We thank Rob Eifert for his assistance in designing and optimizing the laser-cut stainless steel grids. This work was supported by the CSHL Cancer Center (P30-CA045508) and funds for M.E. from the National Institutes of Health (NIH) (1R01CA2374135 and 5P01CA013106-49); CSHL and Northwell Health; the Thompson Family Foundation; Swim Across America; and a grant from the Simons Foundation to CSHL. M.S. was supported by the National Institute of General Medical Sciences Medical Scientist Training Program Training Award (T32-GM008444) and the National Cancer Institute of the NIH under award number 1F30CA253993-01. L.M. is supported by a James S. McDonnell Foundation Postdoctoral Fellowship. J.M.A. is the recipient of a Cancer Research Institute/Irvington Postdoctoral Fellowship (CRI Award #3435). D.A.T. is supported by the Lustgarten Foundation Dedicated Laboratory for Pancreatic Cancer Research and the Thompson Family Foundation. Cartoons were created with Biorender.com.

All procedures described were performed in accordance with the Cold Spring Harbor Laboratory Surgical Guidelines and had been approved by the Institutional Animal Care and Use Committee at Cold Spring Harbor Laboratory.
1. Casting the silicone window 
<ol>
	<li>Prepare the silicone polymer (PDMS) by mixing the base elastomer and curing agent in a 10:1 (v/v) ratio.</li>
	<li>Cast a window by depositing a small quantity of PDMS on a sterile, smooth surface and adjust the volume-to-area ratio to the desired thickness. 
	NOTE: Using 200 mg of polymer solution for a 22 mm diameter circle on the lid of a 24 well-plate lid results in a good compromise between window sturdiness and optical clarity.</li>
	<li>Optional: To provide landmarks for repeated imaging of the same tissue regions, lightly press a stainless-steel grid into the silicone after the PDMS is on the desired casting surface.</li>
	<li>To remove air bubbles, place the coated surface in a vacuum desiccator for 45 min to degas the polymer.</li>
	<li>Cure the silicone windows in an oven at 80 &#176;C for 45 min.</li>
	<li>Score the polymer at the edges of the mold and gently peel the cured windows from the surface used for casting with forceps.</li>
	<li>Before surgery, sterilize the windows by autoclaving.</li>
</ol>
2. Preparing the mouse for insertion of the silicone window 
<ol>
	<li>Anesthetize the mouse in an induction chamber using 4% (v/v) isoflurane. Move the mouse to a warming pad on the surgical table, place the mouse in the anesthesia nose mask, and lower the isoflurane concentration to 2% for maintenance throughout the surgery.</li>
	<li>Check the depth of anesthesia by pinching the toes of the hind limbs. Do not proceed until the mouse is inactive and does not show toe pinch reflex response.</li>
	<li>Apply ophthalmic lubricant to the eyes to keep them from drying and preventing trauma.</li>
	<li>Administer pre-emptive analgesia (buprenorphine 0.05 mg/kg) subcutaneously.</li>
	<li>Shave the surgical site and completely remove the hair with a hair removal cream.</li>
	<li>Apply povidone-iodine solution (10% v/v) and ethanol (70% v/v) consecutively 3x to prevent the surgical site infection.</li>
</ol>
3. Inserting a ventral window for imaging in the liver; adaptable for other abdominal organs (Figure 1) 
<ol>
	<li>Place the mouse in the supine position.</li>
	<li>Make a 10 mm incision starting 3 mm down from the xiphoid process using sterile scissors and forceps.</li>
	<li>Remove a 1&#8211;1.5 cm2 section of skin along the midline.</li>
	<li>Use a second pair of sterile scissors and forceps to remove a section of the peritoneum slightly smaller than the section of skin.</li>
	<li>Optional: To visualize a larger portion of the liver, use sterile cotton swabs moistened with sterile saline to push the liver down from the diaphragm revealing the falciform ligament connecting the liver to the diaphragm. Sever the ligament taking care not to cut the inferior vena cava.</li>
	<li>Withdraw surgical adhesive with a 31 G syringe, apply a small quantity of it on the surface of the liver around the edges of the area to be imaged. This adhesive creates a circular seal, leaving the center area intact for imaging. 
	CAUTION: Limit the adhesive to small droplets forming a circular pattern around the imaging area. Tissue immediately in contact with the adhesive cannot be imaged. It is not necessary to dry the tissue before applying the adhesive. 
	NOTE: Any cyanoacrylate-based adhesives can be used successfully for this procedure. Surgical adhesive ensures sterility. For terminal procedures, all-purpose super glues yield good results.</li>
	<li>Position the window using forceps and hold it firmly against the liver until the adhesive has dried (~2 min).</li>
	<li>Fold the edges of the window under the peritoneum.</li>
	<li>With a syringe, deposit a small quantity of surgical adhesive on the edges of the window that are now under the peritoneum. Push down on the peritoneum with forceps to secure it to the window.</li>
	<li>Similarly, deposit a small quantity of surgical adhesive onto the peritoneum before pushing&#160;down on the skin with forceps to secure it to the peritoneum. 
	NOTE: If performed correctly, a circular area of liver tissue should now be visible through the window.</li>
	<li>Apply glue around the edges of the window to create a rim that will help prevent the skin from growing back over the window. 
	CAUTION: If the procedure is carried out using sterile surgical techniques and the window is sterilized before insertion, sealing the wound with surgical adhesive is sufficient to avoid infections. However, failure to follow proper aseptic techniques during surgical insertion or imperfect closure can lead to overt or subclinical infection and drying out of the tissue.</li>
</ol>
4. Inserting a lateral window for imaging in the liver; compatible with the concurrent injection of cancer cells in the portal vein (Figure 2)
<ol>
	<li>Place the mouse on the left lateral decubitus position</li>
	<li>Make a 10 mm incision on the right flank 3 mm below the costal arch using sterile scissors and forceps.</li>
	<li>Remove a 1 cm2 section of skin.</li>
	<li>Use a second pair of sterile scissors and forceps to remove a section of the peritoneum slightly smaller than the section of skin.</li>
	<li>Withdraw surgical adhesive with a 31 G syringe, apply a small quantity of it on the surface of the liver around the edges of the area to be imaged. 
	CAUTION: Limit the adhesive to small droplets forming a circular pattern around the imaging area. Tissue immediately in contact with the adhesive cannot be imaged. 
	NOTE: Any cyanoacrylate-based adhesives can be used successfully for this procedure. Surgical adhesive ensures sterility. For terminal procedures, all-purpose super glues yield good results.</li>
	<li>Position the window using forceps and hold it firmly against the liver until the adhesive has dried (~2 min).</li>
	<li>Fold the edges of the window under the peritoneum.</li>
	<li>With a syringe, deposit a small quantity of surgical adhesive on the edges of the window that are now under the peritoneum. Push down on the peritoneum with forceps to secure it to the window.</li>
	<li>Similarly, deposit a small quantity of surgical adhesive onto the peritoneum before&#160;pushing&#160;down on the skin with forceps to secure it to the peritoneum. 
	NOTE: If performed correctly, a circular area of liver tissue should now be visible through the window.</li>
	<li>Apply glue around the edges of the window to create a rim that will help prevent the skin from growing back over the window.</li>
	<li>If a portal vein injection is required:
	<ol>
		<li>Place the mouse in the supine position.</li>
		<li>Make a 10 mm incision starting down from the xiphoid process in the skin.</li>
		<li>Using a second pair of sterile scissors and forceps, make a 10 mm incision in the peritoneum.</li>
		<li>Dip two cotton swabs in sterile saline.</li>
		<li>Use the cotton swabs to gently pull the intestine onto sterile gauze moistened with sterile saline, taking care not to twist or otherwise upset the orientation.</li>
		<li>Keep displacing the intestine from the abdominal cavity until the portal vein is visible on the right side of the abdomen.</li>
		<li>Insert a 31&#8211;33 G needle 5&#8211;7 mm caudal to the point of entry of the portal vein into the liver, making sure to proceed within the blood vessel and not through it.</li>
		<li>Slowly inject the cancer cells (resuspended in 100 &#956;L of sterile PBS). 
		NOTE: For this experiment amurine pancreatic cancer cell line (e.g., KPC-BL/6-1199) can be cultured in complete DMEM media and 1 x 105 cells injected in 100 &#956;L of sterile PBS.</li>
		<li>To prevent bleeding, immediately after withdrawing the needle, apply gentle pressure on the injection site with a small piece of surgical sponge for 1&#8211;2 min.</li>
		<li>After the pressure is released, monitor the site for 1&#8211;2 min to ensure hemostasis.</li>
		<li>Using moistened cotton swabs, return the intestine into the abdominal cavity following the physiological orientation of the organ.</li>
		<li>Using sterile forceps, insert the window immediately under the peritoneum, on the left side of the abdominal cavity of the mouse.</li>
		<li>Suture with 4-0 silk sutures and staple the midline incision with 7 mm stainless steel wound clips.</li>
		<li>Move the mouse to the left lateral decubitus position</li>
		<li>Make a 10 mm incision on the right flank 3 mm under the costal arch through the skin using sterile scissors and forceps.</li>
		<li>Remove a 1 cm2 section of skin around the incision.</li>
		<li>Use a second pair of sterile scissors and forceps to remove a section of the peritoneum slightly smaller than the section of skin.</li>
		<li>Pull the window into place over the liver before gluing it in place, as described under steps 4.8&#8211;4.10.</li>
	</ol>
	</li>
</ol>
5. Inserting the window for imaging in the pancreas (Figure 3) 
<ol>
	<li>Place the mouse on the right lateral decubitus position.</li>
	<li>Make a 10 mm incision on the left flank 3 mm below the costal arch using sterile scissors and forceps.</li>
	<li>Remove a 1 cm2 section of skin around the incision.</li>
	<li>Use a second pair of sterile scissors and forceps to remove a section of the peritoneum slightly smaller than the section of skin.</li>
	<li>Using sterile cotton swabs soaked with sterile saline, gently pull on the spleen to visualize the pancreas. Proceed to reposition the pancreas with the moistened cotton swabs to make more surface area visible through the incision. 
	NOTE: At this point, pancreatic cancer cells can be orthotopically injected into the pancreas if it is a part of the experimental design.</li>
	<li>Withdraw surgical adhesive with a 31 G syringe, apply a small quantity of it on the surface of the pancreas around the edges of the area to be imaged. 
	CAUTION: Limit the adhesive to small droplets forming a circular pattern around the imaging area. Tissue immediately in contact with the adhesive cannot be imaged.</li>
	<li>Position the window using forceps and hold it firmly against the pancreas until the adhesive has dried (~2 min).</li>
	<li>Fold the edges of the window under the peritoneum.</li>
	<li>With a syringe, deposit a small quantity of surgical adhesive on the edges of the window that are now under the peritoneum. Push down on the peritoneum with forceps to secure it to the window.</li>
	<li>Similarly, deposit a small quantity of surgical adhesive onto the peritoneum before pushing down on the skin with forceps to secure it to the peritoneum. 
	NOTE: If performed correctly, a circular area of pancreas tissue should now be visible through the window.</li>
	<li>Apply glue around the edges of the window to create a rim that will help prevent the skin from growing back over the window.</li>
</ol>
6. Inserting the window for imaging in the mammary gland (Figure 4)
<ol>
	<li>Place the mouse on the supine position.</li>
	<li>Make a 10 mm incision medial to one of the inguinal nipples using sterile scissors and forceps.</li>
	<li>Remove a 0.5 cm2 section of skin above the mammary gland.</li>
	<li>Use a second pair of sterile scissors to carefully separate the mammary gland from the skin by spreading the scissors between the two surfaces to disrupt adherence. 
	NOTE: To facilitate imaging of the inguinal mammary gland area, be careful to dissect a portion of skin above the mammary gland but superior to the hind leg, so the window does not limit the mobility of the mouse.</li>
	<li>Withdraw surgical adhesive with a 31 G syringe, apply a small quantity of it on the surface of the mammary gland around the edges of the area to be imaged. 
	CAUTION: Limit the adhesive to small droplets forming a circular pattern around the imaging area. Tissue immediately in contact with the adhesive cannot be imaged.</li>
	<li>Position the window using forceps and hold it firmly against the mammary gland until the adhesive has dried (~2 min). 
	NOTE: A circular area of mammary gland tissue should now be visible through the window if performed correctly.</li>
	<li>Fold the edges of the window under the skin.</li>
	<li>With a syringe, deposit a small quantity of surgical adhesive on the edges of the window that are now under the skin. Push down on the skin with forceps to secure the skin to the window.</li>
	<li>Apply glue around the edges of the window to create a rim that will help prevent the skin from growing back over the window.</li>
</ol>
7. Post-surgical recovery 
<ol>
	<li>Place the mouse in a clean recovery cage with ample nesting material, ensuring that part of the cage is resting on a heating pad.</li>
	<li>Monitor the mouse continuously until it is conscious and mobile.</li>
	<li>If needed, provide an additional dose of analgesic 12 h after the pre-emptive dose. 
	NOTE: Additional analgesia is generally unnecessary, but consult with a veterinarian if the mouse shows signs of distress (such as hunched back, unkempt fur, or lost interest in food). Monitor the mouse daily for the first 3 days after surgery for signs of infection or other adverse effects. Less than 2% of mice require veterinary attention or euthanasia, typically due to partial detachment of the window. It is important to note that for male mice with ventral liver imaging windows, fighting between mice can result in detachment of windows. This is avoided by housing the mice separately.</li>
</ol>
8. Imaging through the window
<ol>
	<li>Anesthetize the mouse in an induction chamber using 4% (v/v) isoflurane.</li>
	<li>Apply ophthalmic lubricant to the eyes to keep them from drying and preventing trauma.</li>
	<li>Move the mouse from the induction chamber to the microscope stage. Place the mouse in the anesthesia nose mask, and lower the isoflurane concentration to approximately 1&#8211;1.5% for maintenance anesthesia throughout the imaging procedure.</li>
	<li>Place a pressure pad sensor below the mouse to monitor breath rate and fix the mouse to the stage using soft surgical tape.</li>
	<li>Insert a rectal thermometer to monitor body temperature throughout the imaging session.</li>
	<li>Turn on the heated pad, monitoring the mouse closely to ensure that body temperature does not exceed 37 &#176;C. &#160;</li>
	<li>Utilize software to monitor the breath rate of the mouse. The optimal rate is ~1 breath/second. Adjust anesthesia as required.</li>
	<li>Before each imaging session, clean the window from any residual lens immersion medium and debris by gently wiping it with a cotton swab dipped in 70% ethanol (v/v).</li>
	<li>When utilizing a water immersion lens, apply ultrasound gel to the window, avoiding bubbles. 
	NOTE: Distilled water can also be used but might require reapplication during imaging.</li>
	<li>To find the optimal depth for imaging, first, locate the grid and place it in focus.</li>
	<li>Determine the approximate tissue imaging depth by setting the bottom of the grid to 0. This information is necessary to locate the corresponding z plane in subsequent imaging sessions.</li>
	<li>To identify the same location over multiple imaging sessions, utilize the squares on the grid as a reference point. During imaging, note the orientation of the grid and location within the grid of each field of view imaged (e.g., 2nd row from the top, 4th square from the left).</li>
	<li>During successive imaging sessions, use the grid to navigate back to specific grid areas - and thus imaging fields.</li>
	<li>Following each imaging session, remove any residual lens immersion medium and debris by gently wiping the window with a cotton swab dipped in 70% ethanol (v/v).</li>
</ol>

<ol>
	<li>Dondossola, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+osteolytic+progression+and+therapy+response+of+cancer+lesions+in+the+bone.">Intravital microscopy of osteolytic progression and therapy response of cancer lesions in the bone.</a> Science Translational Medicine. 10 (452), (2018).</li><li>Haeger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cancer+invasion+forms+an+integrin-dependent+radioresistant+niche.">Collective cancer invasion forms an integrin-dependent radioresistant niche.</a> Journal of Experimental Medicine. 217 (1), 20181184 (2020).</li><li>Harper, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+early+dissemination+and+metastasis+in+Her2(+)+mammary+cancer.">Mechanism of early dissemination and metastasis in Her2(+) mammary cancer.</a> Nature. 540 (7634), 588-592 (2016).</li><li>Eickhoff, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+anti-viral+immunity+requires+multiple+distinct+T+cell-dendritic+cell+interactions.">Robust anti-viral immunity requires multiple distinct T cell-dendritic cell interactions.</a> Cell. 162 (6), 1322-1337 (2015).</li><li>Engelhardt, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginating+dendritic+cells+of+the+tumor+microenvironment+cross-present+tumor+antigens+and+stably+engage+tumor-specific+T+cells.">Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells.</a> Cancer Cell. 21 (3), 402-417 (2012).</li><li>Sammicheli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+monocytes+hinder+antiviral+B+cell+responses.">Inflammatory monocytes hinder antiviral B cell responses.</a> Science Immunology. 1 (4), (2016).</li><li>Entenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+permanent+window+for+the+murine+lung+enables+high-resolution+imaging+of+cancer+metastasis.">A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.</a> Nature Methods. 15 (1), 73-80 (2018).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mice+for+long-term+intravital+imaging+of+the+mammary+gland.">Preparation of mice for long-term intravital imaging of the mammary gland.</a> Cold Spring Harbor Protocols. 2011 (2), 5562 (2011).</li><li>Ritsma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implantation+of+an+abdominal+imaging+window+for+intravital+microscopy.">Surgical implantation of an abdominal imaging window for intravital microscopy.</a> Nature Protocols. 8 (3), 583-594 (2013).</li><li>Pittet, M. J., Garris, C. S., Arlauckas, S. P., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+the+wild+lives+of+immune+cells.">Recording the wild lives of immune cells.</a> Science Immunology. 3 (27), (2018).</li><li>Alieva, M., Ritsma, L., Giedt, R. J., Weissleder, R., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+windows+for+long-term+intravital+imaging:+General+overview+and+technical+insights.">Imaging windows for long-term intravital imaging: General overview and technical insights.</a> Intravital. 3 (2), 29917 (2014).</li><li>Heo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+soft,+transparent,+freely+accessible+cranial+window+for+chronic+imaging+and+electrophysiology.">A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology.</a> Scientific Reports. 6, 27818 (2016).</li><li>Anderson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fracture Mechanics: Fundamental and Applications. , (2005).</li><li>Nakasone, E. S., Askautrud, H. A., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+drug+responses+in+the+tumor+microenvironment+in+mouse+models+of+breast+cancer.">Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer.</a> Journal of Visualized Experiments: JoVE. (73), e50088 (2013).</li><li>Sasmono, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Cole, R. W., Jinadasa, T., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+interpreting+point+spread+functions+to+determine+confocal+microscope+resolution+and+ensure+quality+control.">Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control.</a> Nature Protocols. 6 (12), 1929-1941 (2011).</li><li>Sobolik, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+novel+murine+mammary+imaging+windows+to+examine+wound+healing+effects+on+leukocyte+trafficking+in+mammary+tumors+with+intravital+imaging.">Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.</a> Intravital. 5 (1), 1125562 (2016).</li><li>Jacquemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+high-resolution+imaging+through+a+flexible+intravital+imaging+window.">Longitudinal high-resolution imaging through a flexible intravital imaging window.</a> Science Advances. 7 (25), (2021).</li></ol>

M.E. is a member of the research advisory board for brensocatib for Insmed, Inc.; a member of the scientific advisory board for Vividion Therapeutics, Inc.; a consultant for Protalix, Inc.; and holds shares in Agios Pharmaceuticals, Inc. D.A.T. is co-founder of Mestag Therapeutics, and is on the scientific advisory board and holds shares in Mestag Therapeutics, Leap Therapeutics, Surface Oncology, and Cygnal Therapeutics. The other authors declare no competing interests.

Intravital imaging windows are important tools for directly visualizing physiological and pathological processes at cellular resolution as they unfold over time. The novel procedure described for casting and inserting flexible, silicone imaging windows in mice overcomes some of the most common issues with currently used imaging windows (exudate, breaking, and interference with normal mobility), provides additional safety for the mouse, and increases the accessibility of this technique.
The mos...

Intravital microscopy (IVM), the imaging of tissues in anesthetized animals, offers insights into the dynamics of physiological and pathological events at cellular resolution in intact tissues. The applications of this technique vary widely, but IVM has been instrumental in the cancer biology field to help elucidate how cancer cells invade tissues and metastasize, interact with the surrounding microenvironment, and respond to drugs1,2,3. In addition, IVM has been key to advancing the understanding of the complex mechanisms governing immune responses by providing insights complementary to ex vivo profiling approaches (e.g., flow cytometry). For instance, intravital imaging experiments have revealed details about immune functions as they relate to cell migration and cell-cell contact&#160;and have offered a platform to quantitate spatiotemporal dynamics in response to injury or infection4,5,6,7. Many of these processes can also be studied through histological staining, but only IVM allows the tracking of dynamic changes. In fact, whereas a histological section offers a snapshot of the tissue at a given time, intravital imaging can track intercellular and subcellular events within the same tissue over time. In particular, progress in fluorescence labeling and the development of molecular reporters have allowed molecular events to be correlated with cellular behaviors, such as proliferation, death, motility, and interaction with other cells or the extracellular matrix. Most IVM techniques are based on fluorescence microscopy, which due to light scattering, makes imaging deeper tissues challenging. The tissue of interest, therefore, often needs to be surgically exposed with an often invasive and terminal procedure. Thus, depending on the organ site, the tissue can be imaged continuously for a period varying from a few to 40 h8. Alternatively, the surgical insertion of a permanent imaging window permits the imaging of the same tissue sequentially over a period of days to weeks7,9.
The development of new imaging windows has been highlighted as a technological need to further improve intravital imaging approaches10. The prototypical intravital imaging window is a metal ring containing a glass coverslip secured to the skin with sutures11. Interference with free movement, the accumulation of exudate, and damage to the glass coverslip are common problems seen with using such windows. Moreover, the prototypical window requires specialized production, and the surgical procedure can require extensive training. To address these issues, polydimethylsiloxane (PDMS), a silicone polymer, which has previously been used in cranial windows for long-term imaging in the brain12, was adapted for use in abdominal organ and mammary gland imaging. Here, a detailed method for generating PDMS-based silicone windows is presented, including how to cast the window around a stainless-steel grid to provide landmarks for repeated imaging of the same tissue regions. Furthermore, a simple, stitch-free surgical procedure for inserting the window over abdominal organs or the mammary gland is described. This new approach overcomes some of the most common issues with currently used imaging windows and increases the accessibility of longitudinal intravital imaging.

<table><tbody><tr><td>3M Medipore Soft Cloth Surgical Tape</td><td>3M</td><td>70200770819</td><td></td></tr><tr><td>Silk suture 4-0 PERMA HAND BLACK 1 x 18&quot; RB-2</td><td>Ethicon&amp;nbsp;</td><td>N267H</td><td></td></tr><tr><td>ACTB-ECFP mice</td><td>Jackson Laboratory</td><td>22974</td><td></td></tr><tr><td>AEC Substrate Kit, Peroxidase (HRP), (3-amino-9-ethylcarbazole)</td><td>Vector Laboratories&amp;nbsp;</td><td>SK-4200</td><td></td></tr><tr><td>Alcohol swabs</td><td>BD&amp;nbsp;</td><td>326895</td><td></td></tr><tr><td>Anesthesia system</td><td>Molecular Imaging Products Co.</td><td></td><td></td></tr><tr><td>Acq&lt;em&gt;knowledge&lt;/em&gt; software and sensors&amp;nbsp;</td><td>BIOPAC</td><td>ACK100W, ACK100M, TSD110</td><td></td></tr><tr><td>Betadine spray&amp;nbsp;</td><td>LORIS</td><td>109-08</td><td></td></tr><tr><td>c-&lt;em&gt;fms&lt;/em&gt;-EGFP (MacGreen) mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Jackson Laboratory</td><td>664</td><td></td></tr><tr><td>CD45 Monoclonal Antibody (30-F11)</td><td>Invitrogen</td><td>14-0451-82</td><td></td></tr><tr><td>CD68 Antibody</td><td>Abcam</td><td>ab125212</td><td></td></tr><tr><td>Curity gauze sponges&amp;nbsp;</td><td>Covidien</td><td></td><td></td></tr><tr><td>Donkey Anti-Goat IgG H&amp;amp;L (HRP)&amp;nbsp;</td><td>Abcam</td><td>ab6885</td><td></td></tr><tr><td>Donkey Anti-Rabbit IgG H&amp;amp;L (HRP)&amp;nbsp;</td><td>Abcam</td><td>ab97064</td><td></td></tr><tr><td>Donkey Anti-Rat IgG H&amp;amp;L (HRP)&amp;nbsp;</td><td>Abcam</td><td>ab102182</td><td></td></tr><tr><td>Dow SYLGARD 184 Silicone Encapsulant Clear</td><td>Electron Microscopy Sciences</td><td>24236-10</td><td>Two-part, 10:1 mixing ratio</td></tr><tr><td>Round Cover Glass, 8mm Diameter, #1.5 Thickness&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>72296-08</td><td></td></tr><tr><td>Ender-3 Pro 3D printer</td><td>Shenzhen Creality 3D Technology Co., LTD</td><td></td><td></td></tr><tr><td>Far Infrared Heated blanket</td><td>Kent Scientific</td><td>RT-0520</td><td></td></tr><tr><td>Fc Receptor Blocker</td><td>Innovex Biosciences</td><td>NB309</td><td></td></tr><tr><td>Fiji imaging processing package</td><td></td><td></td><td>https://imagej.net/software/fiji/</td></tr><tr><td>FluoroSpheres carboxylate, 0.04&amp;micro;m, yellow-green (505/515)</td><td>Invitrogen</td><td>F8795</td><td></td></tr><tr><td>Gating system:</td><td>BIOPAC Systems Inc.</td><td></td><td>The components together allow monitoring mouse vitals during imaging and gating image acquisition on mouse respiration. All were acquired from BIOPAC systems.</td></tr><tr><td>Acq&lt;em&gt;knowledge&lt;/em&gt; software&amp;nbsp;</td><td></td><td>ACK100W, ACK100M</td><td></td></tr><tr><td>Diff. Amp. Module, C Series&amp;nbsp;</td><td></td><td>DA100C</td><td></td></tr><tr><td>Dual Gating Sys small animal</td><td></td><td>DTU200&amp;nbsp;</td><td></td></tr><tr><td>MP160 for Windows - Analysis system</td><td></td><td>MP160WSW&amp;nbsp;</td><td></td></tr><tr><td>MouseOx Plus 120V&amp;nbsp;</td><td></td><td>MOX-120V;015000&amp;nbsp;</td><td></td></tr><tr><td>Pressure Pad&amp;nbsp;</td><td></td><td>TSD110&amp;nbsp;</td><td></td></tr><tr><td>Gelfoam</td><td>Pfizer</td><td>9031508</td><td>Absorbable gelatin sponge</td></tr><tr><td>Hardened fine scissors</td><td>Fine Science Tools</td><td>14090-11</td><td>Two pairs; stainless steel, sharp-sharp&lt;br/&gt; tips, straight tip, 26 mm&lt;br/&gt; cutting edge, 11 cm length</td></tr><tr><td>Human/Mouse Myeloperoxidase/MPO Antibody</td><td>R&amp;amp;D Systems</td><td>AF3667</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Turn on approximately 30 min&lt;br/&gt; before use; sterilize tools at &gt;200&lt;br/&gt; &amp;deg;C for 30 s</td></tr><tr><td>Imaris&amp;nbsp;</td><td>Bitplane</td><td>www.bitplane.com</td><td></td></tr><tr><td>Immersion medium Immersol W 2010</td><td>Zeiss</td><td>444969-0000-000&amp;nbsp;</td><td></td></tr><tr><td>Insulin Syringes with BD Ultra-Fine needle 6mm x 31G 1 mL/cc</td><td>BD</td><td>324912</td><td></td></tr><tr><td>Isoflurane (Fluriso)</td><td>VetOne</td><td>502017</td><td></td></tr><tr><td>Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight&amp;reg; 594</td><td>Vector Laboratories&amp;nbsp;</td><td>DL-1177-1</td><td></td></tr><tr><td>&lt;em&gt;LysM&lt;/em&gt;-eGFP mice</td><td>www.mmrrc.org</td><td>012039-MU</td><td></td></tr><tr><td>Micro dissecting forceps</td><td>Roboz</td><td>RS-5135</td><td>Serrated, slight curve, 0.8 mm tip width; 4&quot; length</td></tr><tr><td>Micro dissecting forceps</td><td>Roboz</td><td>RS-5153</td><td>1 x 2 teeth, slight curve, 0.8 mm tip&lt;br/&gt; width, 4&quot; length</td></tr><tr><td>MTS MiniBionix II 808</td><td>MTS Systems</td><td></td><td>Servohydraulic material testing machine</td></tr><tr><td>Neutrophil Elastase 680 FAST probe</td><td>PerkinElmer</td><td>NEV11169</td><td></td></tr><tr><td>Nitrogen</td><td>General Welding Supply Corp.</td><td></td><td></td></tr><tr><td>Oxygen</td><td>General Welding Supply Corp.</td><td></td><td></td></tr><tr><td>Polylactic acid filament</td><td>Hatchbox</td><td></td><td>1.75 mm diameter</td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Invitrogen</td><td>P36970</td><td></td></tr><tr><td>Puralube ophthalmic ointment</td><td>Dechra&amp;nbsp;</td><td>NDC17033-211-38</td><td></td></tr><tr><td>Reflex 7 wound clips</td><td>Roboz Surgical</td><td>RS-9255</td><td></td></tr><tr><td>Stainless steel grid</td><td>Fotofab</td><td></td><td>One grid is 0.200 inches in diameter, with a total of 52 individual grid squares that are 0.016 x 0.016 inches. There is 0.003 inches of space between each square.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Surface Treated SterileTissue Culture Plates</td><td>Fisher Scientific</td><td>FB012929</td><td>Lid used as curing surface for imaging windows</td></tr><tr><td>TriM Scope Multiphoton Microscope&amp;nbsp;</td><td>LaVision BioTec</td><td></td><td>Imaging was done on an upright 2-photon microscope (Trimscope, LaVision BioTec) equipped with two Ti:Sapphire lasers (Mai Tai and InSight, Spectra-Physics) and an optical parametric oscillator. The following Longpass Dichroic Beamsplitters (Chroma) were used to direct the signal towards four photomultipler tubes:&lt;br/&gt; T560LP&lt;br/&gt; T665LPXXR&lt;br/&gt; T495lxpr</td></tr><tr><td>Vetbond</td><td>3M</td><td>70200742529</td><td></td></tr><tr><td>VWR micro cover glass</td><td>VWR</td><td>48404-453</td><td></td></tr></tbody></table>

longitudinal intravital imaging through clear silicone windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

Intravital imaging through imaging windows can be used to observe, track, and quantify a wide array of cellular and molecular events at single-cell resolution over a period of hours to weeks. Ideal features for an imaging window include: a) limited impact on the well-being of the mouse and the physiology of the tissue; b) durability; c) simplicity of insertion; and d) clear landmarks for repeated imaging of the same region. The result is a versatile, inert silicone window that is easily produced and inserted and that can...

Watch this Scientific Journal Video about Longitudinal Intravital Imaging Through Clear Silicone Windows at JoVE.com

Longitudinal Intravital Imaging Through Clear Silicone Windows

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

longitudinal-intravital-imaging-through-clear-silicone-windows

Research

JoVE Journal

Cancer Research

3.5K Views.  Cold Spring Harbor Laboratory. Deze procedure voor het gieten en inbrengen van siliconen ramen verlaagt de technische barrières voor longitudinale intravitale beeldvorming. Ons protocol produceert stevige, gemakkelijk aanpasbare ramen die goed worden verdragen door de dieren. Met behulp van deze techniek hebben we kanker en immuuncellen in de lever, alvleesklier en borstklier in beeld gebracht.Met minimale aanpassingen kon het worden aangepast aan vele anatomische locaties en biologische vragen. Voor degenen die de...

Video: Longitudinal Intravital Imaging Through Clear Silicone Windows

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Medicine

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Neuroscience

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.

This protocol describes the fabrication of an implantable integrated imaging window using 3D laser printing. The window consists of a system of microlenses coupled with micro-scaffolds. The method involves two-photon polymerization (2PP) of the biocompatible photoresist SZ2080 in a continuous sequence, optimizing manufacturing efficiency and alignment between the different components.

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable ...

Bioengineering

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Biology

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It is therefore critical to optimize imaging approaches that facilitate the observation of cell dynamics in situ, where tissue structure and composition remain unperturbed. The epidermis is the body&#39;s outermost barrier, as well as the source of the most prevalent human cancers, namely cutaneous skin carcinomas. The accessibility of skin tissue presents a unique opportunity to monitor epithelial and dermal cell behaviors in intact animals using noninvasive intravital microscopy. Nevertheless, this sophisticated imaging approach has primarily been achieved using upright multiphoton microscopes, which represent a significant barrier for entry for most investigators. This study presents a custom-designed, 3D-printed microscope stage insert suitable for use with inverted confocal microscopes, streamlining the long-term intravital imaging of ear skin in live transgenic mice. We believe this versatile invention, which may be customized to fit the inverted microscope brand and model of choice and adapted to image additional organ systems, will prove invaluable to the greater scientific research community by significantly enhancing the accessibility of intravital microscopy. This technological advancement is critical for bolstering our understanding of live cell dynamics in normal and disease contexts.

Author Spotlight: Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging 

The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...

Implantation of Cranial Imaging Window in Mouse Model: A Surgical Procedure to Implant Glass-based Coverslip for Stable Optical Access to Regions of Murine Brain

Source: Alieva, M. et al. <a href="https://www.jove.com/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.</a>&#160;J. Vis. Exp.&#160; (2019)
This video presents a surgical procedure to implant cranial window in a mouse model for stable optical access to different regions of the brain.

Source: Alieva, M. et al. Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.&#160;J. Vis. Exp.&#160; ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video