached muscles on the medial aspect of the tibia, thereby providing clear exposure of the tibial tuberosity. A high-speed cranial drill was employed to create a bone defect with a diameter ranging from 0.5 to 0.6 mm through a single layer of the bone cortex, targeting the medullary cavity in the plane of the tibial tuberosity, situated above the medullary cavity (Figure 1b). Following the immobilization of the right lower limb with a custom-designed instrument (spinal cord adapter, RWD, China), we utilized a commercial Bruker 2P laser scanning microscope to imaging the bone defects areas illustrated in Figure 1c. Figure 1d provides a detailed schematic of the system and the optical path of the 2P platform, emphasizing the two-color channel that support our imaging methodology. Tibial Structure and Vascular Imaging by TPLSM For visualization fluoresce signal of blood vessels in the tibia, we employed a 920 nm wavelength laser to conduct in vivo imaging of the vascular system in the tibia (Figure 2a) of wild-type mice. The fluorescein isothiocyanate (FITC) were injected into the retro-orbital vein of mouse. The tibia has preserving second harmonic generation (SHG) signals. [19,20] To effectively differentiate between the vascular fluorescence (FITC) signal and SHG, we subsequently imaged the same region with a reduced laser wavelength (800 nm). This adjustment resulted in a significant attenuation of the previously dominant FITC signal, while the SHG signal was markedly enhanced. The vascular structures located in the cortical region of the bone (depicted by the blue dotted line) and the vascular structures within the medullary cavity (indicated by the green dotted line) in Figure 2a are depicted in Figures 2b and 2e, respectively. The cortical vascular structures in Figure 2b exhibit higher density and finer diameters ranging from 5 to 8 μm compared to the bone marrow cavity region, with blood vessels intersecting the bone cortex in a non-specific direction. Magnified imaging clarifies the intricacies of vascular architecture and associated blood flow, as shown in Figure 2c. To visualize the three-dimensional structure of the vessels, we used a Z-stack scan imaging of the fluoresce signals in vessels with 1um step size and reconstructed the three-dimensional vessel structures (Figure 2d). Compared to the vascular areas of the bone cortex (Figure 2b), wide-field, low-magnification imaging reveals the morphology and structure of the vasculature within the bone marrow cavity, which is more regularly distributed primarily along the longitudinal axis of the medullary cavity (Figure 2e). We conducted detailed imaging of the intramedullary vasculature in the green area shown in Figure 2a. The high-resolution imaging of Figure 2f further elucidates the complex structure of the blood vessels depicted in the red box in Figure 2e. Similarly, the vascular structures in the green box area in Figure 2e are also represented in Figure 2g by means of 3D reconstruction. Furthermore, to accurately quantify the velocity of blood flow within an individual vessel, line scan measurements were conducted on the medullary cavity vessels depicted in Figure 2i, as illustrated in Figure 2j, yielding an average velocity of 3.1344 mm/sec (Figure 2k). Macroscopically, natural bone consists of compact cortical and trabecular cancellous layers, each characterized by unique collagen fiber morphologies. Hydroxyapatite nanocrystals are deposited within the interstitial spaces between collagen molecules, resulting in bundles of mineralized collagen fibers approximately 100 nm in diameter. Under enlarged field view, we examined the superficial structure of the bone cortex (indicated by the black area in Figure 2a) and were able to distinctly visualize the bone architecture in vivo. This included the trabecular cavity (denoted by the red arrow) and the trabecular pathway (denoted by the blue arrow) as illustrated in Figure 2h. This detailed visualization is instrumental for subsequent in-depth analyses of osteocyte and blood vessel interactions. Validation the vascular structure with FITC tracer and Tek-Cre;Ai-14 transgenic mice To verify the vascular structure in bone tissue，we imaging the tibia in Tek-Cre;Ai-14 transgenic mice, which overexpress the angiopoietin receptor TEK specific expression in vascular endothelial cell. [31] By injection with the FITC tracer，dual-color imaging were performed to acquire the tracer fluoresce in blood and the endothelial cell fluoresce. Low magnification field of view showed angiographic morphology of the medullary cavity (Figure 3a) and bone cortex (Figure 3d) regions, respectively. The data indicated that the expression patterns and spatial distribution of vascular endothelial signaling (TEK) were largely consistent with those of FITC signaling. At enlarged field view with high magnification objective, Figures 3b and 3e more distinctly reveal the intricate morphological structures and blood flow within the vasculature of the two regions. Consistent with the description of wild-type mice above, the vascular structure in the bone marrow cavity of Tek-Cre; Ai-14 transgenic mice is similarly more defined compared to the cortical region. These findings robustly affirm the efficacy of our two-photon in vivo imaging technique for vascular visualization in the mouse tibia. To further elucidate the functional characteristics of these vessels, we conducted an examination of the region of interest (ROI) demarcated by the yellow dashed line (Figure 3b and e). Our analysis determined that the average flow velocity was 2.6459 mm/s for medullary vessels and 2.4473 mm/s for cortical vessels (Figure 3c and f). TPLSM imaging tibial bone injury recovery for 21 days After successful imaging the bone vascular system in vivo in healthy mouse. Next, we monitor bone defects in the tibiae of mice over a three-week period by two-photon imaging. The objective of this study was to meticulously document the healing process of the bone defect region and the concomitant angiogenesis on a daily basis following surgical intervention. The imaging data obtained at critical time points: 3, 7-, 10-, 14-, and 21-days post-surgery. The bone callus area was visualized at both low and high flied view (Figure 4a and b). During the initial phase of bone defect healing, spanning from days 3 to 14, a significant proliferation of micro-vessels measuring 5-15 μm in diameter is observed. However, as the healing process progresses beyond 14 days, the majority of these micro-vessels regress, resulting in the predominance of larger diameter vessels, approximately 20 μm. The defect site was covered by a hematoma on postoperative day 3. By postoperative day 7, the formation of bone scabs was evident, indicating the progression of the bone healing process (Figure 4c). Following the identification of vascular, the blood vessels morphology within the defect area were quantified by ImageJ software, a progressive increase in vascular density during the initial stages of bone healing were found. This growth reached its zenith at 14 days postoperatively (Figure 4d), underscoring the critical phase of the vascularization process. To further characterize the vascular architecture of the region, we performed the imaging of the defect site at 14 days postoperatively. Using 4X objective, it allows for the simultaneous acquisition of a larger and volume imaging, thereby facilitating direct observation of the vascular architecture at the defect site (Figure 4e). The three-dimensional reconstruction of the vascular images 21 days post-surgery were constructed showed the vessel network were interconnected and recovery. (Figure 4e). Our findings indicate that, by this time, the neovascularization at the defect site had largely interconnected with the surrounding vasculature, exhibiting morphology and structure that were essentially consistent. Functional recovery of vascular flow To further confirm the vessel functional recovery during the functional status of neovascularization in the context of bone defect healing, we conducted a comprehensive analysis of blood flow in quantitatively resolved vessels at various postoperative time points (Figure 5). This evaluation is crucial for understanding the establishment the functional role of neovascularization in bone defect repair. During the process of bone healing, we observed that not all neovascularization formation period had blood cell flow, particularly in the initial stages following surgical intervention (Figure 5a-c). This phenomenon may be attributed to the transient absence of blood flow within the neovessel, which precluded the measurement of blood flow velocity in these nonfunctional neovascularized structures. The recorded blood flow velocities at 12- and 14-days post-surgery were 0.6205 mm/s and 0.9784 mm/s, respectively (Figure 5d-e). Our data indicate that the period of 12 to 14 days post-surgery represents a pivotal transition phase. After 12-14 days, the blood flow velocities can be measured as a slow speed on the neovascularization. This blood flow represented the neovascularization at the site appears to form a connected circuit. This critical observation implies that the vessels have attained a functional state characterized by substantial blood cell flow. As illustrated in Figure 5f, a blood flow velocity of 3.7644 mm/s was recorded at 21 days post-bone defect surgery, which corresponds to the baseline blood flow velocity observed in wild-type mice, as shown in Figure 2k. Which indicated the completed recovery of the vessel structural and functional connection. Within the TPSLM imaging method, we can image and analysis the blood vessel structure and functional information during the bone healing process, which will expand the use of TPSLM in the basic and preclinical research. Conclusion Vascularization and bone formation are closely associated processes, with both endochondral and intramembranous osteogenesis intricately linked to blood vessels development. [ 32 ] While multiphoton laser scanning microscopy (MPLSM) has successfully been used to image calvarial bone, visualizing blood vessels and cells in bone marrow or endosteum of long bones remains a formidable challenge due to technical and anatomical hurdles. [ 12 , 33 ] In our current study, we developed a new methods for visualize and functionally analyze the angiogenesis and its interaction with bone regeneration at the site of tibial defect repair longitudinally and spatiotemporally in vivo. We applied this technology and demonstrated the functional vascular network begins to form at 14 days post-surgery (dps) and the completed recovery at 21 days. Notably, the number of blood vessels peaked at 14 dps, and the blood flow starts after this stage, suggesting that the pre-14 days might be critical stages for recovery. Based on the results of this study, it is suggested that the experimental protocol of this study is suitable for generalization across related experiments. A mature and functional vascular network is critical for bone injury repair. Angiogenesis precedes osteogenesis, [ 34 ] therefore neovascularization ensures the recruitment of osteoclast and osteoblast precursors. [ 35 ] Many scientists have tried to establish the imaging methods in other bone system. Dr. Masaru Ishii’s group pioneered the use of MPLSM to image the endosteal bone cells, including the osteoclast, osteoblasts and their interactions in calvarial bones. [ 32 , 36 ] This technology was later expanded to track the longitudinal changes of angiogenesis and osteogenesis, and their interaction, in calvarial bone defects, including the critical defect. [ 37 ] These studies demonstrated that MPLSM is a valuable intravital imaging platform offering high resolution, deeper tissue penetration, and reduced photobleaching. [ 38 ] However, the heavy mineralization of long bones hinders the application of TPLSM in both physiological and pathological conditions. To address this, Dr. Niesner’s group developed implanted gradient refractive index (GRIN) endoscopic lenses for long bones, enabling the examination of the bone, hematopoitic or tumor cells in bone marrow and on endosteal surface of long bones. [ 29 ] However, this method was limited by the implanted object's interference with local bone regeneration and the associated high cost. Dr. Phan’s group later optimized the TPLSM imaging procedure for tibiae due to their relatively thin front surface. [ 30 ] Dr.Xi, developed a method that combines of near-infrared (NIR)-II fluorescence labeled red blood cells (RBC) with a fast NIR camera and TPLSM, successfully imaging the functional vascular network in deep spinal cord tissues. [ 39 ] But for long bones, we are the first established methods for longitudinal repeated imaging. Prior methods had successful application of transparency reagents to cranial bones anticipates future enhancements in the imaging quality of blood vessels in bone tissue. [ 40 ] The advent of three-photon imaging technology, which utilizes higher-order nonlinear effects to reduce surface tissue background and employs longer wavelength photons for excitation, weakens the scattering of excitation light by biological tissues, allowing for greater penetration depth. [ 41 ] Therefore, the exploration of strategies to enhance multiphoton microscopy for in vivo imaging of blood vessels in bone tissue is not only feasible but also holds immense potential. By refining the capabilities of multiphoton microscopy, we can expect significant advancements in the field of orthopedic research and tissue engineering. Such strategies may include the development of novel vascular dyes with improved depth penetration, the engineering of transgenic animal models with enhanced fluorescent markers, and the refinement of optical techniques to boost imaging clarity. The pursuit of these strategies will undoubtedly provide deeper insights into the complex interplay between vascularization and bone regeneration, ultimately contributing to more effective treatments for bone injuries and diseases. Experimental Section/Methods For animal research, wild-type mice and Tek-Cre;Ai-14 transgenic mice were randomized into groups using a table of random numbers: the Sham group, and the bone-defect group. Animals and Materials: Male C57BL/6 mice aged 11–12 weeks and weighing 25-30 g, were obtained from purchased from the Hangzhou Paisiao Biotechnology Co., Ltd. (Zhejiang, China) and Tek-Cre;Ai-14 transgenic mice were provided by Xi Wang's group at Zhejiang University. The mice were placed in controlled environments (12-h light/dark cycle; 20-26°C; 40-60% humidity) and had free access to bacteria-free water and food. All animal housing and experiments were conducted in accordance with the ethical guidelines formulated by the Animal Ethics Committee of Zhejiang Provincial People's Hospital (20241202554152). Male wt mice and Tek-Cre;Ai-14 transgenic mice were divided into 2 groups of eight each by randomization method: sham-operated group, and bone defect group. bone defect was established according to the experimental protocol described in this paper. All the chemicals and reagents were purchased from chemical sources, and the solvents for chemical reactions were distilled before use. Tetrahydrofuran (THF) was acquired from Sinopharm Chemical Reagent Co., Ltd. Pluronic F-127 (F127) was bought from Sigma-Aldrich. Heavy water (D2O) was used in all of the imaging experiments as the immersion liquid for objectives. Surgical procedure and TPLSM for tibial bone defects in mice C576BL/6J mice and Tek-Cre;Ai-14 transgenic mice were randomized into a bone defect surgery group and a sham surgery control group. Bone defect success criteria could be fully determined during surgery. Control mice were also anesthetized. Mice are anesthetized through an intraperitoneal injection of 2,2,2-tribromoethanol (Nanjing Abbey Biotechnology Co, China) at a dosage of 400 mg/kg. A heating pad is used to maintain the mouse's body temperature at approximately 37°C to prevent hypothermia during the surgery. The surgical area, centered around the knee joint, is shaved to a width of about 1 cm and a length of about 3 cm. The shaved area is then cleaned with an iodine solution to reduce the risk of infection. A longitudinal incision of about 1 cm is made on the anterior medial side of the tibia, extending from the ankle to the knee, using tissue scissors. Fine surgical scissors are used to make a longitudinal incision between the tibia and the triceps muscle of the lower leg. Muscles and fascia attached to the tibia are carefully removed in a circular area centered on the tibial tuberosity with a diameter of 1 cm to expose the tibia's inner surface. Saline mixed with 0.2% cephalosporin is used to rinse the exposed tibial surface to remove any debris and to reduce the risk of infection. Excess muscle and tissue are removed from the area. The tibia is held steady with forceps. A high-speed drill is used to create a bone defect approximately 0.5-0.6 mm in diameter on the posterior side of the tibial tuberosity on the medial side of the tibia (above the bone marrow cavity), taking care not to penetrate the entire tibia. Saline water is used to wash away bone powder from the surface. The wound is inspected to ensure there is no significant bleeding, aside from the bone defect area. The wound is rinsed again with saline and then sutured. Before imaging, mice are re-anesthetized using the same method. The previous incision is reopened to expose the tibial bone defect. Any remaining soft tissue is carefully removed using forceps and fine surgical scissors. The area is rinsed with saline and any fine bleeding is controlled. Dental cement is used to fill in around the bone defect to create a stable imaging chamber; adhesive may be used if necessary to ensure a secure bond with the underlying soft tissue. Special clips are used to stabilize the mice, providing the necessary stability for the two-photon imaging process. Treatment of mice and sample collection were not blinded as they were performed by one single investigator. Mouse samples were coded and experimental measurements and analysis blinded during the assessment and data analysis process. Which was performed by two other investigators. The bone defect surgeries in mice were all performed by the same person to ensure homogeneity of the procedure. Blinding was not feasible during the procedure; however, when possible, we analyzed the results blindly. 2P Microscopy Imaging Mice with tibial imaging windows were secured on a specialized fixation frame to ensure stability during imaging. Mice were anesthetized to ensure they remained still throughout the procedure. Just before imaging, mice were injected retro-orbitally with FITC (fluorescein isothiocyanate) dextran conjugate (2000 kDa, Invitrogen), a fluorescent marker that binds to plasma proteins and outlines the vasculature. A customized two-photon microscope (Ultima 2P，Bruker) was coupled with a femtosecond laser (ChameleonUltra II, Coherent Inc. model-locked Ti: Sapphire laser) for high-resolution imaging. A 920 nm laser pulse with a pulse width of less than 200 femtoseconds and a repetition rate of 80 MHz was used for excitation. FITC served as the angiography agent, enabling the visualization of blood vessels. A Pockels Cell (EO-PC, Thorlabs Corporation) was utilized to modulate the laser power, ensuring that it was optimized for imaging without causing damage to the sample. Emission light from the fluorescent signal was filtered using a bandpass filter of 525/50 nm to match the emission spectrum of FITC. The filtered light was then detected using GaAsP photomultiplier tubes (PMTs, Hamamatsu, model H10770), which are sensitive and fast detectors suitable for capturing the weak signals from two-photon excited fluorescence. High-quality images were captured using a high-numerical aperture objective lens (Olympus XLUMPLFLN20XW, 16×, N.A. = 1.0), which provided the necessary resolution for detailed imaging of the tibial vasculature. The average laser power was adjusted according to the imaging depth, with the maximum power kept below 50 mW to prevent phototoxicity or thermal damage to the tissue. Images were acquired at varying depths, with adjustments made to the laser power to accommodate the increased scattering and absorption as the imaging depth increased. Imaging Analysis: MATLAB (R2020b; MathWorks) was the primary software used for data analysis, benefiting from its robust suite of built-in toolboxes for image processing and computational biology. The Simple Non-Local Means (NLM) Filter was applied to reduce noise in the images. This filter is designed to preserve edges while removing noise, which is crucial for clear vascular imaging. The Jerman Enhancement Filter, which utilizes hessian eigenvalues to enhance image structures, was used to improve the visibility of vascular structures in 2D images. ImageJ (NIH, USA) was employed for preliminary image processing tasks, including the application of a one-pixel-radius median filter and background subtraction using a 50-pixel rolling ball radius. These techniques help to reduce noise and enhance the contrast of the vascular structures in the images. Imaris (Bitplane) was used for the three-dimensional reconstruction of the vascular network. This software specializes in the visualization and analysis of biological image data, allowing for the precise mapping of the 3D vascular network. Vascular Network Analysis: MATLAB's built-in toolboxes were utilized to extract the vascular network and measure various parameters such as cross-sectional area, intensity, and length of each vascular structure at different depths. RBC Speed Measurement Line scans parallel to the center of the vessels were defined in the imaging stacks captured at 1000 frames per second (fps) to track the flow path of RBCs. LS-PIV (Lagrangian Single-Particle Image Velocimetry) in MATLAB was employed to calculate the velocity of RBCs. LS-PIV is a technique used to measure fluid velocity fields by tracking the displacement of particles (in this case, RBCs) in a fluid over time. To track the movements of bright points within the capillaries, the original images were subtracted from the average image of all frames, revealing the vascular structure in each frame. Data collection and processing Mice were acclimated for 1 weeks and simultaneously randomized to two groups following simple randomization: sham or bone defect group. Data were not included if values were excluded by outlier test. Data were not included if mis surgery occurred during surgery, or if values were excluded by outlier test. Declarations Author Contribution Jiongnan Xu, Liang Zhu and Tingxiao Zhao wrote the main manuscript tex and prepared figures 3-5 and Weiyi Wu, Keyi Chen and Wangjie Fu prepared figures 1-2, Hengwei Zhang and Jun Zhang revised the manuscript, Wang Xi provided technical support for two-photon microscopy. Acknowledgements The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Jiongnan Xu, Liang Zhu and Tingxiao Zhao contributed equally to this work. This work was funded by the National Natural Science Foundation of China (No. 82460423 to Dr. Jun Zhang), Zhejiang Administration of Traditional Chinese Medicine (GZY-ZJ-KJ-23057 to Dr. Jun Zhang), Guizhou Provincial Administration of Traditional Chinese Medicine (QZYY-2024-109 to Dr. Jun Zhang), Guizhou Provincial Office of Science and Technology (Qiankehe Foundation-[2024] Youth 385 to Dr. Jun Zhang), Bijie Science and Technology Bureau (BKH [2023] No. 55 to Dr. Jun Zhang), Health Commission of Guizhou Province (gzwkj2024-159 to Dr. Jun Zhang) and National Institutes of Health (R01 grant AG084707 to Dr. Hengwei Zhang). A statement to confirm that all experimental protocols were approved by a named institutional and/or licensing committee All the procedure of the study is followed by the ARRIVE guidelines. The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff)) Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.All the procedure of the study is followed by the ARRIVE guidelines. References O'Keefe RJ. Fibrinolysis as a Target to Enhance Fracture Healing. N Engl J Med . 2015;373(18):1776-1778. Taitsman LA, Lynch JR, Agel J, Barei DP, Nork SE. Risk factors for femoral nonunion after femoral shaft fracture. J Trauma . 2009;67(6):1389-1392. Carano RA, Filvaroff EH. 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Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis [published correction appears in Nature. 2010 Jun 17;465(7300):966]. Nature . 2009;458(7237):524-528. Sano H, Kikuta J, Furuya M, Kondo N, Endo N, Ishii M. Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo. Bone . 2015;74:134-139. Marsell R, Einhorn TA. The biology of fracture healing. Injury . 2011;42(6):551-555. Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat Rev Rheumatol . 2015;11(1):45-54. Kikuta J, Wada Y, Kowada T, Wang Z, Sun-Wada GH, Nishiyama I, Mizukami S, Maiya N, Yasuda H, Kumanogoh A, Kikuchi K, Germain RN, Ishii M. Dynamic visualization of RANKL and Th17-mediated osteoclast function. J Clin Invest . 2013;123(2):866-873. Schilling K, Zhai Y, Zhou Z, Zhou B, Brown E, Zhang X. High-resolution imaging of the osteogenic and angiogenic interface at the site of murine cranial bone defect repair via multiphoton microscopy. Elife . 2022;11:e83146. Fernández A, Vendrell M. Smart fluorescent probes for imaging macrophage activity. Chem Soc Rev . 2016;45(5):1182-1196. Zhang, H., Zhu, L., Gao, D.S., Liu, Y., Zhang, J., Yan, M., Qian, J., & Xi, W. Imaging the Deep Spinal Cord Microvascular Structure and Function with High-Speed NIR-II Fluorescence Microscopy. Small Methods . 2022;6(8):e2200155. Li, D., Hu, Z., Zhang, H., Yang, Q., Zhu, L., Liu, Y., Yu, T., Zhu, J., Wu, J., He, J., Fei, P., Xi, W., Qian, J., & Zhu, D. A Through-Intact-Skull (TIS) chronic window technique for cortical structure and function observation in mice. eLight 2 , 15 (2022). He, M., Li, D., Zheng, Z., Zhang, H., Wu, T., Geng, W., Hu, Z., Feng, Z., Peng, S., Zhu, L., Xi, W., Zhu, D., Tang, B.Z., & Qian, J. Aggregation-induced emission nanoprobe assisted ultra-deep through-skull three-photon mouse brain imaging. Nano Today , 45 , 101536. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6196928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":440684127,"identity":"9845a032-7d89-4c71-a499-91de2a9804dc","order_by":0,"name":"Jiongnan Xu","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiongnan","middleName":"","lastName":"Xu","suffix":""},{"id":440684128,"identity":"32c557a0-d781-4be5-9894-3ad464f56a7d","order_by":1,"name":"Liang Zhu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zhu","suffix":""},{"id":440684129,"identity":"8aa5f606-425f-4330-8098-c9283246c4c6","order_by":2,"name":"Tingxiao Zhao","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tingxiao","middleName":"","lastName":"Zhao","suffix":""},{"id":440684130,"identity":"d2dac8c7-84c4-4c3b-a8b4-fce894765531","order_by":3,"name":"Weiyi Wu","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiyi","middleName":"","lastName":"Wu","suffix":""},{"id":440684131,"identity":"19e58e42-33b4-419b-914b-5ee69b7b9ea2","order_by":4,"name":"Keyi Chen","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Keyi","middleName":"","lastName":"Chen","suffix":""},{"id":440684133,"identity":"cf63f74c-51eb-45f6-bf77-5cea41f3af52","order_by":5,"name":"Wangjie Fu","email":"","orcid":"","institution":"Hangzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wangjie","middleName":"","lastName":"Fu","suffix":""},{"id":440684135,"identity":"d3468f8c-aa41-4b53-999e-90547a0abb4d","order_by":6,"name":"Hengwei Zhang","email":"","orcid":"","institution":"University of Rochester Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hengwei","middleName":"","lastName":"Zhang","suffix":""},{"id":440684136,"identity":"66f95e95-ff22-4d57-8cad-25660c6efd83","order_by":7,"name":"Wang Xi","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Xi","suffix":""},{"id":440684137,"identity":"cdd6e141-d3d1-4fd4-b21d-0a1b2eb94dd7","order_by":8,"name":"Jun Zhang","email":"data:image/png;base64,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","orcid":"","institution":"Zhejiang Provincial People’s Hospital Bijie Hospital Bijie","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-10 15:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6196928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6196928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80808118,"identity":"32cd910a-2108-4141-ad4a-bc5484276ef4","added_by":"auto","created_at":"2025-04-17 09:43:03","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104015,"visible":true,"origin":"","legend":"\u003cp\u003eSurgical protocol and two-photon imaging procedure for tibial bone defects window of mouse in vivo. a). Basic steps of the surgical protocol for opening the window of tibial bone defects in mice. b) White imaging of mouse tibia surface. c) Schematic diagram of mouse tibia in TPM imaging. The tibial bone defect Window for two-photon microscope system monitoring in mouse model. The site (5 mm below the tibial tuberosity above the medullary cavity) and size (≈0.6 mm) of the tibial bone defect were shown. A sink was created around the bone defect using dental cement to facilitate water storage. Imaging was performed using retro-orbital injection of vascular dye. d). The 2P fluorescent microscopy system and the light paths of the system were shown. L –lens, DM – dichroic mirror, PMT –photomultiplier tube.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196928/v1/a579769c38d9a48eb51ac7ec.jpeg"},{"id":80808120,"identity":"953da492-5626-438c-8add-842475d5b1ea","added_by":"auto","created_at":"2025-04-17 09:43:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":244810,"visible":true,"origin":"","legend":"\u003cp\u003eStructural images of tibial vessels and s of blood flow velocity measurement in WT mice. a) Mouse tibia surface imaging. The blue area is the bone surface; the green area is the medullary cavity. b, Images of intracortical vascular structures within the bone cortex on the surface of the tibia. c) enlarged vessel images in yellow box in b）. d) Three-dimensional images of intracortical vascular structures reconstructed from c). e) Images of vascular structures within the bone marrow in the tibia. f) enlarged vessel images in red box in e）. g) Three-dimensional images of vascular structures reconstructed from f). h) SHG Image of tibial surface structure. Colored arrows indicate different bone trabecular structures. i-k) Measurement of blood flow velocity in the marrow cavity of the tibia. The colored arrows indicate the lines along the vessel where the blood flow velocity is measured.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196928/v1/5cb65c1b4e2462ae17edb742.jpeg"},{"id":80808537,"identity":"0f0c6443-1ce7-4c0c-b97a-a621f1ed357f","added_by":"auto","created_at":"2025-04-17 09:51:03","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":261068,"visible":true,"origin":"","legend":"\u003cp\u003eImaging of tibial vascular structure and blood flow velocity in Ai-14; Tek-Cre mice by TPM. a, b) Images of vascular structures in the marrow cavity of the tibia at 4X and 16X objective lens. c) Measurement of blood flow velocity in the medullary cavity. Colored arrows indicate the lines along the vessel where blood flow velocity is measured. d, e) Images of intracortical vascular structures within the bone cortex on the surface of the tibia. f) Measurement of blood flow velocity within the bone cortex. Colored arrows indicate lines measuring blood flow velocities along different vessels.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196928/v1/8a2a20820f0c8d40bfb26dde.jpeg"},{"id":80808122,"identity":"9ef95e99-9000-4021-ab86-af3397ae671d","added_by":"auto","created_at":"2025-04-17 09:43:03","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":224395,"visible":true,"origin":"","legend":"\u003cp\u003eImaging of tibial bone defects healing at different time points. a, b) TPM images of mice at 3, 7, 11, 14, and 21 days after tibial bone defects at 4X and 16X. c) Surface pictures \u0026nbsp;of tibial bone defects after 3 and 7 days. d) Percentage of vascular area at different time points after bone defect. e) Vascular TPM imaging in mice 14 days after tibial bone defect by 4X objective. And three-dimensional images of vascular structures reconstructed from PSD21 in b) by 16X objective.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196928/v1/5cb478f19a21a8cc6e4a78e1.jpeg"},{"id":80808539,"identity":"f5ed277b-48cb-4eef-b4af-7fc7197ca37a","added_by":"auto","created_at":"2025-04-17 09:51:04","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":193361,"visible":true,"origin":"","legend":"\u003cp\u003eVascular blood flow velocity measurement of at different time points after bone defects. a-f) Blood flow velocities were measured within the bone callus at 3, 7, 11, 12, 14, and 21 days after the bone defect. Blood flow velocity was 0 mm/s at 3 and 11 days postoperatively. The blood flow velocity was measured at 0.2925 mm/s after 12 days postoperatively, at which time a more complete circuit may have been formed by the vessels within the osseous crust.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196928/v1/71f1fd134eef545769743740.jpeg"},{"id":86915621,"identity":"a3dd82cc-c8cc-45af-9e78-0d0325eb4886","added_by":"auto","created_at":"2025-07-17 06:23:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1677620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6196928/v1/728871b1-fb99-48e6-87df-8a97112ee2f3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intravital imaging of neovascularization by two-photon laser scanning microscopy in tibial bone defects","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe skeleton is one of the most commonly injured tissues, with more than 16\u0026nbsp;million fractures of long bones occurs in the United States annually.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e Although bones have excellent regenerative potential, the rate of delayed or non-union healing of fractures ranges is up to 15%.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e The healing of bone injury includes a dynamic process of tissue regeneration driven by progenitor cells, necessitating the coordinated processes of osteogenesis and angiogenesis at the site of injury.\u003csup\u003e[3.4]\u003c/sup\u003e During the healing of bone injury, the vasculature plays a crucial role by recruiting mesenchymal stem cells (MSCs) and regulating the differentiation of perivascular MSCs into bone-forming cells.\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e While the structure and function of blood vessels within bones has been somewhat mysterious until recently, largely due to the technical challenges associated with imaging on calcified tissues.\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e The technical breakthrough in imaging of murine bones now has provided insights into the heterogeneity of blood vessels in bone.\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTwo-photon laser scanning microscopy (TPLSM) has emerged as an invaluable and invasive imaging for in vivo surveillance of cell or organ function at a cellular or subcellular level temporally and on live-animals.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e TPLSM enables the sophisticated morphological and functional investigation of new blood vessels.\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e It offers the exclusive advantages comparing with conventional imaging methods, such as the high spatiotemporal resolution, minimal invasiveness, and the ability to capture 3D images.\u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e In addition to capturing nonlinear fluorescence by using transgenic mice or labeling cells/structures with fluorescent dyes, TPLSM is also capable of visualizing the bone matrix through detecting the autofluorescence of collagen in bone known as second harmonic generation (SHG).\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e Owing to its substantial penetrating capability and high microscopic resolution, TPLSM is capable of measuring hemodynamic parameters, such as the flow rate within blood vessels.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e With those unique advantages, the intravital TPLSM allows us to simultaneously visualize cells, the extracellular matrix (ECM), and the surrounding vascular networks and dynamically analyze the osteogenesis and angiogenesis spatiotemporal on live-animals during the whole repair and regenerative process of bone injury.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTPLSM has been initially used in studies of osteoclasts and hematopoietic stem cells images.\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e Later, TPLSM was used to image the interaction of angiogenesis with osteoblasts and bone regeneration during the bone repair in cranial bone defect.\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e However, the non-weight-bearing bone differs from the weight-bearing bone, including the bone cell functions, blood vessel regeneration and bone mineral composition.\u003csup\u003e[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e To overcome the disadvantages, investigators implanted a gradient refractive index (GRIN) endoscopic lenses and visualize the blood vessels and bone cells in bone marrow of long bones.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e Recently, Dr. Phan\u0026rsquo;s group developed a minimally invasive SOP for longitudinal imaging of endosteal bone cells and hematopoiesis in the place of intact tibiae with acceptable bone thickness.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e However, the longitudinal intravital imaging method for visualizing the angiogenesis and bone regeneration long bone during bone injury repair is still lacking.\u003c/p\u003e \u003cp\u003eThe present study aims are to develop a TPLSM-based longitudinal imaging platform that enables the non-invasive and high-resolution analysis of osteogenesis and angiogenesis, particularly the function of newly formed vessels throughout the repair and regeneration process of tibial bone defects in mice. By employing Tek-Cre;Ai-14 mice, we are presenting for the first time, to visualize and functionally analyze the angiogenesis and its interaction with bone regeneration at the site of tibial defect repair in vivo longitudinally and spatiotemporally. Our research represents a unique and innovative tool, ideally suited for examining the temporal and spatial dynamics and functional aspects of angiogenesis throughout the process of bone defect repair.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eSurgical procedure and TPLSM imaging protocol for tibial bone defects in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAngiogenesis is an important integral part of the fracture healing process. Due to technical limitations, traditional research study the physiologic function of blood vessels by examining their morphology, number, or composition of different subtypes of endothelial cells in vitro. However, there is a significant gap in testing blood vessels\u0026apos; function to transport blood in vivo. Therefore, there is urgently needed to conduct both qualitative and quantitative research on vessel function during the fracture healing process in live animals.\u003c/p\u003e\n\u003cp\u003eTo achieve repeated long-term observations of neovascularization in healing bone defects in live mice, we devised a detailed surgical protocol for creating tibial bone defects, shown in Figure 1a. Initially, consistent with standard protocols for murine surgical interventions, the mice were anesthetized via intraperitoneal injection. Subsequently, hair was removed from the right lower limb, and the area was sterilized to ensure a clean and intact surgical field. A scalpel blade was then employed to create a 1-cm vertical incision along the anterior border of the tibia. Ophthalmic forceps were utilized to carefully detach the surface-attached muscles on the medial aspect of the tibia, thereby providing clear exposure of the tibial tuberosity. A high-speed cranial drill was employed to create a bone defect with a diameter ranging from 0.5 to 0.6 mm through a single layer of the bone cortex, targeting the medullary cavity in the plane of the tibial tuberosity, situated above the medullary cavity (Figure 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing the immobilization of the right lower limb with a custom-designed instrument (spinal cord adapter, RWD, China), we utilized a commercial Bruker 2P laser scanning microscope to imaging the bone defects areas illustrated in Figure 1c. Figure 1d provides a detailed schematic of the system and the optical path of the 2P platform, emphasizing the two-color channel that support our imaging methodology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTibial Structure and Vascular Imaging by TPLSM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor visualization fluoresce signal of blood vessels in the tibia, we employed a 920 nm wavelength laser to conduct in vivo imaging of the vascular system in the tibia (Figure 2a) of wild-type mice. The fluorescein isothiocyanate (FITC) were injected into the retro-orbital vein of mouse. The tibia has preserving second harmonic generation (SHG) signals.\u003csup\u003e\u0026nbsp;[19,20]\u003c/sup\u003e To effectively differentiate between the vascular fluorescence (FITC) signal and SHG, we subsequently imaged the same region with a reduced laser wavelength (800 nm). This adjustment resulted in a significant attenuation of the previously dominant FITC signal, while the SHG signal was markedly enhanced. The vascular structures located in the cortical region of the bone (depicted by the blue dotted line) and the vascular structures within the medullary cavity (indicated by the green dotted line) in Figure 2a are depicted in Figures 2b and 2e, respectively. The cortical vascular structures in Figure 2b exhibit higher density and finer diameters ranging from 5 to 8 \u0026mu;m compared to the bone marrow cavity region, with blood vessels intersecting the bone cortex in a non-specific direction. Magnified imaging clarifies the intricacies of vascular architecture and associated blood flow, as shown in Figure 2c. To visualize the three-dimensional structure of the vessels, we used a Z-stack scan imaging of the fluoresce signals in vessels with 1um step size and reconstructed the three-dimensional vessel structures (Figure 2d). Compared to the vascular areas of the bone cortex (Figure 2b), wide-field, low-magnification imaging reveals the morphology and structure of the vasculature within the bone marrow cavity, which is more regularly distributed primarily along the longitudinal axis of the medullary cavity (Figure 2e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe conducted detailed imaging of the intramedullary vasculature in the green area shown in Figure 2a. The high-resolution imaging of Figure 2f further elucidates the complex structure of the blood vessels depicted in the red box in Figure 2e. Similarly, the vascular structures in the green box area in Figure 2e are also represented in Figure 2g by means of 3D reconstruction. Furthermore, to accurately quantify the velocity of blood flow within an individual vessel, line scan measurements were conducted on the medullary cavity vessels depicted in Figure 2i, as illustrated in Figure 2j, yielding an average velocity of 3.1344 mm/sec (Figure 2k).\u003c/p\u003e\n\u003cp\u003eMacroscopically, natural bone consists of compact cortical and trabecular cancellous layers, each characterized by unique collagen fiber morphologies. Hydroxyapatite nanocrystals are deposited within the interstitial spaces between collagen molecules, resulting in bundles of mineralized collagen fibers approximately 100 nm in diameter. Under enlarged field view, we examined the superficial structure of the bone cortex (indicated by the black area in Figure 2a) and were able to distinctly visualize the bone architecture in vivo. This included the trabecular cavity (denoted by the red arrow) and the trabecular pathway (denoted by the blue arrow) as illustrated in Figure 2h. This detailed visualization is instrumental for subsequent in-depth analyses of osteocyte and blood vessel interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation the vascular structure with FITC tracer and Tek-Cre;Ai-14 transgenic mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify the vascular structure in bone tissue，we imaging the tibia in Tek-Cre;Ai-14 transgenic mice, which overexpress the angiopoietin receptor TEK specific expression in vascular endothelial cell.\u003csup\u003e[31]\u003c/sup\u003e By injection with the FITC \u0026nbsp;tracer，dual-color imaging were performed to acquire the tracer fluoresce in blood and the endothelial cell fluoresce. Low magnification field of view showed angiographic morphology of the medullary cavity (Figure 3a) and bone cortex (Figure 3d) regions, respectively. The data indicated that the expression patterns and spatial distribution of vascular endothelial signaling (TEK) were largely consistent with those of FITC signaling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt enlarged field view with high magnification objective, Figures 3b and 3e more distinctly reveal the intricate morphological structures and blood flow within the vasculature of the two regions. \u0026nbsp;Consistent with the description of wild-type mice above, the vascular structure in the bone marrow cavity of Tek-Cre; Ai-14 transgenic mice is similarly more defined compared to the cortical region. These findings robustly affirm the efficacy of our two-photon in vivo imaging technique for vascular visualization in the mouse tibia. To further elucidate the functional characteristics of these vessels, we conducted an examination of the region of interest (ROI) demarcated by the yellow dashed line (Figure 3b and e). Our analysis determined that the average flow velocity was 2.6459 mm/s for medullary vessels and 2.4473 mm/s for cortical vessels (Figure 3c and f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTPLSM imaging tibial bone injury recovery for 21 days\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter successful imaging the bone vascular system in vivo in healthy mouse. Next, we monitor bone defects in the tibiae of mice over a three-week period by two-photon imaging. The objective of this study was to meticulously document the healing process of the bone defect region and the concomitant angiogenesis on a daily basis following surgical intervention. The imaging data obtained at critical time points: 3, 7-, 10-, 14-, and 21-days post-surgery. The bone callus area was visualized at both low and high flied view (Figure 4a and b). During the initial phase of bone defect healing, spanning from days 3 to 14, a significant proliferation of micro-vessels measuring 5-15 \u0026mu;m in diameter is observed. However, as the healing process progresses beyond 14 days, the majority of these micro-vessels regress, resulting in the predominance of larger diameter vessels, approximately 20 \u0026mu;m.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe defect site was covered by a hematoma on postoperative day 3. By postoperative day 7, the formation of bone scabs was evident, indicating the progression of the bone healing process (Figure 4c).\u003c/p\u003e\n\u003cp\u003eFollowing the identification of vascular, the blood vessels morphology within the defect area were quantified by ImageJ software, a progressive increase in vascular density during the initial stages of bone healing were found. This growth reached its zenith at 14 days postoperatively (Figure 4d), underscoring the critical phase of the vascularization process. To further characterize the vascular architecture of the region, we performed the imaging of the defect site at 14 days postoperatively. Using 4X objective, it allows for the simultaneous acquisition of a larger and volume imaging, thereby facilitating direct observation of the vascular architecture at the defect site (Figure 4e). The three-dimensional reconstruction of the vascular images 21 days post-surgery were constructed showed the vessel network were interconnected and recovery. (Figure 4e). Our findings indicate that, by this time, the neovascularization at the defect site had largely interconnected with the surrounding vasculature, exhibiting morphology and structure that were essentially consistent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional recovery of vascular flow\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further confirm the vessel functional recovery during the functional status of neovascularization in the context of bone defect healing, we conducted a comprehensive analysis of blood flow in quantitatively resolved vessels at various postoperative time points (Figure 5). This evaluation is crucial for understanding the establishment the functional role of neovascularization in bone defect repair.\u003c/p\u003e\n\u003cp\u003eDuring the process of bone healing, we observed that not all neovascularization formation period had blood cell flow, particularly in the initial stages following surgical intervention (Figure 5a-c). This phenomenon may be attributed to the transient absence of blood flow within the neovessel, which precluded the measurement of blood flow velocity in these nonfunctional neovascularized structures. The recorded blood flow velocities at 12- and 14-days post-surgery were 0.6205 mm/s and 0.9784 mm/s, respectively (Figure 5d-e). Our data indicate that the period of 12 to 14 days post-surgery represents a pivotal transition phase. After 12-14 days, the blood flow velocities can be measured as a slow speed on the neovascularization. This blood flow represented the neovascularization at the site appears to form a connected circuit. This critical observation implies that the vessels have attained a functional state characterized by substantial blood cell flow. As illustrated in Figure 5f, a blood flow velocity of 3.7644 mm/s was recorded at 21 days post-bone defect surgery, which corresponds to the baseline blood flow velocity observed in wild-type mice, as shown in Figure 2k. Which indicated the completed recovery of the vessel structural and functional connection. Within the TPSLM imaging method, we can image and analysis the blood vessel structure and functional information during the bone healing process, which will expand the use of TPSLM in the basic and preclinical research.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eVascularization and bone formation are closely associated processes, with both endochondral and intramembranous osteogenesis intricately linked to blood vessels development.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e While multiphoton laser scanning microscopy (MPLSM) has successfully been used to image calvarial bone, visualizing blood vessels and cells in bone marrow or endosteum of long bones remains a formidable challenge due to technical and anatomical hurdles.\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e In our current study, we developed a new methods for visualize and functionally analyze the angiogenesis and its interaction with bone regeneration at the site of tibial defect repair longitudinally and spatiotemporally in vivo. We applied this technology and demonstrated the functional vascular network begins to form at 14 days post-surgery (dps) and the completed recovery at 21 days. Notably, the number of blood vessels peaked at 14 dps, and the blood flow starts after this stage, suggesting that the pre-14 days might be critical stages for recovery. Based on the results of this study, it is suggested that the experimental protocol of this study is suitable for generalization across related experiments.\u003c/p\u003e \u003cp\u003eA mature and functional vascular network is critical for bone injury repair. Angiogenesis precedes osteogenesis,\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e therefore neovascularization ensures the recruitment of osteoclast and osteoblast precursors.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e Many scientists have tried to establish the imaging methods in other bone system. Dr. Masaru Ishii\u0026rsquo;s group pioneered the use of MPLSM to image the endosteal bone cells, including the osteoclast, osteoblasts and their interactions in calvarial bones.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e This technology was later expanded to track the longitudinal changes of angiogenesis and osteogenesis, and their interaction, in calvarial bone defects, including the critical defect.\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e These studies demonstrated that MPLSM is a valuable intravital imaging platform offering high resolution, deeper tissue penetration, and reduced photobleaching.\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e However, the heavy mineralization of long bones hinders the application of TPLSM in both physiological and pathological conditions. To address this, Dr. Niesner\u0026rsquo;s group developed implanted gradient refractive index (GRIN) endoscopic lenses for long bones, enabling the examination of the bone, hematopoitic or tumor cells in bone marrow and on endosteal surface of long bones.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e However, this method was limited by the implanted object's interference with local bone regeneration and the associated high cost. Dr. Phan\u0026rsquo;s group later optimized the TPLSM imaging procedure for tibiae due to their relatively thin front surface.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDr.Xi, developed a method that combines of near-infrared (NIR)-II fluorescence labeled red blood cells (RBC) with a fast NIR camera and TPLSM, successfully imaging the functional vascular network in deep spinal cord tissues.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e But for long bones, we are the first established methods for longitudinal repeated imaging. Prior methods had successful application of transparency reagents to cranial bones anticipates future enhancements in the imaging quality of blood vessels in bone tissue.\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e The advent of three-photon imaging technology, which utilizes higher-order nonlinear effects to reduce surface tissue background and employs longer wavelength photons for excitation, weakens the scattering of excitation light by biological tissues, allowing for greater penetration depth.\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e Therefore, the exploration of strategies to enhance multiphoton microscopy for in vivo imaging of blood vessels in bone tissue is not only feasible but also holds immense potential. By refining the capabilities of multiphoton microscopy, we can expect significant advancements in the field of orthopedic research and tissue engineering. Such strategies may include the development of novel vascular dyes with improved depth penetration, the engineering of transgenic animal models with enhanced fluorescent markers, and the refinement of optical techniques to boost imaging clarity. The pursuit of these strategies will undoubtedly provide deeper insights into the complex interplay between vascularization and bone regeneration, ultimately contributing to more effective treatments for bone injuries and diseases.\u003c/p\u003e"},{"header":"Experimental Section/Methods","content":"\u003cp\u003eFor animal research, wild-type mice and Tek-Cre;Ai-14 transgenic mice were randomized into groups using a table of random numbers: the Sham group, and the bone-defect group.\u003c/p\u003e\n\u003cp\u003eAnimals and Materials: Male C57BL/6 mice aged\u0026nbsp;11\u0026ndash;12\u0026nbsp;weeks and weighing 25-30 g, were obtained from purchased from the Hangzhou Paisiao Biotechnology Co., Ltd. (Zhejiang, China)\u0026nbsp;and Tek-Cre;Ai-14 transgenic mice were provided by Xi Wang\u0026apos;s group at Zhejiang University.\u0026nbsp;The mice were placed in controlled environments (12-h light/dark cycle; 20-26\u0026deg;C; 40-60% humidity) and had free access to bacteria-free water and food. All animal housing and experiments were conducted in accordance with the ethical guidelines formulated by the Animal Ethics Committee of Zhejiang Provincial People\u0026apos;s Hospital (20241202554152).\u003c/p\u003e\n\u003cp\u003eMale wt mice and Tek-Cre;Ai-14 transgenic mice were divided into 2 groups of eight each by randomization method: sham-operated group, and bone defect group. bone defect was established according to the experimental protocol described in this paper.\u003c/p\u003e\n\u003cp\u003eAll the chemicals and reagents were purchased from chemical sources, and the solvents for chemical reactions were distilled before use. Tetrahydrofuran (THF) was acquired from Sinopharm Chemical Reagent Co., Ltd. Pluronic F-127 (F127) was bought from Sigma-Aldrich. Heavy water (D2O) was used in all of the imaging experiments as the immersion liquid for objectives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgical procedure and TPLSM for tibial bone defects in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC576BL/6J mice and Tek-Cre;Ai-14 transgenic mice were randomized into a bone defect surgery group and a sham surgery control group. Bone defect success criteria could be fully determined during surgery. Control mice were also anesthetized.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMice are anesthetized through an intraperitoneal injection of 2,2,2-tribromoethanol (Nanjing Abbey Biotechnology Co, China) at a dosage of 400 mg/kg. A heating pad is used to maintain the mouse\u0026apos;s body temperature at approximately 37\u0026deg;C to prevent hypothermia during the surgery. The surgical area, centered around the knee joint, is shaved to a width of about 1 cm and a length of about 3 cm. The shaved area is then cleaned with an iodine solution to reduce the risk of infection. A longitudinal incision of about 1 cm is made on the anterior medial side of the tibia, extending from the ankle to the knee, using tissue scissors. Fine surgical scissors are used to make a longitudinal incision between the tibia and the triceps muscle of the lower leg. Muscles and fascia attached to the tibia are carefully removed in a circular area centered on the tibial tuberosity with a diameter of 1 cm to expose the tibia\u0026apos;s inner surface. Saline mixed with 0.2% cephalosporin is used to rinse the exposed tibial surface to remove any debris and to reduce the risk of infection. Excess muscle and tissue are removed from the area. The tibia is held steady with forceps. A high-speed drill is used to create a bone defect approximately 0.5-0.6 mm in diameter on the posterior side of the tibial tuberosity on the medial side of the tibia (above the bone marrow cavity), taking care not to penetrate the entire tibia. Saline water is used to wash away bone powder from the surface. The wound is inspected to ensure there is no significant bleeding, aside from the bone defect area. The wound is rinsed again with saline and then sutured. Before imaging, mice are re-anesthetized using the same method. The previous incision is reopened to expose the tibial bone defect. Any remaining soft tissue is carefully removed using forceps and fine surgical scissors. The area is rinsed with saline and any fine bleeding is controlled. Dental cement is used to fill in around the bone defect to create a stable imaging chamber; adhesive may be used if necessary to ensure a secure bond with the underlying soft tissue. Special clips are used to stabilize the mice, providing the necessary stability for the two-photon imaging process.\u003c/p\u003e\n\u003cp\u003eTreatment of mice and sample collection were not blinded as they were performed by one single investigator. Mouse samples were coded and experimental measurements and analysis blinded during the assessment and data analysis process. Which was performed by two other investigators. The bone defect surgeries in mice were all performed by the same person to ensure homogeneity of the procedure. Blinding was not feasible during the procedure; however, when possible, we analyzed the results blindly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2P Microscopy Imaging\u0026nbsp;\u003c/strong\u003eMice with tibial imaging windows were secured on a specialized fixation frame to ensure stability during imaging. Mice were anesthetized to ensure they remained still throughout the procedure. Just before imaging, mice were injected retro-orbitally with FITC (fluorescein isothiocyanate) dextran conjugate (2000 kDa, Invitrogen), a fluorescent marker that binds to plasma proteins and outlines the vasculature. A customized two-photon microscope (Ultima 2P，Bruker) was coupled with a femtosecond laser (ChameleonUltra II, Coherent Inc. model-locked Ti: Sapphire laser) for high-resolution imaging. A 920 nm laser pulse with a pulse width of less than 200 femtoseconds and a repetition rate of 80 MHz was used for excitation. FITC served as the angiography agent, enabling the visualization of blood vessels. A Pockels Cell (EO-PC, Thorlabs Corporation) was utilized to modulate the laser power, ensuring that it was optimized for imaging without causing damage to the sample. Emission light from the fluorescent signal was filtered using a bandpass filter of 525/50 nm to match the emission spectrum of FITC. The filtered light was then detected using GaAsP photomultiplier tubes (PMTs, Hamamatsu, model H10770), which are sensitive and fast detectors suitable for capturing the weak signals from two-photon excited fluorescence. High-quality images were captured using a high-numerical aperture objective lens (Olympus XLUMPLFLN20XW, 16\u0026times;, N.A. = 1.0), which provided the necessary resolution for detailed imaging of the tibial vasculature. The average laser power was adjusted according to the imaging depth, with the maximum power kept below 50 mW to prevent phototoxicity or thermal damage to the tissue. Images were acquired at varying depths, with adjustments made to the laser power to accommodate the increased scattering and absorption as the imaging depth increased.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging Analysis:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMATLAB (R2020b; MathWorks)\u0026nbsp;\u003c/strong\u003ewas the primary software used for data analysis, benefiting from its robust suite of built-in toolboxes for image processing and computational biology. The Simple Non-Local Means (NLM) Filter was applied to reduce noise in the images. This filter is designed to preserve edges while removing noise, which is crucial for clear vascular imaging. The Jerman Enhancement Filter, which utilizes hessian eigenvalues to enhance image structures, was used to improve the visibility of vascular structures in 2D images. \u003cstrong\u003eImageJ (NIH, USA)\u003c/strong\u003ewas employed for preliminary image processing tasks, including the application of a one-pixel-radius median filter and background subtraction using a 50-pixel rolling ball radius. These techniques help to reduce noise and enhance the contrast of the vascular structures in the images.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaris (Bitplane)\u0026nbsp;\u003c/strong\u003ewas used for the three-dimensional reconstruction of the vascular network. This software specializes in the visualization and analysis of biological image data, allowing for the precise mapping of the 3D vascular network.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVascular Network Analysis:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMATLAB\u0026apos;s built-in toolboxes were utilized to extract the vascular network and measure various parameters such as cross-sectional area, intensity, and length of each vascular structure at different depths.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRBC Speed Measurement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLine scans parallel to the center of the vessels were defined in the imaging stacks captured at 1000 frames per second (fps) to track the flow path of RBCs. LS-PIV (Lagrangian Single-Particle Image Velocimetry) in MATLAB was employed to calculate the velocity of RBCs. LS-PIV is a technique used to measure fluid velocity fields by tracking the displacement of particles (in this case, RBCs) in a fluid over time. To track the movements of bright points within the capillaries, the original images were subtracted from the average image of all frames, revealing the vascular structure in each frame.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were acclimated for 1 weeks and simultaneously randomized to two groups following simple randomization: sham or bone defect group. Data were not included if values were excluded by outlier test. Data were not included if mis surgery occurred during surgery, or if values were excluded by outlier test.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiongnan Xu, Liang Zhu and Tingxiao Zhao wrote the main manuscript tex and prepared figures 3-5 and Weiyi Wu, Keyi Chen and Wangjie Fu prepared figures 1-2, Hengwei Zhang and Jun Zhang revised the manuscript, Wang Xi provided technical support for two-photon microscopy.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Jiongnan Xu, Liang Zhu and Tingxiao Zhao contributed equally to this work. This work was funded by the National Natural Science Foundation of China (No. 82460423 to Dr. Jun Zhang), Zhejiang Administration of Traditional Chinese Medicine (GZY-ZJ-KJ-23057 to Dr. Jun Zhang), Guizhou Provincial Administration of Traditional Chinese Medicine (QZYY-2024-109 to Dr. Jun Zhang), Guizhou Provincial Office of Science and Technology (Qiankehe Foundation-[2024] Youth 385 to Dr. Jun Zhang), Bijie Science and Technology Bureau (BKH [2023] No. 55 to Dr. Jun Zhang), Health Commission of Guizhou Province (gzwkj2024-159 to Dr. Jun Zhang) and National Institutes of Health (R01 grant AG084707 to Dr. Hengwei Zhang).\u003c/p\u003e \u003cp\u003eA statement to confirm that all experimental protocols were approved by a named institutional and/or licensing committee\u003c/p\u003e \u003cp\u003eAll the procedure of the study is followed by the ARRIVE guidelines.\u003c/p\u003e \u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e \u003cp\u003eReceived: ((will be filled in by the editorial staff))\u003c/p\u003e \u003cp\u003eRevised: ((will be filled in by the editorial staff))\u003c/p\u003e \u003cp\u003ePublished online: ((will be filled in by the editorial staff))\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.All the procedure of the study is followed by the ARRIVE guidelines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eO\u0026apos;Keefe RJ. 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Aggregation-induced emission nanoprobe assisted ultra-deep through-skull three-photon mouse brain imaging. \u003cem\u003eNano Today\u003c/em\u003e, \u003cem\u003e45\u003c/em\u003e, 101536.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Two-photon laser scanning microscopy, in vivo, Weight-bearing bone, Neovascularization, Blood flow velocity","lastPublishedDoi":"10.21203/rs.3.rs-6196928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6196928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeovascularization plays a critical role in bone regeneration and skeletal development. Our understanding of weight-bearing bone healing has been hindered by the lack of a reliable method that allows tracking neovascularization at a high spatiotemporal resolution in living model. Thus, we employed two-photon laser scanning microscopy (TPLSM) for longitudinal analysis of angiogenesis of tibial bone defects in mice. In this study, we established an effective model for long-term visualization and longitudinal analyses of angiogenesis in tibial bone defect healing. The vessel structural can be imaged and analyzed in healthy and tibial bone defects mice for over 3 weeks. Blood flow could be tracked for 21 days post-surgery. During this tibial bone healing process imaging, we found the blood flow start at 12\u0026ndash;14 days after surgery and the velocity reach 0.6205 mm/sed and 0.9784 mm/sed. After 21 days recovery, the vessel structural and functional recovered to normal with velocity of 3.7644 mm/s which corresponding to baseline. The establishment of a in vivo imaging platform provides a unique tool to better understand angiogenesis in tibial bone defects repair, enabling further investigation of structure and function of vascularization during weight-bearing bone healing.\u003c/p\u003e","manuscriptTitle":"Intravital imaging of neovascularization by two-photon laser scanning microscopy in tibial bone defects","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 09:42:59","doi":"10.21203/rs.3.rs-6196928/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"faf5fdeb-6f01-4290-95a6-6d9db9d9aa8a","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46918947,"name":"Biological sciences/Biological techniques/Microscopy/Multiphoton microscopy"},{"id":46918948,"name":"Biological sciences/Physiology/Bone"}],"tags":[],"updatedAt":"2025-07-17T06:23:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 09:42:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6196928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6196928","identity":"rs-6196928","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}