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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+metastatic+pulmonary+environment.">Intravital microscopy of the metastatic pulmonary environment.</a> Methods in Molecular Biology. , 383-396 (2023).</li><li>Nal Chen, ., 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=Neutrophils+promote+glioblastoma+tumor+cell+migration+after+biopsy.">Neutrophils promote glioblastoma tumor cell migration after biopsy.</a> Cells. 11 (14), 2196 (2022).</li><li>Courson, J. A., Langlois, K. W., Lam, F. W. <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+to+study+platelet-leukocyte-endothelial+interactions+in+the+mouse+liver.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+imaging+of+breast+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+to+visualize+murine+small+intestinal+intraepithelial+lymphocyte+migration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+hair+follicle+regeneration+in+the+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+to+study+cancer+progression+and+metastasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+techniques+to+image+wound+healing+in+mouse+skin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+3D+intravital+look+at+the+juxtaglomerular+renin-cell-niche+reveals+an+individual+intra/extraglomerular+feedback+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+conversion+to+a+tumor-killing+phenotype+underpins+effective+microbial+therapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+for+intracutaneous+imaging+of+stem+cell+activity+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+imaging+depth+of+two-photon+autofluorescence+microscopy+in+epithelial+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+and+risks+of+multiphoton+microscopy+in+physiology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+comparison+between+confocal+and+multiphoton+microscopy+for+rapid+histopathological+evaluation+of+unfixed+human+breast+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-induced+cell+death+and+epithelial+phagocytosis+regulate+hair+follicle+stem+cell+pool.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pdgfra+marks+a+cellular+lineage+with+distinct+contributions+to+myofibroblasts+in+lung+maturation+and+injury+response.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+epithelial+stem+cell+niche+in+skin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+microscopy.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#34;K14-H2B-mCherry&#34;</td>
		</tr>
		<tr>
			<td>Mouse: pTRE-H2B-GFP: STOCK 
			Tg(tetO-HIST1H2BJ/GFP)47Efu/J</td>
			<td>The Jackson Laboratory</td>
			<td>RRID: IMSR_JAX:005104</td>
			<td>&#160;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&#160;</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 ...

streamlined-intravital-imaging-approach-for-long-term-monitoring

Research

JoVE Journal

Biology

1.5K 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 Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...