Name: Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow (Video) | JoVE Journal
Uploaded: 2025-04-11T13:00:00.000+00:00
Duration: 10 min 49 s
Description: Explore intravital microscopy for vascular dynamics in calvarial bone marrow using two-photon imaging and transgenic mouse models. This longitudinal method reveals blood vessel responses to hematopoiesis and leukemia progression.

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The authors would like to thank all the staff of the IMAG&#8217;IC and Animal facilities at Institut Cochin for their support with microscopy experiments and mouse housing. Tg(Nes-EGFP)33Enik and Tg(Pdgfb-icre/ERT2)1Frut mice were a kind gift of Dr Bonnet (The Francis Crick Institute, London). Tg(Cdh5-cre/ERT2)1Rha and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mice were a kind gift of Dr Rafii (Weill Cornell Medicine, New York). The work described has been supported by CNRS, INSERM and Universit&#233; de Paris Cite, and grants from ATIP-AVENIR, Fondation ARC pour la recherche sur le cancer (R19084KS - RSE20008KSA), Ville de Paris &#8220;Emergence&#8221; (R20192KK - RPH20192KKA), Laurette Fugain (R23197KK), Canc&#233;rop&#244;le IDF (RPH23177KKA), INCA PLBIO (RPH21162KKA), Fondation de France (RAF23152KKA), Ligue contre le cancer (282273/807251), Institut du cancer Paris Carpem, European Hematology Association (RAK23130KKA) and European Research Council ERC-STG (EEA24092KKA). IMAG&#8217;IC core facility is supported by the National Infrastructure France BioImaging (grant ANR-10-INBS-04). Passaro lab is affiliated to the &#8220;Institut Hors Murs des Sciences Cardiovasculaires&#8221; and to the &#8220;Leukemia Institute Paris Saint-Louis.

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Jozsua Fodor is the founder of the YMETRY company (register No 888312352). The other authors have no conflicts of interest.

The primary strength of intravital microscopy is its ability to capture dynamic cellular processes in real-time within their native environment, overcoming the limitations of traditional histological and ex vivo imaging methods. By directly observing the calvarial BM using the advanced imaging techniques coupled with fluorescent labeling as described in this protocol, researchers can study not only vascular functional parameters8,14, but also longitudinal processes such as leukemic cell engraftment and migration6,15, proliferation, and metabolic activity16,17 preserving physiological relevance and avoiding artifacts associated with tissue fixation and processing.
The advantages of the BM calvaria intravital imaging over alternative bone sites include easy accessibility, transparency of the skull bone allowing for better visualization without bone damage, stability of the calvarium bone minimizing tissue motion artifacts, and reproducibility across experimental animals facilitating comparison between different experimental groups. The possibility to recover the animals and perform longitudinal imaging allows as well to reduce the number of animals used in a study. It is worth mentioning that because strain-specific differences in skull vessel growth patterns and consequent osteogenesis have been observed18, it is important to take this into account when designing the specific imaging implant for the desired mouse strain to allow perfect anatomical compatibility and access to the desired imaging site.
Another important point to mention is the potential differences in vascular and hematopoietic properties within the calvarial BM compared to other bones, a poorly explored question until recent years. Novel studies suggest localized functions for different bones, with the calvarial BM differing from other bones in terms of hematopoiesis, bone and vascular structure and function19,20,21,22,23, as well as response to neurological pathologies24. These differences need to be further explored and taken into account before generalizing specific calvaria findings.
The design and construction of an imaging implant is a key step for this experimental setup, starting from the choice of the material. Biocompatible implants play a pivotal role in biomedical research, facilitating a wide array of applications ranging from tissue engineering and regenerative medicine to drug delivery systems and in vivo monitoring devices. The choice of material for a head implant adapted for intravital imaging is critical. Ideally, the material should exhibit excellent biocompatibility, mechanical properties suitable for ensuring minimal flexibility and overall stability, and finally, the ability to integrate within the skull without inducing inflammation. Titanium is ideal for its good tolerability within the animal body, without induction of adverse reactions in contact with biological tissues, as well as for its resistance to corrosion, ensuring long-term stability even if exposed to body fluids or experimental solutions. Moreover, its mechanical strength makes it resistant to deformation and fracture. Despite its strength, titanium has a relatively low density, resulting in lightweight intravital imaging devices that minimize the burden on experimental animals and researchers. Finally, its versatility in fabrication allows for the customization of intravital imaging devices to suit specific experimental requirements, such as size, shape, and functionality.
Here, we designed a biocompatible head fixation implant using parametric modeling CAD software with built-in Finite Element Analysis (FEA) capabilities, specifically SolidWorks. This approach enables precise, iterative adjustments to the implant&#39;s structural and spatial requirements, ensuring both anatomical compatibility and mechanical resilience. Free alternatives, such as FreeCAD, offer similar modeling and simulation functionalities. The primary inputs for the design process include a high-resolution anatomical model of the mouse skull, a model of the microscope objective, and a model of the fixation system, which together inform the implant&#39;s dimensions and configuration. The final output is a platform-independent mesh file, typically in STL or STEP format, compatible with most 3D printing software.
The initial step involves capturing detailed anatomical features of the mouse&#39;s head using high-resolution imaging techniques, such as in vivo micro-CT scanning. This approach provides the highest anatomical detail and accuracy, capturing the skull&#39;s microstructures. 3D Scanning of a Prepared, Naked Skull could be also an option. This method, using laser or structured light scanners, is commonly employed on a prepared skull and provides accurate surface contours, though with less internal detail compared to micro-CT. Otherwise, anatomy models can be downloaded from open source publications and databases25 or DigiMorph {https://www.digimorph.org/specimens/Mus_musculus/}. While convenient, these models may lack specimen-specific details, so adjustments are often needed for the specific animal in the study such as scaling to actual Bregma-Lambda distance. The acquired data are then used to create a precise 3D model of the mouse&#39;s skull, serving as the foundational template for the implant design.
To secure the implant onto the skull, a cementing structure is designed to cover the remaining skull surface not occupied by the observation window. This structure must provide robust attachment points while avoiding critical anatomical features. The cementing structure has multiple openings to ensure optimal polymerization of the cement under the implant through diffusion. Additionally, the walls of the cementing structure have a small draft angle, which allows the cement to anchor securely against these angled walls. The tail of the implant, which extends from the main body to the fixation system, is designed. This component is crucial for aligning and stabilizing the implant during observation, and its design must consider the available space and the anatomical constraints of the mouse&#39;s head. Finally, a dovetail mechanism is integrated into the implant design for easy attachment and detachment of the implant from the fixation system. This feature enhances the practicality and usability of the implant during repeated observations. The dovetail mechanism provides repeatable fixation for easy retrieval of observed tissues for repeated imaging sessions.
Readers considering the adoption of intravital microscopy imaging of the calvarial BM should carefully evaluate their research goals and experimental requirements to determine whether this method is appropriate for their studies. While intravital microscopy offers unparalleled insights into hematopoiesis, tissue microenvironment regulation, and vascular dynamics in vivo, it also presents certain technical challenges and limitations. Researchers should be prepared to address these challenges through careful experimental design, optimization of imaging parameters, and utilization of appropriate controls. Additionally, researchers should consider the availability of specialized imaging equipment, expertise in fluorescent labeling techniques, and computational resources for image analysis. Overall, intravital microscopy imaging of the calvarial BM holds tremendous potential for advancing our understanding of hematopoiesis and vascular biology, offering a unique window into the dynamic processes occurring within the BM microenvironment.

Intravital microscopy imaging of the calvarial bone marrow (BM) serves as a powerful and indispensable technique for investigating the dynamic processes of hematopoiesis, tissue microenvironment regulation, and vascular dynamics within the BM niche. The primary purpose of this methodological approach is to enable real-time visualization and analysis of cellular behaviors, interactions, and spatial organization within the BM microenvironment in vivo. By directly observing the BM calvaria using advanced imaging techniques coupled with fluorescent labeling, researchers can elucidate the complex interplay between hematopoietic stem cells (HSCs), stromal cells, and the surrounding vasculature, thereby providing crucial insights into the regulation of hematopoiesis and immune responses.
The development and utilization of intravital microscopy imaging stem from the limitations of traditional histological and ex vivo imaging methods, which often fail to capture the dynamic nature of cellular behaviors and interactions within a tissue. Unlike static imaging techniques, intravital microscopy enables researchers to observe cellular dynamics in real time, allowing for the study of longitudinal processes such as cell migration, proliferation, and differentiation within their native niche, without sacrificing experimental animals. Furthermore, intravital microscopy provides the unique advantage of studying functional behaviors in vivo, such as vascular functionality (e.g., perfusion, permeability, hypoxia), thereby preserving the physiological relevance and avoiding artifacts associated with tissue fixation and processing. Pioneering studies in the field have demonstrated the tremendous advantages of this approach1,2, and their findings have been corroborated and expanded by more recently refined approaches3,4,5 that utilized intravital microscopy to track endogenous HSC localization, migration, and interactions with the vasculature within the BM niche. Moreover, intravital microscopy has been instrumental in elucidating the mechanisms underlying hematopoietic disorders, such as leukemia and BM failure syndromes, offering new insights into leukemic cell motility6,7, disease-associated vascular implications8, and drug response9.
There are several advantages of intravital microscopy imaging of the calvarial BM cover alternative bone sites. First, the BM contained within the skull bone provides easy accessibility for intravital imaging compared to deeper bones such as the femur or tibia. This accessibility facilitates the direct observation of the tissue microenvironment, including the bone itself, via second harmonic generation (SHG) visualization10, without the need for invasive surgical procedures. Second, the skull is relatively thin and translucent, allowing for better visualization of cellular dynamics within the BM niche. This transparency facilitates high-resolution imaging with two-photon microscopy, confocal microscopy, and light-sheet microscopy, without the need for bone thinning or clearing techniques. The calvarial bone offers a stable, flat, and rigid platform for intravital imaging experiments, minimizing tissue motion artifacts and ensuring consistent imaging conditions over prolonged observation periods. This stability is particularly advantageous for longitudinal studies tracking cellular behaviors and responses over time. Reproducibility is another great advantage, given the relatively small and spatially defined structure of the calvarial bone across experimental animals. This uniformity facilitates comparison between different experimental groups and enables robust statistical analysis of imaging data.
Here we describe a method to image the mouse calvarial BM via intravital two-photon microscopy by introducing a newly developed head fixation implant11, 3D-printed using biocompatible Grade 23 titanium alloy (Ti6Al4V), which features a dedicated and easily positioned hard cover made of the same titanium alloy, allowing safe closure of the wound to avoid infections or damage of the surgery site. The implant fixes the mouse head firmly and stably to the microscope stage via an implant holder, minimizing breathing artifacts and allowing longitudinal imaging of the same areas over time. Some examples are provided of multi-color images depicting cells and structures from the BM microenvironment (SHG+ bone surface; nestin-GFP+ mesenchymal cells; cdh5-DSRED+, cdh5-GFP+, or pdgfb-GFP+ endothelial cells) and the malignant hematopoietic compartment (tdTOMATO+ AML cells), as well as fluorescent contrast agents depicting the lumen of the vessels (dextran-TRITC). Vascular parameters measured over hours or days, including vessel length, straightness, and diameter, as well as permeability in different vascular regions, can provide important information on tissue behavior and health.

<table><tbody><tr><td>Amira</td><td>Thermo ScientificTM</td><td></td><td></td></tr><tr><td>Anesthesia</td><td></td><td></td><td>Isoflurane 2% to 3%</td></tr><tr><td>Anesthesic mask (animal detection)</td><td>Minerve</td><td></td><td></td></tr><tr><td>Anesthesic unit</td><td>Minerve</td><td></td><td></td></tr><tr><td>B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze</td><td>Jackson laboratories</td><td>MGI:3809523</td><td></td></tr><tr><td>CalvariaVue</td><td>Ymetry</td><td>https://ymetry.com/site/head-fixation-implants/44-mouse-head-fixation-implant-for-calvaria.html</td><td></td></tr><tr><td>Dental cement</td><td>Kemdent</td><td>SUN527</td><td></td></tr><tr><td>Dextran 500 kDa-Cy5&amp;nbsp;</td><td>Tebu-bio</td><td>DX500-S5-1</td><td>3 mg/mouse</td></tr><tr><td>Dextran 70 kDa-TRITC</td><td>Sigma</td><td>T1162</td><td>3 mg/mouse</td></tr><tr><td>Disinfectant</td><td>MP-Labo</td><td>Dermidine-60ml</td><td></td></tr><tr><td>Electric razor</td><td>Aescular</td><td>Isis</td><td></td></tr><tr><td>Eye gel</td><td>Ocry-gel</td><td>10g</td><td></td></tr><tr><td>Fiji&amp;nbsp;</td><td>https://imagej.net/software/fiji/downloads</td><td>v 17 May 30</td><td></td></tr><tr><td>Fiji plugings to bridge with Imaris</td><td>https://imagej.net/software/fiji/downloads</td><td>Imaris_Bridge96.jar ; ImarisBridgeUtils.jar</td><td></td></tr><tr><td>Heating box&amp;nbsp;</td><td>Datesand</td><td>Thermacage</td><td></td></tr><tr><td>Heating pad for surgery</td><td>Minerve</td><td></td><td></td></tr><tr><td>Imaging heating pad &amp;amp; rectal probe</td><td>F. Haer</td><td></td><td></td></tr><tr><td>Imaris v9.6.0</td><td>Oxford instruments</td><td></td><td></td></tr><tr><td>Intrasite gel</td><td>Chinoxia</td><td>2390766</td><td></td></tr><tr><td>LAS AF Software</td><td>Leica</td><td>LAS X 3.5.7.23225</td><td></td></tr><tr><td>Medication</td><td></td><td></td><td>Buprecare, 0.01 mg/kg</td></tr><tr><td>Objective HCX IRAPO L 25x/0.95 WATER</td><td>Leica</td><td>506374</td><td></td></tr><tr><td>Saline buffer (PBS 1x)</td><td>Sigma</td><td>P4417</td><td>Sterilize by autoclave</td></tr><tr><td>SP8 DIVE FALCON Multiphoton Microscope&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Stereotoxic mask</td><td>Minerve</td><td>1201261</td><td></td></tr><tr><td>Sterilizator beads</td><td>Sigma</td><td>Z742555</td><td></td></tr><tr><td>Surgery tools</td><td>Moria</td><td>4877A; 2183</td><td></td></tr><tr><td>Survival blanket</td><td>SECURIMED</td><td>11006</td><td></td></tr><tr><td>Swabs / Tissues</td><td></td><td></td><td>Sterilize by autoclave</td></tr><tr><td>Syringe 1 mL 26 G</td><td>BD Plastipak</td><td>305501</td><td></td></tr><tr><td>Temperature controller</td><td>F. Haer</td><td>40-90-5D-02</td><td></td></tr><tr><td>Tg(Nes-EGFP)33Enik mice&amp;nbsp;</td><td>Jackson laboratories</td><td>MGI:5523870</td><td></td></tr><tr><td>Tg(Pdgfb-icre/ERT2)1Frut mice</td><td>Jackson laboratories</td><td>MGI:3793852</td><td></td></tr><tr><td>Tg(Cdh5-cre/ERT2)1Rha mice</td><td>Jackson laboratories</td><td>MGI:3848982</td><td></td></tr><tr><td>Ultrasound gel&amp;nbsp;</td><td>Parker laboratories</td><td>Aquasonic 100</td><td></td></tr></tbody></table>

intravital longitudinal imaging of vascular dynamics in the calvarial bone marrow

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the context of tissue regeneration, tumor development, and therapeutic responses. Particularly, the calvarial bone marrow is a highly dynamic tissue, where the hematopoietic fate is interconnected with the surrounding microenvironment, with specialized vessels responding to normal and pathologic hematopoiesis. Traditional imaging of fixed tissues offers static information, often limiting a comprehensive understanding of these processes. 
The integration of transgenic animals expressing cell-specific markers, live cell tracers, advancements in imaging equipment, and the use of specialized chambers has elevated intravital microscopy to a pivotal tool for gaining insights into dynamic biological phenomena. One application of intravital imaging is the investigation of tumor vessel behavior and therapeutic effects. A newly designed 3D-printed titanium head fixation implant can be stably connected to the mouse skull and is suitable for longitudinal imaging during multiple sessions. 
The proposed protocol allows for the spatial and temporal examination of vascular dynamics in the calvarial bone marrow, including visualization and quantification of vascular heterogeneity, interaction with stromal and hematopoietic cells, and measurement of vascular functional parameters. Additionally, the technique enables the visualization of established vascular beds and the monitoring of therapeutic effects, stem cell mobilization, and the localization of chemotherapeutic compounds over time using two-photon microscopy. Overall, this intravital longitudinal imaging protocol provides a comprehensive platform for investigating both tumor vessel behavior and hematopoietic cell dynamics, offering valuable insights into the intricate processes governing these biological phenomena.

In Figure 1 and Figure 2, the CAD model is shown of a titanium head fixation implant positioned on a scanned mouse skull, which is designed to follow the anatomical structure of the skull and provide a lightweight and biocompatible device able to hold firmly to the microscope stage ensuring cellular level stability. By following this step-by-step protocol, the implant is stably attached to the mouse skull and can be firmly secured to the microscope holder by its dovetail, allowing a flat imaging area for liquid retention and intravital observation over time. It can be closed with a cover to minimize any damage or infection of the wound, allowing repeated imaging of the same tissue area over weeks. Once awake, the mouse wearing a head implant can freely walk around, feed, and have a regular routine.
Figure 3 shows a tile scan view of the calvarial BM vasculature made of heterogeneous capillaries, including arterioles, transition capillaries, and sinusoids. Vessels are embedded into a complex tissue microenvironment in close contact with the bone surface and perivascular mesenchymal cells. During leukemia development, single isolated leukemic cells can be detected within the BM microenvironment in close proximity with vessels, and their engraftment increases over time, filling up the calvaria at late stages of the disease.
Figure 4 shows how images obtained with this protocol can provide quantitative data, which can be analyzed with statistical methods. We show how to segment vessels with the IMARIS filament tool and measure the length and diameter of vascular fragments, as well as their straightness. Correlation of these parameters can also be evaluated.
Figure 5 shows longitudinal imaging acquisition of two different positions of the calvarial BM during AML progression at days 4, 7, and 10, with day 10 being associated with a ~50% engraftment of the BM with leukemic cells, as measured via flow cytometry (not shown). We can observe an important remodeling of the size of preexisting vessels, as well as formation of new vessels in specific areas associated with local bone loss.
Finally, in Figure 6, we show how vascular permeability can be measured as a dynamic parameter with time lapse imaging showing the ability of different vascular barriers to retain a fluorescent dye over time.
<img alt="Biomechanical armature and microscope setup showing stress analysis, components, and assembly diagram." class="xfigimg" src="/files/ftp_upload/67332/67332fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Design and production of a titanium-based biocompatible head holder. (A) Parts of the implant in situ: 1 observation ring, 2 cementing feature, 3 stabilizing anchor, 4 tail, 5 dovetail, 6 threaded hole, 7 Bregma. (B) Connection of the head implant to the holder: 8 fixation body, 9 clamp, 10 eccentric lever, 11 structure, 12 microscope objective. (C) Deformation of implant against load by FEM simulation where maximum displacement is 0.23 µm against 0.04 N force. (D) Protecting cover and its screw. <a href="https://www.jove.com/files/ftp_upload/67332/67332fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="mouse brain imaging setup diagram with attachment showing imaging area, experiment study" class="xfigimg" src="/files/ftp_upload/67332/67332fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Preparation of the mouse for intravital imaging. (A) View of the head implant and the imaging area surgically exposed before imaging. (B) Head implant firmly attached to the mouse skull. (C) Mouse awake in the recovery cage with the closed cover on the head implant. <a href="https://www.jove.com/files/ftp_upload/67332/67332fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="VE-CAD immunofluorescence in bone vasculature, microscopy image, cellular structure analysis." class="xfigimg" src="/files/ftp_upload/67332/67332fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Intravital imaging of calvaria vasculature. (A) z-projection of tile scan view of the calvaria vasculature labeled by cdh5-DSRED. (B) Zoom into depicted areas to describe different types of vessels depicted by arrows, arterioles by red arrows, transition capillaries by orange arrows, sinusoids by yellow arrows. (i) and (ii) z-projections of X µm tissue; (iii) single slice. (C) Single slices of several fields of view of BM vessels, showing the bone surface (SHG), the perivascular cells (nes-GFP+) and the vascular lumen (dextran-TRITC). (D) Vascular niche associated with AML progression. Representative slices of early (top) and late (bottom) time points of AML development. MLL-AF9 leukemia is labeled with tdTOMATO (red arrows), while vessels are labeled with pdgfb-GFP (green arrows), bone surface with SHG, and macrophages in yellow (autofluorescence, yellow asterisk). Scale bars = 200 µm (A), 40 µm (B,D-lower panel), 50 µm (C). Abbreviations: BM = bone marrow; GFP = green fluorescent protein; AML = acute myeloid leukemia; EC= Endothelial cells. <a href="https://www.jove.com/files/ftp_upload/67332/67332fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microvasculature analysis; vessel diameter, length, straightness; microscopy and scatter plot data." class="xfigimg" src="/files/ftp_upload/67332/67332fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Vascular parameter quantification. (A) Measurement of vascular parameters via IMARIS filament tool in a representative z-projection of bone marrow vessels labeled with TRITC-dextran. Line and cone representations are shown. (B) Quantification of vessel parameters in the image shown in A. (C) Correlation between vascular parameters showing an opposite correlation between vessel straightness and length (negative, Spearman r = -3523; p &lt; 0.0001; R2 = 0.2102) vs diameter (positive; Spearman r = 0.4110; p &lt; 0.0001; R2 = 0.1299). Scale bars = 100 µm. <a href="https://www.jove.com/files/ftp_upload/67332/67332fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy results of VE-cad+EC in bone, time-lapse z-stack/slice analysis for Day 4, 7, 10." class="xfigimg" src="/files/ftp_upload/67332/67332fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Longitudinal imaging of two different positions of the calvaria BM over AML development. Endothelial cells lining vessels are labeled with cdh5-GFP, bone surface with SHG, and macrophages in yellow (autofluorescence). Remodeling of preexisting vessels (red arrows) and formation of new vessels (yellow arrows) are shown. Scale bars = 100 µm. <a href="https://www.jove.com/files/ftp_upload/67332/67332fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Vascular leaky vessels analysis with microscopy images, segmentation diagram, and leaky graph results." class="xfigimg" src="/files/ftp_upload/67332/67332fig06.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Vascular permeability. (A) Schematics of vascular permeability measurement via IMARIS surface tool. (B) Z-projection of the same area imaged longitudinally over 1 h. (C) Quantification of vascular permeability within areas as depicted in A. <a href="https://www.jove.com/files/ftp_upload/67332/67332fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Prepared skull. In the median plane, create an axis tangential to the calvaria, then save the prepared skull. <a href="https://www.jove.com/files/ftp_upload/67332/Sup_fig_1_JoVE67332R1_screenfile5_prepared_skull.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Plans 2 mm spaced. How to create a set of equally spaced planes (2 mm spacing) across the skull. <a href="https://www.jove.com/files/ftp_upload/67332/Sup_fig_2_JoVE67332R1_screenfile4_plans_2mm_spaced.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure S3: Observable surface contour. Create a sketch in the calvarial plane and create a pear-shaped spline from AP +6.5 to -2, 6 mm width at AP 0.0. <a href="https://www.jove.com/files/ftp_upload/67332/Sup_fig_3_JoVE67332R1_screenfile3_Observable_Surface_Contour.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure S4: Observation vindow. Create a sketch in the calvarial plane and draw a 0.5 mm-thick C shape connecting to the Observation window. <a href="https://www.jove.com/files/ftp_upload/67332/Sup_fig_4_JoVE67332R1_screenfile1_1.4.1.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure S5: Click new study. Navigate to the Simulation tab (if visible) or go to Simulation | Study. In the Study dialog box, choose Static as the study type. Click OK to create the new study. <a href="https://www.jove.com/files/ftp_upload/67332/Sup_fig_5_JoVE67332R1_screenfile2_clic_new_study.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure S6: Right click to create mesh. Refine the mesh in areas where high stress or deformation is expected. <a href="https://www.jove.com/files/ftp_upload/67332/Sup_fig_6_JoVE67332R1_screenfile6_right_clic_create_mesh.jpg" target="_blank">Please click here to download this File.</a>

Intravital microscopy allows the study of dynamic biological processes such as tissue regeneration and tumor development. The calvarial bone marrow, a highly dynamic tissue, offers insights into hematopoiesis and vascular function. Using a biocompatible 3D-printed head fixation implant allows for repetitive longitudinal imaging, enhancing our understanding of tissue dynamics and the tumor microenvironment.

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the ...

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Research

JoVE Journal

Biology

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Tracking Mouse Bone Marrow Monocytes In Vivo

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer's disease.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Explore intravital microscopy for vascular dynamics in calvarial bone marrow using two-photon imaging and transgenic mouse models. This longitudinal method reveals blood vessel responses to hematopoiesis and leukemia progression.

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

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