This work is supported by NIH R21 AI143892, New Jersey Health Foundation Grant, Busch Biomedical Grant (KLE). We thank Madeleine Hu for her assistance in editing the manuscript and providing the data shown in the representative results.

All studies were conducted in an Association of the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility according to protocols approved by Rutgers New Jersey Medical School Comparative Medicine Resources.
1. Mouse Preparation
NOTE: The following procedure, including animal preparation and surgery, will take 30&#8211;40 min. Prior to the surgery, turn on the microscope and warm up the enclosed incubator on the microscope to 37 &#176;C.
<ol>
	<li>Perform experiments on 8&#8211;10 weeks old TcrdEGFP mice on a C57BL/6 background, which were obtained from Bernard Malissen (INSERM, Paris, France). According to IACUC-approved procedure, anesthetize the mouse by i.p. injection of ketamine (120 mg/kg) and xylazine (10 mg/kg) based on the weight of the animal. Evaluate the level of anesthesia by respiration rate (55&#8211;65 breaths per minute)21, toe pinch and palpebral reflex.</li>
	<li>Once surgical plane anesthesia is reached, secure the hind legs of the mouse using tape to a heating pad to maintain body temperature, which can be monitored by rectal thermometer. If there are signs that the anesthesia is wearing off (e.g., increased respiration rate, blinking and/or eye muscle twitching response to palpebral reflex), re-administer 50&#160;&#956;L of ketamine/xylazine at a time until the mouse is fully sedated.</li>
	<li>Prepare the nuclear dye by diluting 50 &#181;L of Hoechst 33342 (10 mg/mL) with 315 &#181;L of 0.9% saline; the final concentration is 37.5 mg/kg based on the weight of the mouse (200 &#181;L approximate volume). Once the mouse is anesthetized, slowly inject the Hoechst using a tuberculin syringe through the retro-orbital venous sinus. 
	NOTE: Wait 1&#8211;2 min before proceeding to the next step to allow the mouse to acclimate to the change in circulating volume following retro-orbital injection.</li>
	<li>Place a small amount of lubricant over the eyes to prevent them from drying out.</li>
</ol>
2. Mouse Surgery: Laparotomy to Expose Intestinal Mucosa
<ol>
	<li>Make a 2 cm vertical incision through skin and peritoneum along the midline of the lower abdomen to externalize the region of intestine to be imaged.</li>
	<li>Using angled forceps, carefully pull the cecum out of the peritoneal cavity and identify the area of small intestine to be imaged. Be careful not to tear mesenteric blood vessels during this process. To minimize potential damage or bleeding, avoid grabbing or pinching the intestine with the forceps.</li>
	<li>Locate a suitable region of small intestine (2&#8211;3 cm) for imaging that contains minimal fecal contents. Carefully return the cecum and remaining small intestine into the peritoneal cavity, while leaving the segment of interest externalized. 
	NOTE: Avoid puncturing the cecum or disrupting the mesenteric vasculature during this step.</li>
	<li>Place two pairs of forceps on either side of the underlying mesentery between blood vessels, and gently rub the tips of the forceps together to create a hole in the membrane (Figure 1)22. This will allow the needle to pass through the mesentery when closing the peritoneal cavity beneath the intestine.</li>
	<li>Close the incision while keeping the loop of intestine externalized. Using angled forceps and a curved, taper point needle attached to a 5 cm suture, penetrate one side of the peritoneum, go through the hole made in the previous step, and up through the other side of the peritoneal lining. Place one suture at the top, and another near the bottom of the incision. 
	NOTE: Avoid damaging the vasculature during this step.</li>
	<li>Repeat this process to close the skin beneath the intestinal loop, by placing one suture in the middle of the incision, between the previous sutures in the underlying peritoneum. 
	NOTE: It is important to only externalize the area that is to be imaged; this will reduce extraneous motion during imaging. Be sure to avoid tying off the mesenteric arteries.</li>
	<li>Use an electrocautery to make a line of perforation along the anti-mesenteric border. Immediately after cauterization, apply a few drops of water to the surface of the intestine to prevent additional heat-induced tissue damage. Blot with a Kimwipe to remove residual water.</li>
	<li>Cut a small horizontal slit at the distal edge of the cauterized tissue using Vannas scissors and proceed to cut along the length of the cauterized line toward the proximal end of the externalized tissue segment (approximately 1.5 cm in length) to expose the mucosal surface. 
	NOTE: If necessary, use forceps to gently remove any excess fecal content remaining on the exposed mucosal surface.</li>
	<li>Cover the abdomen of the mouse with a moist Kimwipe to prevent the tissue from becoming dehydrated. Transport the mouse to the microscope in a covered vessel.</li>
</ol>
3. Image Acquisition by Spinning Disk Confocal Microscopy
<ol>
	<li>Pipette 150 &#181;L of 1 &#181;M AlexaFluor 633 in HBSS onto the glass coverslipped bottom of a 35 mm dish. Position the mouse so that the opened mucosal surface directly contacts the coverslip.</li>
	<li>Tilt the head of the microscope back and place the mouse on the coverslipped dish on the imaging stage in a pre-warmed incubator. Alternatively, cover the mouse by a heating blanket to maintain body temperature.</li>
	<li>Launch imaging software. The excitation intensity and exposure time for each laser should not exceed 10&#8211;15 mW or 120&#8211;150 ms, respectively, to avoid photobleaching and to minimize the interval between time points. Adjust the frame average to &#8220;2&#8221; and turn on the EM gain function to reduce background noise. Select 63x objective calibration to ensure the correct measurement of pixel size. 
	NOTE: The settings detailed above are meant to provide an initial reference, but will vary depending on individual microscope configurations.</li>
	<li>Using the 405 nm laser and the 20x air objective, visualize the nuclei to locate a field of villi that lack noticeable movement or drift by eye. Avoid areas of artifactual movement due to respiration, peristalsis or heartbeat.</li>
	<li>Using the XY scan, record the XY coordinate of the field of interest and switch to the glycerol immersion 63X objective.</li>
	<li>Confirm that the villi in the selected field are stable by acquiring a live image on the 405 nm channel for up to 1 min.</li>
	<li>While acquiring a live image on the 405 nm channel, adjust the focus to find the orthogonal plane just beneath the villous tip. Acquire Z-stacks starting from the just below the villous tip epithelium down the villus until it is difficult to resolve the nuclei, about 15&#8211;20 &#181;m, using a 1.5 &#181;m step. 
	NOTE: When imaging with three channels (405, 488, 640 nm) using the exposure times described above, it is possible to acquire all three channels sequentially at approximately 20 s intervals.</li>
	<li>Open the software in analysis mode, 3&#8211;5 min after beginning the acquisition, to confirm the image stability and &#947;&#948; IEL motility. Continue to acquire images for 30&#8211;60 min for each field of villi. Record 2&#8211;3 fields for each mouse. While imaging, monitor the mouse every 5 min. 
	NOTE: If the anesthesia begins to wear off, use forceps to gently scruff the neck of the mouse to administer 50 &#181;L of ketamine/xylazine at a time. Continue to monitor anesthesia levels and re-administer when necessary.</li>
	<li>It is not advisable to image a single mouse for longer than 4 h. Once image acquisition is complete, sacrifice the mice by cervical dislocation followed by bilateral pneumothorax.</li>
</ol>
4. 4D Analysis of Images
<ol>
	<li>Directly import imaging files to 4D rendering software.</li>
	<li>Create individual Surfaces for IELs and the lumen, then track the surfaces over time using the parameters outlined in Table 1 as an initial reference. For the IELs, enable Split touching objects using the seed point diameter indicated.</li>
	<li>Use the autoregressive tracking algorithm to determine IEL motility. Although the tracking algorithm is relatively accurate, it is imperative to visually inspect tracks for each individual IEL. If a track is incorrect, correct it using the Disconnect and Connect functions under the Edit Tracks tab.</li>
	<li>To determine the distance from each IEL to the lumen, select the lumen Surface and perform Distance Transformation function under the Tool menu. When prompted, select Outside Surface. Once calculated, the distance transformation will be displayed as the 4th channel.</li>
	<li>Filter the Intensity min of the 4th channel to select &#947;&#948; IELs that are within the lateral intercellular space (LIS). This value should be close to 15 &#181;m, based on the height of a columnar epithelial cell. The percent of total &#947;&#948; IELs within this range are reported as the frequency of &#947;&#948; IELs in the LIS.</li>
	<li>For further analysis, export statistical data including: IEL track speed, track displacement, track straightness and track length. Alternatively, analyze data using the Vantage module. Depending on the experiment, obtain additional metrics from the acquired data, including dwell time within the LIS or IEL/epithelial interactions.</li>
</ol>

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The authors have nothing to disclose.

The development of intravital microscopy techniques has provided an unprecedented opportunity to observe the reorganization of subcellular structures8,9,22, cell-cell interactions12,25 and cell migratory behavior13,14,15,16,26 in otherwise inaccessible tissues. There has been a general lack of tools to study the regulation and function of &#947;&#948; IELs in vivo18,27, and as a result, the use of intravital imaging has opened new avenues of investigation regarding the molecular regulation and functional characteristics of IEL populations13,14,15,16,20. &#947;&#948; IELs were previously presumed to be stationary within the intestinal mucosa28. However, further analysis demonstrated that these cells are highly dynamic, but migrate more slowly than lymphocytes in secondary lymphoid organs due to the tight confines of the epithelial compartment13. Moreover, the use of in vivo imaging has provided additional insight into the spatiotemporal dynamics of &#947;&#948; IEL surveillance behavior, including the crosstalk between IELs and epithelial cells that drives &#947;&#948; IEL motility and function13,14,15,16. Analysis of this migratory behavior has defined new metrics for IEL cellular responses that would not have been accurately quantifiable using ex vivo or in vitro experiments.
Throughout the entire procedure, it is essential to continuously monitor the sedation level of the mouse. Continuous administration of gas anesthesia (isoflurane) can be used as an alternative to ketamine/xylazine. When performing the laparotomy, it is critical to keep the neurovascular supply intact to provide an accurate evaluation of the biology. Tying off mesenteric blood vessels can lead to hypoxia and gradual necrosis of the tissue. In contrast, severing blood vessels will result in bleeding into the exposed mucosa, which can reduce the fluorescence intensity of some channels during imaging. If the IELs develop a rounded morphology and stop moving during image acquisition, this may be due to a slight decrease in the body temperature of the mouse. Placing temperature sensor close to the microscope stage ensures that the temperature at the site of the exposed mucosa is maintained at 37 &#176;C. Raising the temperature to 38&#8211;39 &#176;C may be necessary depending on the temperature stability of the incubation chamber.
Once the mouse has been positioned on the microscope stage, it can be difficult to identify fields of villi that lack excessive movement due to peristalsis or contraction of adjacent blood vessels. If this occurs, reposition the mouse within the dish or manipulate the mouse&#8217;s flanks externally in an effort to tightly pack villi against the coverslip. Alternatively, the imaging field may become more stable a few minutes after placing the mouse on the stage. If there is gradual XY drift during the acquisition, this can be corrected using drift correction in the 4D rendering software by selecting 4&#8211;5 nuclei that are present in different locations of the image throughout the time course and tracking a Spot for each nucleus. Once the Spots have been generated, select Correct Drift under the Edit Track menu to correct translational drift. It is important that the drift correction be done prior to any other analysis, since this will alter the coordinates used to track other objects in the image.
Although two-photon laser scanning microscopy allows for a wide depth of imaging that is useful for penetrating deep into tissues29, spinning disk laser confocal microscopy has its own unique advantages for intravital imaging applications. Two-photon laser scanning microscopy relies on photomultiplier tubes (PMT) that can detect only one pixel at a time with a quantum efficiency of 40&#8211;50%. In contrast, spinning disk confocal laser microscopy uses an electron multiplication (EM)CCD camera to directly detect up to a million pixels simultaneously, thus increasing the quantum efficiency to 95%. As a result, the potential for photobleaching and photodamage is significantly reduced, while the increased acquisition speed maximizes temporal resolution when imaging cell migration in more superficial tissues. Regardless of the imaging modality, this surgical procedure is ideal for exposing the intestinal mucosa for imaging on an inverted microscope.
With the increasing availability of mouse strains expressing fluorescent reporters, fluorescently-labeled probes or fluorescent strains of microorganisms that have been generated in a multitude of wavelengths, the combinations to evaluate in vivo intestinal responses in real-time are endless. Not only can intravital imaging provide novel insight into cell-cell and host-microbe interactions, but it can also highlight dynamic molecular and cellular processes in both epithelial and immune cells under homeostatic or pathological conditions.

Intraepithelial lymphocytes (IEL) are located within the intestinal epithelium, and are found both along the basement membrane and between adjacent epithelial cells in the lateral intercellular space1. There is approximately one IEL for every 5-10 epithelial cells; these IELs serve as sentinels to provide immune surveillance of the large expanse of the intestinal epithelial barrier2. IELs expressing the &#947;&#948; T cell receptor (TCR) comprise up to 60% of the total IEL population in the murine small intestine. Studies in &#947;&#948; T-cell-deficient mice demonstrate a largely protective role of these cells in response to intestinal injury, inflammation and infection3,4,5. Despite the generation of the Tcrd knockout mouse6, our understanding of &#947;&#948; IEL biology remains limited due in part to the fact that ligands recognized by the &#947;&#948; TCR have yet to be identified7. As a result, the lack of tools to study this cell population has made it difficult to investigate the role of &#947;&#948; TCR activation and function under physiological and pathological conditions. To fill this gap, we have developed live imaging techniques to visualize &#947;&#948; IEL migratory behavior and interactions with neighboring enterocytes as a means to provide additional insight into &#947;&#948; IEL function and responsiveness to external stimuli in vivo.
Over the last decade, intravital imaging has significantly expanded our understanding of the molecular events involved in multiple facets of intestinal biology, including epithelial cell shedding8, regulation of epithelial barrier function9,10, myeloid cell sampling of luminal contents11,12, and host-microbe interactions11,13,14,15,16. In the context of IEL biology, the use of intravital microscopy has shed light on the spatiotemporal dynamics of IEL motility and the factors mediating their surveillance behavior13,14,15,16. The development of TcrdH2BeGFP (TcrdEGFP) reporter mice, which labels &#947;&#948; IELs by nuclear GFP expression17, revealed that &#947;&#948; IELs are highly motile within the epithelium and exhibit a unique surveillance behavior that is responsive to microbial infection17,13,14. Recently, another &#947;&#948; T cell reporter mouse was developed (Tcrd-GDL) which expresses GFP in the cytoplasm to allow visualization of the entire cell18. Similar methodology has been used to investigate the requirement of specific chemokine receptors, such as G protein-coupled receptor (GPCR)-18 and -55, on the dynamics of IEL motility19,20. In the absence of a cell-specific reporter, fluorescent conjugated antibodies against CD8&#945; were used to visualize and track IEL motility in vivo19,20. Although two-photon laser scanning microscopy is commonly used for intravital imaging, the use of spinning disk confocal laser microscopy provides unique advantages to capture high speed and high-resolution multi-channel images with minimal background noise. This technology is ideal to elucidate the spatiotemporal dynamics of immune/epithelial interactions within the complex microenvironment of the intestinal mucosa. Moreover, through the use of various transgenic and/or knockout mouse models, these studies can provide insight into the molecular regulation of intestinal immune and/or epithelial cell function.

<table><tbody><tr><td>35 mm dish, No. 1.5 Coverslip</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Alexa Fluor 633 Hydrazide</td><td>Invitrogen</td><td>A30634</td><td></td></tr><tr><td>BD PrecisionGlide Hypodermic needles - 27 G</td><td>Thermo Fisher Scientific</td><td>14-826-48</td><td></td></tr><tr><td>BD Slip Tip Sterile Syringe - 1 mL</td><td>Thermo Fisher Scientific</td><td>14-823-434</td><td></td></tr><tr><td>BD Tuberculin Syringe</td><td>Thermo Fisher Scientific</td><td>14-829-9</td><td></td></tr><tr><td>Dissecting scissors</td><td>Thermo Fisher Scientific</td><td>08-940</td><td></td></tr><tr><td>Electrocautery</td><td>Thermo Fisher Scientific</td><td>50822501</td><td></td></tr><tr><td>Enclosed incubation chamber</td><td>OKOLAB</td><td></td><td>Microscope</td></tr><tr><td>Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length</td><td>Roboz</td><td>RS-7983-3</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>55037C</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>Imaris (v. 9.2.1) with Start, Track, XT modules</td><td>Bitplane</td><td></td><td>Software</td></tr><tr><td>Inverted DMi8</td><td>Leica</td><td></td><td>Microscope</td></tr><tr><td>IQ3 (v. 3.6.3)</td><td>Andor</td><td></td><td>Software</td></tr><tr><td>Ketamine</td><td>Putney</td><td></td><td>Anesthesia</td></tr><tr><td>Kimwipes</td><td>VWR</td><td>21905-026</td><td></td></tr><tr><td>McPherson-Vannas scissors 3&amp;rdquo; (7.5 cm) Long 5 x 0.15 mm Straight Sharp</td><td>Roboz</td><td>RS-5600</td><td></td></tr><tr><td>Non-absorbable surgical suture, Silk Spool, Black Braided</td><td>Fisher Scientific</td><td>NC0798934</td><td></td></tr><tr><td>Nugent Forceps 4.25&amp;rdquo; (11 cm) Long Angled Smooth 1.2 mm Tip</td><td>Roboz</td><td>RS-5228</td><td></td></tr><tr><td>Puralube Vet Ointment</td><td>Dechra</td><td></td><td>Lubricating Eye Ointment</td></tr><tr><td>Spinning disk Yokogawa CSU-W1 with a 63x 1.3 N.A. HC PLAN APO glycerol immersion objective, iXon Life 888 EMCCD camera, 405 nm diode laser, 488 nm DPSS laser, 640 nm diode laser</td><td>Andor</td><td></td><td>Confocal system</td></tr><tr><td>Xylazine</td><td>Akorn</td><td></td><td>Anesthesia</td></tr></tbody></table>

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.

Using intravital imaging of TcrdEGFP reporter mice, we have previously shown that &#947;&#948; IELs exhibit a dynamic surveillance behavior, in which they patrol the epithelium by migrating along the basement membrane and into the lateral intercellular space (LIS) at steady state (Figure 2, Movie 1).
This approach can also be used to evaluate how the inhibition of specific cell signaling pathways and/or receptors influences &#947;&#948; IEL migratory behavior. For example, interleukin (IL)-15 is a pleiotropic cytokine that is essential for &#947;&#948; IEL homeostasis23,24. To determine whether IL-15 signaling through IL-2R&#946; receptor contributes to the kinetics of &#947;&#948; IEL motility at steady state, TcrdEGFP reporter mice were imaged 2 h following administration of anti-IL-2R&#946; antibody (TM-&#946;1) or isotype IgG2b control. The colored tracks indicate the migratory paths of individual &#947;&#948; IELs over the course of 30 min (Figure 3A). Although the frequency of &#947;&#948; IELs in the LIS was increased in mice treated with TM-&#946;1 (Figure 3A,B), more than 30% of these &#947;&#948; IELs exhibited an idling behavior (Figure 3A,C). Idle &#947;&#948; IELs were defined by imposing upper limits on track straightness and track displacement length. This idling phenotype was confirmed by a significant reduction in both the instantaneous speed and confinement ratio of &#947;&#948; IELs following IL-2R&#946; blockade relative to control (Figure 3D,E). Further, the idle &#947;&#948; IELs have longer dwell times and were more frequently localized within the LIS compared to motile &#947;&#948; IELs (Figure 3F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59853/59853fig1.jpg" /> 
Figure 1: Using forceps to create a hole in the mesentery. Place forceps on either side of the membrane to create a hole for the sutures. <a href="https://www.jove.com/files/ftp_upload/59853/59853fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59853/59853fig2.jpg" /> 
Figure 2: Representative 3D image of &#947;&#948; IELs in the jejunum. A single time point was selected from a time-lapse video of taken of the jejunal mucosa of a TcrdEGFP reporter mouse at steady-state. &#947;&#948; IELs are shown in green, nuclei in white and the lumen in red. Scale bar = 30 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59853/59853fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59853/59853fig3.jpg" /> 
Figure 3: Inhibition of IL-2R&#946; induces &#947;&#948; IEL idling in the lateral intercellular space. This figure has been adapted from &#8220;Epithelial IL-15 Is a Critical Regulator of &#947;&#948; Intraepithelial Lymphocyte Motility within the Intestinal Mucosa.&#8221; by Hu, M. D. et al. 2018, J Immunol, 201 (2), p. 747-756.15 Copyright 2018 by the American Association of Immunologists, Inc. (A) Time-lapse images showing migrating GFP &#947;&#948; IELs within the jejunal villous epithelium following 2 h treatment with 0.45 mg of IgG2b or TM-&#946;1. The motility of individual &#947;&#948; IELs (green) over the course of 30 min are shown with colored tracks. Nuclei are shown in white, and the lumen is shown in red. Scale bars = 20 &#181;m. (B) Frequency of &#947;&#948; IELs in the LIS (n = 3 mice per treatment, n = 6-7 videos). (C) Percentage of &#947;&#948; IELs that were idle in IgG2b- or TM-&#946;1-treated mice. (D) Instantaneous speed (n = 13,299 and 9,600 time points). (E) Track confinement ratios (n = 350 and 278 tracks) are shown. (F) Distance of idle and motile &#947;&#948; IELs from the lumen. Mean + SEM is shown (+). *p &#60; 0.05, **p &#60; 0.01, #p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/59853/59853fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>IEL</td>
			<td>lumen</td>
		</tr>
		<tr>
			<td>Surface detail (&#956;m)</td>
			<td>1.2</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>Background subtraction (&#956;m)</td>
			<td>1.67</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Seed points diameter (&#956;m)</td>
			<td>7.5</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Filter: number of voxels</td>
			<td>250</td>
			<td>6,500</td>
		</tr>
		<tr>
			<td>Tracking: max distance (&#956;m)</td>
			<td>10</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Tracking: max gap</td>
			<td>2</td>
			<td>2</td>
		</tr>
	</tbody>
</table>
Table 1: Representative settings for generating &#947;&#948; IEL and luminal &#8220;Surfaces&#8221; in Imaris. These settings can be used as a starting point when generating and tracking Surfaces of the 488 nm channel (IEL) and 640 nm channel (lumen). Depending on experimental conditions, these settings may need to be modified.
Movie 1: Intravital imaging of GFP &#947;&#948; IEL migratory behavior in mouse jejunum. Time-lapse intravital microscopy of &#947;&#948; IEL motility in jejunum from a TcrdEGFP mouse treated with 0.45 mg of IgG2b. &#947;&#948; IELs are shown in green, nuclei in blue, and the lumen in red. Scale bar, 20 &#956;m. Frames were collected every 30 s for approximately 30 min. Movie is adapted from Hu, M. D. et al. 201815. <a href="https://www.jove.com/files/ftp_upload/59853/Movie1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine at JoVE.com

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

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 ...

intravital-imaging-intraepithelial-lymphocytes-murine-small

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Rutgers New Jersey Medical School. We describe a method to visualize GFP-labeled γδ 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.  

Video: Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

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. ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Biology

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...