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 ...

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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,<sup clas...

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>35mm 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 - 27g</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&#39;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&#8221; (7.5 cm) Long 5X0.15mm 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&#8221; (11 cm) Long Angled Smooth 1.2mm 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 migrat...

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 microscopy of the small intestinal mucosa can provide insight into the spatiotemporal dynamics of cellular interactions within the epithelium between different immune cell types, or between these two compartments. The main advantage of this technique is that it allows the acquisition of high-speed and high-resolution images within intestinal mucosa in realtime. Live imaging of the intestine can provide additional insight into the cellular interactions, or cell signaling involved in mucosal immunity, epithelial biology or cancer biology.This is a challenging method that requires practice of the surgical technique, as well as patience and optimal positioning of the mouse for image-acquisition. After confirming a lack of response to toe pinch in an anesthetized transgenic mouse, expressing an enhanced green fluorescent protein, or EGFP, Gamma-delta T-cell receptor, use a tuberculin syringe to slowly inject 200 microliters of freshly prepared Hoechst 33342 Dye Solution into the retro-orbital venous sinus. One to two minutes after the injection, apply ointment to the animal's eyes to prevent them from drying out.Then, make a 2CM vertical incision through skin and peritoneum, along the midline of the lower abdomen and use angled forceps to carefully pull the caecum out of the peritoneal cavity. Identify a 2-3CM area of the small intestine that contains minimal fecal contents, taking care not to tear the mesenteric blood vessels, and carefully return the caecum and remaining small intestine to the peritoneal cavity, leaving the segment of interest externalized. Placing two pairs of forceps on either side of the underline mesentery between the blood vessels, gently rub the tips of the forceps together to create a hole in the membrane.Using angled forceps and a curved taper-point needle attached to a 5CM suture, penetrate one side of the peritoneum through the hole in the membrane, and up through the other side of the peritoneal lining, placing one suture at the top, and another near the bottom of the incision to close the incision, while keeping the loop of intestine externalized. To increase the likelihood of success, do not tie off or tear any blood vessels while placing the abdominal suture and limit any extraneous handling of the intestine. Then, close the skin beneath the intestinal loop in the same manner, placing one suture in the middle of the incision between the previous sutures in the underline peritoneum.Using an electrocautery, make a line of perforation along the antimesenteric border and immediately 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. Use vanna scissors to cut an approximately 1.5CM horizontal slit at the distal edge of the cauterized tissue, along the length of the cauterized line, toward the proximal end of the externalized tissue segment.When the mucosal surface is exposed, cover the abdomen with a moist kimwipe to keep the tissue hydrated. For Spinning Disk Confocal Microscopy Imaging, transport the mouse to the microscope in a covered vessel. Launch the imaging software and tilt the head of the microscope back, and add 150 microliters of 1 micromolar flea Alexa Fluor dye and Hank's Buffered Saline Solution onto the glass-cover's slipped bottom.Position the mouse so that the opened mucosal surface directly contacts the coverslip. Place the mouse and the dish onto the imaging stage in a pre-warmed incubator. Set the Excitation Intensity and Exposure Time for each laser to no more than 10-15 milliwatts and 120-150 milliseconds, respectively, and adjust the Frame Average to 2.Turn on the Electron-Multiplying Gain function to reduce the background noise and select the 63X Objective Calibration to ensure a correct measurement of the pixel size. Using the 405-nanometer laser and the 20X air objective, manually visualize the nuclei to locate a field of villi that lack noticeable movement or drift, avoiding areas of artifactual movement due to respiration, peristalsis or heartbeat. Using the X-Y Scan, record the X-Y co-ordinate of the field of interest and switch to the glycerol immersion 63X objective.Acquire a live image on the 405-nanometer channel for up to 1 minute to confirm that the villi in the selected field are stable, while adjusting the focus to find the orthogonal plane just beneath the villus tip. Then acquire z-stacks, starting from just below the villus tip epithelium, about 15-20 micrometers down to the villus until it is difficult to resolve the nuclei, using 1.5 micrometer-steps. 3-5 minutes after beginning the acquisition, confirm the image stability and the Gamma-delta Intraepithelial Cell Motility, and continue acquiring images for 30-60 minutes for each field of villi.Gamma-delta Intraepithelial Lymphocytes exhibit a dynamic surveillance behavior, in which they patrol the epithelium by migrating along the basement membrane and into the lateral intercellular space at study state. In these representative images, the colored tracks indicate the migratory paths of individual Gamma-delta Intraepithelial Lymphocytes over the course of 30 minutes. Although the frequency of Intraepithelial Lymphocytes in the lateral intercellular space was increased in mice treated with anti-interleukin-2 receptor beta antibody, more than 30%of these Gamma-delta Intraepithelial Lymphocytes exhibited an idling behavior.This idling phenotype was confirmed by a significant reduction in both the instantaneous speed and the confinement ratio of the Gamma-delta Intraepithelial Lymphocytes, following interleukin-2-receptor blockade, relative to controls. Further, the idle Gamma-delta Intraepithelial Lymphocytes had longer dwell-times, and were more frequently localized within the lateral intercellular space, compared to motile Gamma-delta Intraepithelial Lymphocytes. It can be difficult to identify fields of villi that lack excessive movement due to peristalsis.Repositioning the mouse may help in locating a more stable field. Further analysis of the cell-tracking data may help to identify specific motility behavior, or to provide additional metrics for measuring cellular interactions. Intravital Imaging and analysis of IEL-motility has become a standard read-out of IEL-function, providing insight into how innate immune signaling regulates Gamma-delta IEL-function.

intravital-imaging-intraepithelial-lymphocytes-murine-small

Research

JoVE Journal

Immunology and Infection

7.7K Views.  Rutgers New Jersey Medical School. La microscopia intravitale della piccola mucosa intestinale può fornire informazioni sulla dinamica spaziotemporale delle interazioni cellulari all'interno dell'epitelio tra diversi tipi di cellule immunitarie o tra questi due compartimenti. Il principale vantaggio di questa tecnica è che consente l'acquisizione di immagini ad alta velocità e ad alta risoluzione all'interno della mucosa intestinale in tempo reale. L'imaging vivo dell'intestino può fornire ulteriori informazioni sulle inte...