We thank Valentina Greco for the K14-mCherry-H2B mice. We are grateful to the Emory University Physics Department Machine Shop for generating the glass coverslip disks. This work was funded by Career Development Award #IK2 BX005370 from the US Department of Veterans Affairs BLRD Service to LS, NIH Awards RF1-AG079269 and R56-AG072473 to MJMR, and I3 Emory SOM/GT Computational and Data Analysis Award to MJMR.

This research was performed in compliance with Emory University and Atlanta Veterans Affairs Medical Center animal care and use guidelines and has been approved by the Institutional Animal Care and Use Committee (IACUC).
1. Installing the live imaging insert on the inverted microscope stage
<ol>
	<li>Construct the insert using .stl files (Supplementary File 1, Supplementary File 2, and Supplementary File 3)&#160;specifying the 3D dimensions and design (see Figure 1 and Figure 2A), a 3D printer, and polylactic acid (PLA).</li>
	<li>Carefully place the insert (Figure 2B,C) into the large microscope stage groove (Figure 2C and&#160;Figure 3A). Use screws to secure the insert via four screw holes located in each corner of the insert (Figure 2B-C). 
	NOTE: The insert is bidirectional and can be rotated 180&#176; depending on the orientation of the microscope&#160;stage, and anesthesia apparatus.</li>
	<li>Slide the heat plate into the insert plug side-down (Figure 2D)&#160;so the unit lays atop the lower insert grooves with the plug port passing underneath the stage (Figure 3B).</li>
	<li>Align the grooved circular opening in the insert with a 40x silicone oil immersion objective (Figure 3C) and apply silicone oil to the center of the objective.
	<ol>
		<li>If imaging over longer time periods (&#62;1 h), apply a generous dollop of oil that will persist at the coverslip/objective interface throughout imaging. Do not allow oil to spill over the edges of the lens.</li>
	</ol>
	</li>
	<li>Use a syringe to apply a small quantity of vacuum grease along the grooved circular opening and lay the coverslip disk atop to seal onto the insert (Figure 3D). 
	NOTE: The coverslip disk is created by removing the walls of a 35 mm x 10 mm glass-bottom cell culture dish (performed by a machine shop).</li>
	<li>Raise the 40x objective until the oil kisses the bottom of the glass coverslip.</li>
</ol>
2. Isoflurane configuration and mouse prep
<ol>
	<li>Position the low-flow electronic vaporizer components (anesthesia chamber, tubing, vaporizer, charcoal canister) to allow the nose cone and attached tubing to reach the insert (Figure 3A). 
	CAUTION: Isoflurane is an inhalation anesthetic and should be handled with care to avoid spills and minimize human exposure.</li>
	<li>Measure the weight of the isoflurane bottle prior to use and log. Attach the cap from the electronic vaporizer to the isoflurane bottle.</li>
	<li>Connect the power cord to the electronic vaporizer. Close the blue nose cone clips and open the white induction chamber clips to allow airflow through the chamber into the charcoal canister. Remove the red ambient air cap from the left side of the machine to allow air flow.</li>
	<li>Allow the system to warm up for 5 min at 200 mL/min (low flow) and 2% isoflurane. 
	NOTE: Although the nose cone setting is selected, the blue nose cone line should remain closed, and the white induction chamber line should remain open.</li>
	<li>Once the system is properly equilibrated, purge the lines to remove remaining isoflurane from the chamber.</li>
	<li>Place the mouse in the induction chamber and select High Flow. Complete all mouse preparation (i.e., hair removal, topical drug delivery, eye ointment application, etc.) prior to laying the animal&#39;s body atop the insert. Confirm the mouse is fully anesthetized using the toe pinch reflex method.
	<ol>
		<li>With the leg extended, use a fingernail to firmly pinch the toe without causing physical damage. If the mouse exhibits a positive reaction to the stimulus (i.e., leg retraction, foot twitch, etc.), continue administering anesthetic within the chamber until no reaction is observed. Once appropriately anesthetized, the mouse breathing rate&#160;should slow to ~55-65 breaths per minute18.</li>
	</ol>
	</li>
	<li>When the mouse is fully anesthetized, select High Flow again to stop isoflurane delivery. Purge the chamber before opening and adjust the clips (blue: open; white: closed) to deliver isoflurane through the nose cone. Select Low Flow while the nose cone is attached to the mouse to continue isoflurane delivery.</li>
	<li>Thread isoflurane tubing with the attached nose cone through the corner tubing bracket on the insert (Figure 3A and Figure 5).</li>
</ol>
3. Mouse placement on the insert for intravital imaging
<ol>
	<li>Plug the heat plate into the controller (containing an attached anal probe), power on, and allow the plate to reach 36 &#176;C (Figure 4A).</li>
	<li>Remove the anesthetized mouse from the induction chamber and lay across the heat plate (Figure 4B and Figure 5A). Perform the transfer from chamber to insert rapidly to minimize the time the mouse is active without inhalant anesthesia.</li>
	<li>Secure the nose cone onto the mouse (Figure 4B,C, and Figure 5). If necessary, use tape to further secure the angle and positioning of the nose cone.</li>
	<li>Insert the anal probe and adjust the controller temperature until the mouse body temperature is stable at ~36 &#176;C (Figure 4A,B). After the mouse is properly positioned, use plastic cling wrap to trap heat, if needed, to further support the appropriate body temperature (Figure 4C).</li>
	<li>Position the mouse so the head aligns with the coverslip disk and immobilize the ear onto the center of the glass coverslip using a metal ear clip (Figure 5A,B) or tape (Figure 5C). 
	NOTE: The pressure of the metal ear clip on the ear can be adjusted by loosening the screw that secures the clip to the insert. A metal spring can be added for increased clip tightening flexibility.
	<ol>
		<li>To change the ear clip location, unscrew the bolt with a 2.5 mm Allen wrench and transfer to the secondary site (Figure 2B,C). When reassembling the ear clip, place the clip against the insert, followed by the washer on top. Use a bolt to securely fasten the clip with slight force to pivot the clip.</li>
	</ol>
	</li>
	<li>Adjust the objective z-positioning until the cells are within the focal place (Figure 6A,D). Set the z-stack and time-lapse parameters according to the experimental goals and commence image acquisition. 
	NOTE: If the correct setup is achieved, the mouse ear can be imaged for at least 3 consecutive h (Figure 6A&#39;,D&#39;).
	<ol>
		<li>Once the z-stack boundaries are set, adjust the laser power and gain to ensure no z-plane is oversaturated to minimize photobleaching. Determine the time-lapse parameters using the total thickness of the z-stack, step numbers (Nyquist sampling recommended), and time intervals.</li>
		<li>Determine the imaging parameters (time intervals, total imaging time, etc.) using multiple factors, including animal viability under anesthesia, laser-induced photobleaching/phototoxicity, and the goal of live imaging (i.e., cell division dynamics, cell-cell interactions, etc.).</li>
	</ol>
	</li>
</ol>
4. Termination of imaging
<ol>
	<li>Turn on the water-circulating heat pad and set to Continuous Cycle and 35 &#176;C for at least 30 min prior to the termination of image acquisition.</li>
	<li>Upon imaging completion, select Low Flow on the electronic vaporizer to stop isoflurane delivery and move the mouse to a heat pad until ambulatory.</li>
	<li>Once awake, move the mouse back to the transfer container while continuing to maintain on a heat pad until fully ambulatory. Consistently monitor the mouse while on the heat pad until it is responsive.</li>
	<li>On the electronic vaporizer, click Select Menu &#62; Anesthetic Control &#62; Empty. Remove the isoflurane bottle from the system and place the manufacturer&#39;s cap back onto the bottle.</li>
	<li>Measure the weight of the isoflurane bottle and charcoal canister and enter weights into log. Once the charcoal canister weighs 50 g over baseline measurement, dispose of the canister and replace it with a new unit. Return the isoflurane to the lockbox.</li>
	<li>Remove the coverslip disk and wipe clean with lens paper and lens solution. Store properly to avoid scratches for reuse.</li>
	<li>Remove the anesthesia&#160;tubing from the insert bracket and unscrew the insert from the microscope stage.</li>
</ol>

Intravital Imaging Using an Inverted Microscope to Track Skin Cell Dynamics

<ol><li>Babes, L., Yipp, B. G., Senger, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+of+the+metastatic+pulmonary+environment%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Intravital microscopy of the metastatic pulmonary environment.</a> Methods in Molecular Biology. , 383-396 (2023).</li>
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<li>Courson, J. A., Langlois, K. W., Lam, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+to+study+platelet-leukocyte-endothelial+interactions+in+the+mouse+liver%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Intravital microscopy to study platelet-leukocyte-endothelial interactions in the mouse liver.</a> Journal of Visualized Experiments. 188 (188), (2022).</li>
<li>Rios, A. C., van Rheenen, J., Scheele, C. L. G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multidimensional+imaging+of+breast+cancer%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectives+in+Medicine%22%5BJournal%5D)">Multidimensional imaging of breast cancer.</a> Cold Spring Harbor Perspectives in Medicine. 13 (5), (2022).</li>
<li>Fischer, M., Edelblum, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+to+visualize+murine+small+intestinal+intraepithelial+lymphocyte+migration%5BTitle%5D)+AND+%22Current+Protocols%22%5BJournal%5D)">Intravital microscopy to visualize murine small intestinal intraepithelial lymphocyte migration.</a> Current Protocols. 2 (8), (2022).</li>
<li>Pineda, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+of+hair+follicle+regeneration+in+the+mouse%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Intravital imaging of hair follicle regeneration in the mouse.</a> Nature Protocols. 10 (7), 1116-1130 (2015).</li>
<li>Entenberg, D., Oktay, M. H., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+to+study+cancer+progression+and+metastasis%5BTitle%5D)+AND+%22Nature+Reviews.+Cancer%22%5BJournal%5D)">Intravital imaging to study cancer progression and metastasis.</a> Nature Reviews. Cancer. 23 (1), 25-42 (2023).</li>
<li>Turk, M., Biernaskie, J., Mahoney, D. J., Jenne, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy+techniques+to+image+wound+healing+in+mouse+skin%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Intravital microscopy techniques to image wound healing in mouse skin.</a> Methods in Molecular Biology. , 165-180 (2022).</li>
<li>Pal Arndt,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+quantitative+3D+intravital+look+at+the+juxtaglomerular+renin-cell-niche+reveals+an+individual+intra%2Fextraglomerular+feedback+system%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">A quantitative 3D intravital look at the juxtaglomerular renin-cell-niche reveals an individual intra/extraglomerular feedback system.</a> Frontiers in Physiology. 13, 980787(2022).</li>
<li>Oal Yam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+conversion+to+a+tumor-killing+phenotype+underpins+effective+microbial+therapy%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Neutrophil conversion to a tumor-killing phenotype underpins effective microbial therapy.</a> Cancer Research. 83 (8), 1315-1328 (2023).</li>
<li>Huang, S., Rompolas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-photon+microscopy+for+intracutaneous+imaging+of+stem+cell+activity+in+mice%5BTitle%5D)+AND+%22Experimental+Dermatology%22%5BJournal%5D)">Two-photon microscopy for intracutaneous imaging of stem cell activity in mice.</a> Experimental Dermatology. 26 (5), 379-383 (2017).</li>
<li>Durr, N. J., Weisspfennig, C. T., Holfeld, B. A., Ben-Yakar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maximum+imaging+depth+of+two-photon+autofluorescence+microscopy+in+epithelial+tissues%5BTitle%5D)+AND+%22Journal+of+Biomedical+Optics%22%5BJournal%5D)">Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues.</a> Journal of Biomedical Optics. 16 (2), 026008(2011).</li>
<li>Tauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advantages+and+risks+of+multiphoton+microscopy+in+physiology%5BTitle%5D)+AND+%22Experimental+Physiology%22%5BJournal%5D)">Advantages and risks of multiphoton microscopy in physiology.</a> Experimental Physiology. 87 (6), 709-714 (2002).</li>
<li>Yoshitake, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+comparison+between+confocal+and+multiphoton+microscopy+for+rapid+histopathological+evaluation+of+unfixed+human+breast+tissue%5BTitle%5D)+AND+%22Journal+of+Biomedical+Optics%22%5BJournal%5D)">Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.</a> Journal of Biomedical Optics. 21 (12), 126021(2016).</li>
<li>Mesa, K., Rompolas, P., Zito, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Niche-induced+cell+death+and+epithelial+phagocytosis+regulate+hair+follicle+stem+cell+pool%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.</a> Nature. 522, 94-97 (2015).</li>
<li>Ral Li,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pdgfra+marks+a+cellular+lineage+with+distinct+contributions+to+myofibroblasts+in+lung+maturation+and+injury+response%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response.</a> eLife. 7, (2018).</li>
<li>Tal Tumbar,, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+epithelial+stem+cell+niche+in+skin%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Defining the epithelial stem cell niche in skin.</a> Science. 303 (5656), 359-363 (2004).</li>
<li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harbor Protocols. 2 (2), 5563(2011).</li>
<li>Sanderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-photon+microscopy%5BTitle%5D)+AND+%22Current+Protocols%22%5BJournal%5D)">Multi-photon microscopy.</a> Current Protocols. 3 (1), 634(2023).</li>
</ol>

The authors have no conflicts of interest to disclose.

In this study, we present a new tool that facilitates stable, long-term&#160;intravital imaging of intact mouse skin epithelia on inverted confocal microscopes. This invention is made of PLA, which is the most common and inexpensive 3D-printable material; all in-house 3D-printing costs for this insert amount to &#60;$5. The two separate insert pieces (Figure 1, Supplementary File 1, and Supplementary File 2) can be easily assembled using set screws (see <str...

Intravital microscopy is a powerful tool that allows the monitoring of cell behaviors in their unperturbed in vivo environments. This unique method has provided key insights into the inner workings of complex mammalian organ systems, including the lung1, brain2, liver3, mammary gland4, intestine5, and skin6. Furthermore, this approach has revealed cell behavioral alterations during tumor development7, wound healing8,9, inflammation10, and other diverse pathologies in situ. In this study, we focus on enhancing the accessibility of intravital microscopy to image live epithelial and stromal dynamics in intact mouse skin. Understanding cell behaviors in mammalian skin is of high clinical importance due to the remarkable regenerative and tumorigenic capacity of this tissue.
Intravital imaging in mice has&#160;been primarily performed using upright multiphoton microscopes due to their ability to provide high-resolution imaging at tissue depths &#62;100 &#181;m11,12. Nevertheless, these instruments&#160;lack the workhorse versatility and more general accessibility of inverted confocal microscopes, which are more user-friendly and cost-effective, provide the ability to image cultured cells, do not require complete darkness during image acquisition, and are generally safer, among other notable advantages13,14. In this study, we present a new tool that significantly enhances intravital imaging accessibility by adapting this approach for inverted confocal microscopes.
Here, we present a 3D-printed custom stage insert design that incorporates several key features to facilitate stable, long-term intravital imaging of mouse ear skin on an inverted confocal microscope (Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5). These specialized features include an offset objective hole that allows the full body of an adult mouse to lay entirely flat during imaging. This minimizes the vibrational interference of mouse body movements on imaging and eliminates the need to administer ketamine and xylazine to dampen breathing, a practice often coupled with intravital imaging6. In addition, corner brackets on the insert correctly position an isoflurane nose cone to align with the face of the mouse, a metal ear clip immobilizes the mouse ear to a custom-built coverslip disk, and an optional detachable closed-loop biofeedback heat plate lies flush within the insert to support the mouse body temperature during long imaging sessions. The custom coverslip disk, which provides a flat surface essential for the mouse head and ear to lay flat, was generated in a machine shop by removing the walls of a generic coverslip-containing cell culture dish. The use of a 40x silicone oil immersion lens (1.25 numerical aperture [N.A.], 0.3 mm working distance) in conjunction with the coverslip disk and custom stage insert provides high resolution images &#62;50 &#181;m deep into the ear dermis.
To test the functionality of this new inverted microscope stage insert, we captured z-stacks spanning all epidermal epithelial layers over a 3 h time course in the ear of a live transgenic K14-H2B-mCherry15 adult mouse (epithelial nuclei in this mouse line contain a red fluorescent label) (Figure 6A-A&#39;). We also captured z-stacks spanning several fibroblast layers within the skin dermis over a 3 h time course in the ear of a live transgenic Pdgfra-rtTA16; pTRE-H2B-GFP17 adult mouse (fibroblast nuclei in this mouse line contain a green fluorescent label following doxycycline induction) (Figure 6B-D&#39;). Our high-resolution data demonstrate consistent stability by lack of drift in the x-, y-, and z-planes, thus proving the effectiveness of this new intravital imaging tool for use on inverted microscopes. Importantly, the dimensions of this 3D-printed stage insert can be adjusted, as described in Supplementary File 1, Supplementary File 2, and Supplementary File&#160;3, to fit any inverted microscope, and the positioning of the objective opening can be moved to alternative locations within the insert to better suit imaging a particular tissue and/or animal model of interest. This invention can thus empower individual laboratories, or investigators with core facility confocal access, to adapt this tool for their unique intravital imaging needs, thereby streamlining evaluation of diverse in vivo cell biology.

<table><tbody><tr><td>3D Printer</td><td>Qudi Tech</td><td>i-Fast</td><td>3D prints using PLA material</td></tr><tr><td>40x 1.25NA silicone objective lens</td><td>Nikon</td><td></td><td></td></tr><tr><td>AxR Laser Scanning Confocal Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Cotton Tipped Swab</td><td>VWR</td><td>76337-046</td><td>Cream/ointment application</td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891</td><td>Induces GFP labeling of fibroblast nuclei in Pdgfra-rtTA; pTRE-H2B-GFP mice</td></tr><tr><td>Flathead Screwdriver (2.5 mm)</td><td></td><td></td><td>Affiix insert to microscope stage</td></tr><tr><td>Flathead Screws x 4 (#6-32)</td><td>Nikon</td><td></td><td>Screw insert into microscope stage</td></tr><tr><td>Glass Bottom Culture Dish</td><td>chemglass Life Sciences</td><td>CLS-1811-002</td><td>Modified by removing walls of dish for use as coverslip disk compatible with live insert; 35 mm wide disk contains 20 mm wide glass coverslip; dish walls were removed by machine shop</td></tr><tr><td>Heat Plate controller</td><td>Physitemp</td><td>TCAT-2LV</td><td>Animal Temperature Controller - Low Voltage; anal prob attachment for mouse body temperature monitoring</td></tr><tr><td>Hex Wrench (1.5 mm)</td><td></td><td></td><td>For M3 setscrew adjustments</td></tr><tr><td>Hex Wrench (2.5 mm)</td><td></td><td></td><td>Adjust tension on metal ear clip</td></tr><tr><td>Intravital Imaging Insert</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Med-Vet International</td><td>HPA030782-100uL</td><td>Mouse anesthesia</td></tr><tr><td>Labeling Tape (or Scotch Tape)</td><td>VWR</td><td>10127-458</td><td>Alternative to metal ear clip to immobilize ear to coverslip</td></tr><tr><td>Metal fastener</td><td></td><td></td><td>used as ear clip</td></tr><tr><td>Mouse: C57BL/6-Pdgfraem1(rtTA)Xsun/J</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:034459</td><td>Fibrroblast-specific promoter driving doxycycline-inducible rtTA expression</td></tr><tr><td>Mouse: K14-H2BPAmCherry</td><td>Courtesy of Dr. Valentina Greco at Yale University</td><td></td><td>Labels epidermal epithelial cell nuclei with mCherry; referred to in text as &quot;K14-H2B-mCherry&quot;</td></tr><tr><td>Mouse: pTRE-H2B-GFP: STOCK&lt;br/&gt; Tg(tetO-HIST1H2BJ/GFP)47Efu/J</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:005104</td><td>&amp;nbsp;Labels fibroblast nuclei with GFP when combined with Pdgfra-rtTA and induced with doxycycline</td></tr><tr><td>Multipurpose Sealing Wrap</td><td>Glad</td><td></td><td>Enhance mouse warmth</td></tr><tr><td>Optixcare</td><td>VWR</td><td>MSPP-078932779</td><td>Eye lubricant</td></tr><tr><td>Set screws x 3 (M3; 6 mm)</td><td>Thorlabs</td><td>SS3M6</td><td>Attachment for heatplate module</td></tr><tr><td>Silicone Immersion Oil</td><td></td><td></td><td>Applied to 40x silicone objective</td></tr><tr><td>Small Animal Heating Plate</td><td>Physitemp</td><td>HP-4M</td><td>Provides heat to animal</td></tr><tr><td>Somnoflow Low-Flow Electronic Vaporizer</td><td>Kent Scientific</td><td>SF-01</td><td>Mouse anesthesia</td></tr><tr><td>Vacuum Grease</td><td>Flinn Scientific</td><td>AP1095</td><td>Seals coverslip disk to insert</td></tr><tr><td>Veet</td><td></td><td></td><td>hair removal&amp;nbsp;</td></tr><tr><td>Water circulating heat pad</td><td>Stryker Medical</td><td>TP700</td><td>for mouse revival post-imaging</td></tr></tbody></table>

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.

Proper assembly of the live imaging insert on an inverted confocal microscope and appropriate orientation of a transgenic mouse atop the insert is validated by acquiring z-stacks of fluorescently-labeled, live ear tissue over a time course &#8805;1 h with minimal evidence of drift in the x-, y-, and z-axes. Images should be captured at consistent intervals (interval time will depend on the biological question, strength of fluorescence&#160;signal, etc.) so that cell dynamics and image drift can be tracked over time. Thro...

Watch this Scientific Journal Video about Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging at JoVE.com

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.

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 ...

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1.6K Views.  Emory University School of Medicine. The protocol presents a new tool to simplify intravital imaging using inverted confocal microscopy.

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

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 ...

Cancer Research

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Neuroscience

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.

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 ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Medicine

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Immunology and Infection

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

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

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

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.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...