Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

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.