Stabilized Window for Intravital Imaging of the Murine Pancreas

Summary

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

Abstract

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts.

Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days.

The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

Introduction

Benign and malignant pancreatic diseases are potentially life-threatening, with considerable gaps in the understanding of their pathophysiology. Pancreatitis-inflammation of the pancreas-is the third major cause of gastrointestinal disease-related hospital admissions and readmissions in the US and is associated with substantial morbidity, mortality, and socioeconomic burden¹. Ranked as the third leading cause of cancer-related death², pancreatic ductal adenocarcinoma (PDAC) accounts for most pancreatic malignancies³ and portends a poor 5-year survival rate of only 11%². The leading cause of cancer-related mortality in PDAC is overwhelming metastatic burden. Unfortunately, most patients present with metastatic disease. Therefore, understanding the dynamics of metastasis in PDAC is a critical unmet need in the field of cancer research.

The mechanisms underpinning inflammation and the metastatic cascade of the pancreas are poorly understood. A major contributor to this gap in knowledge is the inability to observe pancreatic cellular dynamics in vivo. Direct observation of these cellular dynamics promises to unveil critical targets to leverage and improve the diagnosis and treatment of those with pancreatic disease.

Intravital imaging (IVI) is a microscopy technique that allows researchers to visualize and study biological processes in living animals in real time. IVI allows high-resolution, direct visualization of intracellular and microenvironmental dynamics in vivo and within the native environment of the biological process in question. Therefore, IVI allows in vivo observation of healthy and pathologic processes.

Contemporary whole-body imaging modalities such as MRI, PET, and CT offer excellent views of entire organs and can reveal pathologies, even before the onset of clinical symptoms⁴. They are unable, however, to attain single-cell resolution or reveal the earliest stages of disease-pancreatitis or malignancy.

Previous research has used single-cell resolution IVI to observe benign and malignant diseases of skin^5,6, breast⁷, lung⁸, liver⁹, brain¹⁰, and pancreatic tumors¹¹, leading to insights into mechanisms of disease progression¹². However, the murine pancreas poses significant obstacles to achieving single-cell resolution using IVI, primarily due to its deep visceral location and high compliance. Moreover, it is a branched, diffusely distributed organ within the mesentery that connects to the spleen, small intestine, and stomach, making it challenging to access. The tissue is also highly sensitive to motion caused by adjacent peristalsis and respiration. Minimizing movement of the pancreas is essential for single-cell resolution microscopy, as motion artifacts of even a few microns can blur and distort images, making tracking the dynamics of individual cells impossible¹³.

To perform IVI, an abdominal imaging window (AIW) must be surgically implanted^9,11. To implant the AIW surgically, a metal window frame is sutured into the abdominal wall. Afterward, the organ of interest is attached to the frame using cyanoacrylate adhesive. While this is sufficient for some rigid internal organs (e.g., liver, spleen, rigid tumors), attempts at imaging the healthy murine pancreas are compromised by suboptimal lateral and axial stability due to the tissue's compliant texture and complex architecture¹⁴. To address this limitation, Park et al.¹⁴ developed an imaging window specifically designed for the healthy pancreas. This Pancreas Imaging Window (PIW) minimizes the influence of intestinal movement and breathing by incorporating a horizontal metal shelf within the window frame, just below the coverslip, stabilizing the tissue and maintaining its contact with the cover glass. While the PIW offers increased lateral stability, we found that this window still demonstrates axial drift and additionally prevents the imaging of large solid tumors due to the narrow gap between the metal shelf and coverslip¹⁵.

To address these limitations, we developed the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP), an implantable imaging window capable of achieving stable long-term imaging of both the healthy and diseased pancreas (Figure 1)¹⁵. Here, we provide a comprehensive protocol for the surgical procedure used to implant the SWIP. Although the primary objective was to study the dynamic mechanisms involved in metastasis, this method can also be utilized to explore various aspects of pancreas biology and pathology.

Protocol

All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee (IACUC).

1. Passivation of windows

NOTE: Passivation of stainless steel cleans the metal of contaminants and creates a thin oxide layer that greatly increases the metal's biocompatibility with soft tissues, even beyond that of titanium¹⁶.

1. Start the passivation process by washing the optical window frames with a 1% (w/v) enzymatically-active detergent solution.
2. Submerge the frames in a 5% (w/v) sodium hydroxide solution at 70 °C for 30 min inside a glass jar.
3. Take out the frames and rinse them with deionized water.
4. Immerse the frames in a 7% (w/v) citric acid solution at 55 °C for 10 min inside a new glass jar.
5. Remove the frames and rinse them with deionized water again.
6. Repeat step 1.2, and finally, rinse the window frames with deionized water one last time.

2. Preparation for tumor implantation or window surgery

NOTE: For studies of pancreatic tumors, tumor cells must be implanted and allowed to grow into overt tumors. To visualize the tumor cells in vivo, it is recommended to use cells that have been genetically altered to express fluorescent proteins such as Dendra2. Using fluorescent protein labels that are bright will mitigate potential issues with tissue autofluorescence. Other potential fluorescent proteins, dyes, and genetically encoded fluorescent mouse models that may be used have been discussed elsewhere^17,18. To prevent contamination of the operative field, perform the surgical procedure in a hood or laminar flow cabinet and ensure that distinct areas are used for preparation, surgery, and recovery.

1. Prior to the surgery, sterilize all surgical instruments in an autoclave and, if necessary, use a hot bead sterilizer for subsequent procedures. Ensure that the surgery employs a tips-only technique.
2. Switch on the heated surgical pad and bead sterilizer and wait for it to reach the appropriate operating temperature. The heating pad temperature should be monitored with a surface thermometer to avoid potential burns. Place a sterile cloth over the heating pad if the temperature cannot be adequately controlled.
   NOTE: Body temperature during short procedures (≤20 min), such as tumor and window implantation, is minimally affected while using a heated surgical pad. However, longer periods of anesthesia, such as during extended timelapse imaging, require the mouse to be placed in a heated chamber to maintain body temperature.
3. Anesthetize the mouse with 5% isoflurane in an anesthesia chamber.
4. Critical Step: Lower the anesthesia to 2% once the mouse is unconscious. Carefully monitor the anesthesia level and the mouse's vitals (e.g., using a pulse oximeter)¹⁹.
5. Place a small drop of eye lubricant on each eye of the mouse to prevent corneal drying.
6. Prior to surgery, apply depilatory cream generously to the left upper abdomen to remove hair. After 20 s, use moistened tissue paper to firmly wipe away the hair and depilatory cream. Repeat the process as needed until all hair is removed from the surgical area.
7. Inject 10 µL of buprenorphine (0.1 mg/kg) diluted in 90 µL of PBS subcutaneously to ensure preoperative analgesia.

3. Pancreas tumor implantation

1. Prepare aliquots of tumor cells at the desired concentration (based on tumor cell doubling time). Place the cell suspension in an insulin syringe and keep it on ice. To follow this protocol, use 10⁶ syngeneic KPC tumor cells²⁰ suspended in a maximum of 50 µL of PBS, following the orthotopic injection protocol adapted from Erstad et al.²¹
   NOTE: This cell line injected at this concentration routinely produced palpable or appropriately large tumors by 10-14 days. Subclones of this cell line and other pancreatic cell lines would need to be evaluated for appropriate concentrations and timelines to produce appropriately sized tumors).
2. Wash hands using antiseptic soap.
3. Prior to each new surgery, put on new sterile gloves.
4. Transfer the mouse to the sterile surgical hood and place it in a partial right lateral decubitus position.
5. Secure the limbs with paper tape.
   NOTE: Proper use of the instruments is important throughout the procedure. Examples of how to hold forceps, Castroviejo scissors, and the vacuum pickup tool are shown in Figure 2A-C.
6. Sterilize the abdomen with antiseptic (Figure 2D).
7. Ensure the animal is fully anesthetized by performing a toe pinch test.
8. Make a 10-15 mm left subcostal incision in the skin using forceps and Castroviejo scissors (Figure 2E).
9. Control hemostasis using cotton swabs or a cautery pen when/where deemed necessary.
10. Carefully divide the underlying muscle with forceps and Castroviejo scissors to enter the peritoneum (Figure 2F).
11. Using sterile cotton swabs, atraumatically externalize the pancreas and spleen.
12. Splay the pancreas out so there are no folds (Figure 2G).
13. Identify the desired tumor injection site in the body or tail of the pancreas (away from blood vessels).
14. Critical step: After careful positioning of the pancreas, use forceps to provide tension to the tissue and insert the insulin syringe tip, with the bevel facing upwards, into the desired site of the pancreas to a depth of 4-5 mm (Figure 2H).
15. Slowly inject the tumor cell solution. Look for a small bubble that confirms a successful injection (Figure 2I).
16. Carefully return the pancreas to the abdomen without disturbing the tumor cell injection bubble (Figure 2J).
17. Using absorbable 5-0 polydioxanone sutures, close the muscle layer first and then the skin with interrupted sutures (Figure 2K-N).
18. Cover the incision with cyanoacrylate glue (Figure 2O), then return the mouse to a clean cage under a heating lamp for recovery. Administer antibiotics in drinking water to prevent infection. Monitor the mice and allow them to completely recover from surgery.
    NOTE: Antibiotics are administered as required by IACUC protocol. All animals are housed individually.
19. Allow the tumor to develop for 10-14 days until it is palpable through the abdominal wall.

4. Pancreas window surgery

1. When the animals are ready for imaging, begin the window implantation surgery. To begin, wash hands with antiseptic soap.
2. Before every new surgery, put on fresh sterile gloves.
3. On the heated surgical stand, place the mouse in the right lateral decubitus position to expose the left abdomen.
4. Anchor the mouse's front and hind limbs to the heated surgical stage cranially and caudally using paper tape. Ensure the spleen (beneath the skin) is visible within the surgical field (Figure 3A).
5. To maintain sterility, unpackage all surgical instruments in the hood.
6. Disinfect the surgical site by swabbing the mouse's skin with a generous application of antiseptic.
7. Ensure the animal is fully anesthetized by performing a toe pinch test.
8. Critical step: Lift the skin of the left upper quadrant of the abdomen with forceps and make a ~10 mm circular incision in the skin and musculature using Castroviejo scissors (Figure 3B,C).
9. Control the bleeding and maintain hemostasis using cotton swabs or the cautery pen, where needed.
10. Localize the pancreas, which is attached to the spleen, and identify the direction the pancreas is laying within the incision to decide where the supporting cross-stitch should be placed.
11. Using 5-0 silk suture, place the first stitch at the desired location in the muscle layer. Tie this end with 3-5 knots. (Figure 3D,E)
12. Continue to stitch directly across the incision. Cut and leave a tail of ~5 cm (Figure 3F).
13. Repeat steps 4.11 and 4.12 perpendicular to the first stitch (Figure 3G,H).
14. Critical step: Gently lift and position the pancreas over the cross-stitch (Figure 3I,J). Take care not to damage the pancreas during manipulation.
15. Critical step: Using the 5-0 silk suture, perform a purse-string stitch ~1 mm from the hole, circumferentially, interlacing the skin and muscle layer (Figure 3K).
16. Position the window frame so the edges of the circular incision are seated within the window's groove (Figure 3L).
17. Fasten the implanted window by firmly tying down the 5-0 silk.
18. Load 100 µL of liquid cyanoacrylate adhesive into the 1 mL syringe.
19. Dry the tissue by applying a delicate flow of compressed air for ~10 s.
20. Clasp the window frame by its outer edge with forceps and gently raise it to ensure separation of the pancreas from the undersurface of the window frame.
21. Critical step: Dispense a thin layer of liquid cyanoacrylate adhesive along the recess of the window (Figure 3M). Be sure not to get any of the adhesive on the pancreas tissue.
22. Using vacuum pickups, lift the 5 mm coverslip.
23. Carefully place the coverslip inside the recess in the center of the optical window frame. Hold with light pressure, allowing the cyanoacrylate adhesive to set (~25 s).
24. Separate the coverslip from the vacuum pickups using forceps.
25. Tighten the cross-stitch sutures to secure the pancreas snugly to the coverslip (Figure 3N,O). Note: Do not overtighten the cross-stitch as it can cause damage and ischemia to the pancreas.
26. Cut the ends of the suture.
27. Strip off the tape from the mouse.
28. Switch off the isoflurane vaporizer.
29. Relocate the mouse to a clean cage or directly to the intravital microscope.
30. House the animals individually following window surgery and monitor them until full recovery.
31. Imaging is then carried out on a two-laser multiphoton microscope as we have previously described ^22,23,24 For long imaging sessions the mouse is placed in a heated chamber to maintain body temperature and provided with supporting fluids as per IACUC standards.

5. Cerulein treatment for the induction of pancreatitis

1. To investigate the onset of pancreatitis, treat healthy mice with cerulein after implantation of the SWIP. Ensure that the mice are fasted for 14-18 h and given ad libitum water before cerulein administration.
2. Inject 50 µg/kg of cerulein in 100 µL of sterile 1x DPBS intraperitoneally at 1 h intervals for up to eight injections. Administer an equivalent volume of 1x DPBS alone, injected intraperitoneally, to the control mice.
3. Following imaging, sacrifice the mice 24 h after the first injection by cervical dislocation as per IACUC standards.
4. Perform imaging on a two-laser multiphoton microscope as previously described^22,23,24. For long imaging sessions, place the mouse in a heated chamber to maintain the body temperature and provide it with supporting fluids as per IACUC standards.

Representative Results

Figure 1, adapted from Du et al.¹⁵, shows image stills from a time-lapse IVI movie of the murine pancreas. Some tissue motion can be observed within the initial settling period (first hour of imaging, Figure 1A). However, with continued imaging after this settling period (>75 min), we observed an increase in lateral and axial stability (Figure 1B). The comparison of the stability of the SWIP with the previous AIW and PIW imaging windows identifies that all windows require an initial period for settling. However, the SWIP exhibited the lowest level of drift overall and is best suited for long-term imaging (Figure 1C-K). The images were generated on a custom-built multiphoton microscope²⁴ illuminating at 880 nm and acquiring a z stack-t lapse (field of view [FOV] size = 340 x 340 µm, pixel size 0.67 µm) with 1.7 min between frames, 11 slices with a 2 µm step size, 20 imaging depth, and ~1 slice/s. Laser power and photomultiplier tube (PMT) gains were chosen to maximize the signal while minimizing photobleaching and photodamage.

The steps of the surgical procedures describing pancreas tumor implantation and subsequent pancreas window insertion are depicted in Figure 2 and Figure 3, respectively. Importantly, both surgeries are survival surgeries. Once correctly implanted, the pancreas will remain apposed to the optical window, which is now integrated within the abdominal wall. This allows for the mouse's comfortable survival and enables continuous imaging for up to 12 h, as permitted by the protocol. Additionally, serial imaging can be conducted over multiple consecutive days (up to the protocol allowance of 2 weeks) to monitor regions of interest over time. Intravital imaging (IVI) can be performed through the window in a manner similar to other windows previously described^22,25,26.

The SWIP can be utilized to investigate the dynamics at the onset of acute pancreatitis induced using cerulein. Cerulein is an oligopeptide with structure and function similar to that of cholecystokinin (CKK) and is widely used to experimentally induce acute pancreatitis in rodents²⁷. Treatment with cerulein results in the contraction of the smooth muscle in the gastrointestinal tract and stimulates gastric and pancreatic secretions²⁸. Furthermore, intraperitoneal administration of cerulein leads to pancreas swelling and enlargement²⁹.

Figure 4 shows serial imaging of the murine pancreas following cerulein treatment, using the SWIP protocol. The murine pancreas is visualized at single-cell resolution using genetic fluorescent labeling (ECFP labeled epithelia-MMTV-iCre/CAG-CAC-ECFP transgenic mice), and administration of high-molecular weight dyes (155 kD dextran-TMR) to define local vasculature, denoted by the solid yellow lines. The window design, used previously for murine lung imaging⁸, includes three etched lines on the frame (Figure 4, inset) which act as fiducial markers that allow the use of microcartography³⁰. Microcartography permits serial imaging of the same region of interest of the murine pancreas over multiple days. Here the same lobule of the pancreas (yellow dashed lines) is visualized on Day 1 and relocalized on Day 2, as evidenced by the presence of the same local blood vessels (Figure 4). Images were acquired each day as a single z-stack with 11 slices and 2 µm step size at ~1 slice/s, taken at 880 nm illumination, and resolution of 0.67 µm/pixel (FOV = 340 x 340 µm). Laser power and PMT gains were chosen to maximize the signal while minimizing photobleaching and photodamage.

In addition to serial imaging, the SWIP is well suited to long-term longitudinal imaging, enabling the accurate tracking and measurement of the dynamics of subcellular structures (such as vacuoles) under control and treatment conditions. Here, vacuoles are regions where cytoplasmic fluorescent proteins are excluded, causing dark unlabeled holes to appear (Figure 5A). Marking of vacuoles using software such as ROI Tracker²⁴, allows visualization of vacuole motility (Figure 5A,B) and quantification of numerous motility parameters. For example, the average speed of vacuoles in the murine pancreas increased by approximately 10% after treatment with cerulein to induce pancreatitis, compared to PBS treatment (0.37 ± 0.07 µm/min vs 0.41 ± 0.09 µm/min, p = 0.02) (Figure 5C). Cerulein treatment also increased the average turning frequency of sub-cellular structures by 10% vs PBS treatment (2.3 ± 0.3 deg/min vs 2.6 ± 0.6 deg/min, p = 0.04) (Figure 5F). However, there was no significant difference (p > 0.05) in net speed, directionality, or cumulative distance traveled between cerulein treatment and PBS treatment (Figure 5D,E and Figure 5G). Images were generated on a custom-built multiphoton microscope²⁴ illuminating at 880 nm and acquiring a z stack-t lapse (FOV size = 340 x 340 µm, pixel size 0.67 µm) with 2.9 min between frames, 11 slices with a 2 µm step size, and ~1 slice/s.

Finally, the SWIP enables visualization and capture of tumor cell migration. Figure 6A shows a still from a time-lapse movie (Supplemental Video S1 and Supplemental Video S2) of the migration within the pancreas of Dendra-2 labeled KPC tumor cells that were orthotopically injected. Both collective migration of cells in clusters (Figure 6B and Supplemental Video S1) and single-cell migration (Figure 6C and Supplemental Video S2) can be observed over short periods (<1 h). Images were generated on a custom-built multiphoton microscope²⁴ illuminating at 880 nm and acquiring a 4 x 4 mosaic-z stack-t lapse with 20% overlap between tiles (tile size = 340 x 340 µm), 3.4 min between frames, 3 slices with a 5 µm step size, and ~1 slice/s. Laser power and PMT gains were chosen to maximize the signal while minimizing photobleaching and photodamage.

Figure 1: Improved stability of imaging due to SWIP. (A) Stills from within the first 72 min of a timelapse movie of the pancreas imaged through the SWIP. Some axial but very little lateral drift can be observed. Scale bars = 50 µm. (B) Continued timelapsed imaging after 72 min shows a high level of axial and lateral stability. (A,B) Red = 155 kDa tetramethylrhodamine dextran-labeled blood serum, Cyan = CFP-labeled pancreatic cells (MMTV-iCre/CAG-CAC-ECFP transgenic mice) (C-E) Comparison of the lateral stability of each of the pancreas imaging windows during the first hour of imaging for the (C) AIW, (D) PIW, and (E) SWIP. (F-H) Comparison of the lateral stability of each of the pancreas imaging during the subsequent 150 min for the (F) AIW, (G) PIW, and (H) SWIP. Insets are zoomed-in views of the corresponding plots. (I-K) Comparison of the axial stability of each of the pancreas imaging windows for the first 120 min of imaging for the (I) AIW, (J) PIW, and (K) SWIP. This figure is from Du et al. Open Biology 2022, DOI:10.1098/rsob.210273 https://royalsocietypublishing.org/doi/full/10.109 8/rsob.210273.¹⁵. Abbreviations: SWIP = stabilized window for intravital imaging of the pancreas; AIW = abdominal imaging window; PIW = pancreas imaging window; CFP = cyan fluorescent protein. Please click here to view a larger version of this figure.

Figure 2: Overview of the surgery protocol of orthotopic injection of pancreatic ductal adenocarcinoma cells (KPC). (A) Illustration of how to hold surgical forceps. (B) Illustration of how to hold Castroviejo scissors. (C) Illustration of how to hold the vacuum pickup tool. (D) Sanitizing the skin. (E) Incision in the skin. (F) Incision in the muscle. (G) Splayed pancreas. (H) Insertion of needle into the desired site of injection. (I) Injection of cancer cell suspension creating a bubble (blue arrow). (J) Pancreas returned to peritoneal cavity. (K,L) Closing of the muscle layer with an interrupted silk suture. (M,N) Closing of the skin with an interrupted silk suture. (O) Cyanoacrylate glue applied to incision. Please click here to view a larger version of this figure.

Figure 3: Overview of the surgery protocol of the Stabilized Window for Imaging of the Pancreas. (A) Identification of spleen and attached pancreas. (B,C) Circular incision in the skin and muscle. Red dashed lines in A-C indicate the outline of the spleen that can be seen through the abdominal wall and skin. (D,E) First stitch of cross-stitch basket placed and tied in the muscle layer (E, yellow arrow). (F) Stitch is continued to the opposing side of the muscle incision, leaving a tail (white arrow). (G,H) Second stitch placed perpendicular to the first, tied at one end (yellow arrows) and leaving a tail (white arrows). (I,J) Placement of the pancreas into the cross-stitch basket. (K) Circumferential purse-string suture through muscle and skin. (L) Implantation of the window frame. (M) Application of glue to the window frame. (N) Attachment of glass coverslip. (O) Tail ends of the cross-stitch tightened. (P) Completely implanted SWIP. Abbreviation: SWIP = stabilized window for intravital imaging of the pancreas. Please click here to view a larger version of this figure.

Figure 4: Microcartography used for serial imaging to relocalize the same areas of interest within the optical window. Intravital imaging of a single region of murine pancreas, showing the same lobule relocated over 2 consecutive days (D1-D2) using microcartography. Yellow dashed lines outline the boundaries of the same lobule. The solid lines highlight the same blood vessels (as evidenced by the presence of red labeled blood serum within the lumen) identified each consecutive day. Red = 155 kDa tetramethylrhodamine dextran-labeled blood serum, Cyan = CFP labeled pancreatic cells (MMTV-iCre/CAG-CAC-ECFP transgenic mice) (Inset) Image of the SWIP with scratches for microcartography. The use of microcartography to relocate regions of interest during serial imaging is permitted by the three etched lines on the window frame (inset). Scale bars = 50 µm. Please click here to view a larger version of this figure.

Figure 5: Subcellular resolution and the measurement of subcellular dynamics using SWIP. (A) Single cells (dashed yellow outlines) and subcellular structures such as vacuoles can be visualized through the SWIP over time in the murine pancreas. Examples of these vacuoles are indicated by yellow arrows. The SWIP thus allows tracking of subcellular structures (colored outlines and tracks overlaid on the fluorescence image) over time. Inset is zoomed-in view of a yellow boxed area showing three tracked vacuoles. Scale bar = 50 µm. (B) Trajectories of subcellular structures, such as nuclei and vacuoles (highlighted in A), after a shift of coordinates to the origin point. Red dashed line shows the average net path in 1 h (3.46 µm). Quantification of the dynamic parameters of (C) average speed, (D) net path, (E) directionality, (F) average turning frequency, and (G) cumulative distance. Red = 155 kDa tetramethylrhodamine dextran-labeled blood serum, Cyan = CFP-labeled pancreatic cells (MMTV-iCre/CAG-CAC-ECFP transgenic mice). Abbreviations: SWIP = stabilized window for intravital imaging of the pancreas; CFP = cyan fluorescent protein. Please click here to view a larger version of this figure.

Figure 6: Capturing cell migration with SWIP. (A) A still image from a timelapsed intravital imaging movie of the pancreas in an orthotopically injected mouse model of PDAC. (B) Still images from Supplemental Video S1 showing an example of tumor cells undergoing collective migration (yellow arrow). (C) Still images from Supplemental Video S2 showing examples of single-cell migration of a tumor cell (yellow arrow) and a macrophage (red arrow). Green = Dendra2-labeled tumor cells, Blue = CFP-labeled macrophages, Red = 155 kDa tetramethylrhodamine dextran-labeled blood serum. Scale bars = 50 µm (A), 15 µm (B,C). Abbreviations: SWIP = stabilized window for intravital imaging of the pancreas; CFP = cyan fluorescent protein; PDAC = pancreatic adenocarcinoma. Please click here to view a larger version of this figure.

Supplemental Video S1: Timelapse intravital imaging movie showing tumor cells undergoing collective migration corresponding to Figure 6B. Green = Dendra2-labeled tumor cells, Blue = CFP-labeled macrophages, Red = 155 kDa tetramethylrhodamine dextran-labeled blood serum. Please click here to download this File.

Supplemental Video S2: Timelapse intravital imaging movie showing tumor cells undergoing single cell migration corresponding to Figure 6C. Green = Dendra2-labeled tumor cells, Blue = CFP-labeled macrophages, Red = 155 kDa tetramethylrhodamine dextran-labeled blood serum. Please click here to download this File.

Discussion

The SWIP protocol described here provides an improved method of pancreas tissue stabilization by utilizing a cross-stitch basket technique. Early abdominal imaging windows (AIWs) enabled intravital imaging (IVI) of internal organs of the abdomen but did not adequately limit the movement of soft tissues such as the pancreas. In response, Park et al. developed a pancreas imaging window (PIW) that incorporates a horizontal metal shelf and allows improved stabilization of the pancreas tissue while maintaining contact with the glass coverslip. While this approach improves lateral stability, it limits imaging of solid pancreatic tumors because their size exceeds the narrow space between the shelf and cover glass. The SWIP addresses this issue by stabilizing the pancreas with a cross-stitch basket, limiting both axial and lateral movement, while also being able to accommodate large (≤10 mm) solid tumors.

Direct comparisons of the SWIP with previous imaging windows such as AIW and PIW were conducted previously¹⁵. This is shown in Figure 1, adapted from Du et al.¹⁵, where lateral and axial shifts were quantified by tracking cellular anatomical features such as nuclei or blood vessels. All imaging windows exhibited a need for a period of settling during approximately the first hour of imaging. During this time, a higher degree of lateral movement was observed with the AIW and PIW compared to the SWIP. A greater axial shift was also seen with the AIW and PIW compared with the SWIP over a 2 h period. Overall, the SWIP displayed the lowest level of drift and is suitable for long-term imaging (≤12 h).

Unfortunately, the pancreas is a highly scattering tissue, and as such, the point spread function for the multiphoton microscope used in IVI is rapidly degraded with penetration into the tissue. Thus, the imaging depth with any of the optical windows is limited to only ~30-60 µm. IVI also carries the risk of light-induced damage to the sample. This can be tested at the beginning of imaging sessions by acquiring a time series of 100 images and looking for signs of photobleaching or photodamage. On our microscope system, we found that a maximum power of ~15 mW at the sample can be used without adversely affecting the tissue.

The SWIP protocol allows stable, high-resolution, single-cell optical IVI of the murine pancreas in both normal healthy conditions, as well as diseased states such as pancreatitis and PDAC. This makes the SWIP particularly useful for long timelapsed imaging, as well as for performing 3D and 4D (3D + time) imaging by capturing multiple z slices (taking advantage of multiphoton and confocal microscopy's inherent optical sectioning capabilities). By enabling in vivo visualization of single cells and their interactions with the cellular constituents of the pancreas, high-resolution IVI will prove invaluable to understanding mechanisms underlying diseases of the pancreas.

A certain level of technical expertise is necessary to perform the SWIP protocol. However, with proper practice and attention to key steps, the procedure can be executed with a high success rate. To image the malignant pancreas, it is crucial to first have a successfully implanted tumor. This is obtained by orthotopic injection of a suspension of murine pancreatic cancer cells into the pancreas of the mouse. A successful injection is observed when the parenchyma of the pancreas inflates into a fluid-filled bubble and has been described in detail previously²¹. To ensure maximum success and survival, it is vital that there is minimal-to-no leakage of the cell suspension, as leakage will dramatically reduce potential tumor size as well as lead to carcinomatosis. Additionally, the pancreas is a highly vascularized organ, with many branched blood vessels. It is critical to avoid lacerating any vessels as this will cause bleeding and subsequent hematoma in the pancreas and inhibit tumor growth. In this model, we have used a syngeneic PDAC cell line derived from KPC mice²⁰. This allows tumor engraftment in immune-competent mice. Other syngeneic PDAC cell lines may be used depending on the strain of the mouse used. Human PDAC cell lines may also be used; however, the mouse strain must be immunecompromised so as to avoid rejection of the tumor implantation.

The size of the tumors undergoing imaging in this protocol can be modified as needed. This can be accomplished by using a higher concentration of cancer cells implanted into the murine pancreas to obtain a larger tumor or by extending the growth time of the tumor before window implantation. In this study, we injected 10⁶ KPC PDAC cells and implanted the SWIP 10-14 days afterward, when the tumors were palpable. Smaller and larger tumors can also be accommodated by this SWIP protocol using the appropriate placement of the tissue into the cross-stitch basket.

Secure placement of the imaging window and pancreas is also crucial to obtain high-quality imaging and to limit motion artifacts. The normal murine pancreas is very compliant and prone to movement from breathing and nearby peristalsis. To address this and increase the stability of the pancreas while imaging, a previously described cross-stitch basket technique is utilized³¹. The cross-stitch basket is designed to mimic the body's use of ligaments. By cradling the tissue against the glass coverslip and applying constant axial pressure, it prevents both lateral and axial motion. When dealing with larger solid tumors, the support point and direction of the cross-stitch can be modified to best accommodate the size and position of the tumor for optimal support.

Suboptimal imaging can also happen when the window frame is inadequately implanted into the abdomen of the mouse. A loosely-fitted window frame can lead to imaging difficulties and motion artifacts. An appropriate purse-string suture can address this issue by securing the window frame to the abdomen through the skin and abdominal wall. To avoid excessive skin folding when tightening the purse string, the stitch steps should be no greater than 5 mm between steps that are placed no greater than 1 mm from the tissue edge, ensuring a snug fit around the window frame. Following the purse-string suture, pancreas placement in the cross-stitch basket, and implantation of the window frame, one last critical step is the placement of adhesive within the window frame's recess. It is crucial that during this step, no adhesive contacts the pancreatic tissue as this will damage the tissue and confound imaging. A potential modification that also can keep the glue from contacting the pancreas tissue is to glue the glass coverslip to the window frame and allow both to dry before surgical implantation into the abdomen.

The SWIP, like all intravital imaging techniques, has limitations in its ability to reveal information about other cells and structures in the tissue that are not explicitly labeled. However, combining the SWIP window with fluorescent reporters (such as ECFP-expressing epithelia and Dendra2-labeled KPC cells as in this study) and controlling protein or cell states with pharmacologic and/or optogenetic tools can eliminate these limitations.

In addition, the SWIP window design includes three etched lines on the window frame, serving as fiducial markers for microcartography to relocalize areas of interest during serial imaging³⁰. This enables locating the same field of view multiple times, even in unmarked tissue.

In summary, the SWIP can be used in normal healthy pancreatic tissue as well as in benign and malignant pancreatic diseases such as pancreatitis and PDAC. Single-cell and sub-cellular dynamics can be captured in these states using the SWIP and can help researchers understand important physiological events such as metastatic dissemination in PDAC. The enhanced quality and stability of IVI have the potential to provide valuable insights into the pathophysiology and cell biology of the pancreas, making it a promising and beneficial tool.

Materials

Name	Company	Catalog Number	Comments
1% (w/v) solution of enzyme-active detergent	Alconox Inc	NA	Concentrated, anionic detergent with protease enzymes for manual and ultrasonic cleaning
5% (w/v) solution of sodium hydroxide	Sigma-Aldrich	S8045	Passivation reagent
5 mm cover glass	Electron Microscopy Sciences	72296-05	Round Glass Coverslips
7% (w/v) solution of citric acid	Sigma-Aldrich	251275	Passivation reagent
28G 1 mL BD Insulin Syringe	BD	329410	Syringe for cell injection
Baytril 100 (enrofloxacin)	Bayer (Santa Cruz Biotechnology)	sc-362890Rx	Antibiotic
Bench Mount Heat Lamp	McMaster-Carr	3349K51	Heat lamp
Buprenorphine 0.3 mg/mL	Covetrus North America	059122	Buprenorphine Analgesia
Castroviejo Curved Scissors	World Precision Instruments	WP2220	Scissor for cutting tissue
C57BL/6J Mouse	Jackson Laboratory	000664	C57BL/6J Mouse
Chlorhexidine solution	Durvet	7-45801-10258-3	Chlorhexidine Disinfectant Solution
Compressed air canister	Falcon	DPSJB-12	Compressed air for drying tissue
Cyano acrylate - Gel Superglue	Staples	234790-6	Skin Glue
Cyano acrylate - Liquid Superglue	Staples	LOC1647358	Coverslip Glue
DPBS 1x	Corning	21-031-CV	DPBS for cerulein/cell injections
Gemini Cautery Kit	Harvard Apparatus	726067	Cautery Pen
Germinator 500	CellPoint Scientific	GER 5287-120V	Bead Sterilizer
Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4" Length	Roboz Surgical	RS-5135	Graefe Micro Dissecting Forceps
Imaging microscope	NA	NA	See Entenberg et al. 2011 [27]
Imaging software	NA	NA	See Entenberg et al. 2011 [27]
Isoethesia (isoflurane)	Henry Schein Animal Health	50033	Isoflurane Anesthesia
Kim Wipes	Fisher Scientific	06-666-A	Kim Wipes
Laboratory tape	Fisher Scientific	159015R	Laboratory Tape
Mouse Dissecting Kit	World Precision Instruments	MOUSEKIT	Surgical Instruments
Mouse Paw Pulse Oximeter Sensor	Kent Scientific Corpo	MSTAT Sensor-MSE	Pulse Oximeter
Mouse Surgisuite	Kent Scientific	SURGI-M04	Heated platform
Nair Hair Removal Lotion	Amazon	B001RVMR7K	Depilatory Lotion
Oxygen	TechAir	OX TM	Oxygen
PERMA-HAND Black Braided Silk Sutures, ETHICON Size 5-0	VWR	95056-872	Silk Suture
Phosphate Buffered Saline 1x	Life Technologies	10010-023	PBS
PhysioSuite System	Kent Scientific	PhysioSuite	Heated Platform Controller
Puralube	Henry Schein Animal Health	008897	Eye Lubricant
Puritan Nonsterile Cotton-Tipped Swabs	Fisher Scientific	867WCNOGLUE	Cotton Swabs
SHARP Precision Barrier Tips, For P-100, 100 µL	Denville Scientific Inc.	P1125	100 µL Pipet Tips
Tetramethylrhodamine isothiocyanate–Dextran	Sigma-Aldrich	T1287-500MG	Vascular Label
Window-fixturing plate	NA	NA	Custom made plate for window placement on microscope stage. Plate is made of 0.008 in stainless steel shim stock. For dimensions of plate see Entenberg et al., 2018 [8].
Window Frame	NA	NA	The window is composed of a steel frame with a central aperture that accepts a 5 mm coverslip. A groove of 1.75 mm around the circumference of the frame provides space for the peritoneal muscle and skin layers to adhere to. See Entenberg et al., 2018 [8].