Longitudinal Intravital Imaging Through Clear Silicone Windows

This procedure for casting and inserting silicone windows lowers the technical barriers to longitudinal intravital imaging. Our protocol produces sturdy, easily adaptable windows that are well-tolerated by the animals. Using this technique, we have imaged cancer and immune cells in the liver, pancreas, and mammary gland.

With minimal adjustments, it could be adapted to many anatomical locations and biological questions. For those trying this technique for the first time, practice releasing small amounts of glue from the syringe. Applying too much glue will cloud the imaging field and make the surgical area difficult to work with.

Start casting the silicone window by depositing a small quantity of the freshly prepared silicone polymer, polydimethylsiloxane, of PDMS, on a clean smooth surface, and adjust the volume to area ratio to get the desired thickness. Once the PDMS is deposited on the desired casting surface, lightly press a stainless steel grid into the silicone to provide landmarks for repeated imaging of the same tissue regions. To remove the air bubbles and degas the silicone polymer, place the coated surface in a vacuum desiccator for 45 minutes.

When done, cure the polymer in an oven at 75 to 80 degrees Celsius for 45 minutes. Score the polymer at the edges of the mold and use forceps to gently peel the cured windows from the surface used for casting. Place the mouse on the left lateral decubitus position.

Prepare the anesthetized mouse for surgery by applying 10%volume by volume povidone iodine solution and 70%volume by volume ethanol three times consecutively on the shaved surgical site. Care must be taken to maintain a sterile surgical field. Next, use sterile scissors and forceps to make a 10 millimeter incision on the right flank at three millimeters below the costal arch.

Remove a one square centimeter section of the skin and use a second pair of sterile scissors and forceps to remove a section of the peritoneum slightly smaller than the section of the skin. Deposit a small quantity of surgical adhesive on a sterile well plate and withdraw it with a 31 gauge syringe to apply on the surface of the liver around the edges of the area to be imaged, then position the window on the desired area, and hold it firmly against the liver for approximately two minutes until the adhesive has dried. After folding the edges of the window under the peritoneum deposit a small quantity of the surgical adhesive on the edges of the window that are now under the peritoneum, push down on the peritoneum with forceps to secure it to the window.

Similarly, seal the peritoneum by depositing a small quantity of the surgical adhesive before pushing down the skin, then apply glue around the edges of the window to create a rim that will prevent the skin from growing back over the window. To insert the window in the pancreas, place the mouse on the right lateral decubitus position, and make the incision on the left flank before removing a section of the peritoneum as demonstrated earlier. Using sterile cotton swabs soaked with sterile saline, gently pulled the spleen to visualize the pancreas.

Reposition the pancreas with the moistened cotton swabs to make more surface area visible through the incision. Once the pancreas is accessed, insert and seal the imaging window on the desired area as explained before. To access the mammary gland, place the mouse in the supine position, and make a 10 millimeter incision medial to one of the inguinal nipples using sterile scissors and forceps.

Remove a 0.5 square centimeter section of the skin above the mammary gland, then gently separate the mammary gland from the skin by spreading the sterile scissors between the two surfaces to disrupt adherence. Use a 31 gauge syringe to apply a small quantity of the surgical adhesive on the surface of the mammary gland around the edges of the area to be imaged. Position and hold the window firmly against the mammary gland until the adhesive dries.

After folding the edges of the window under the skin, deposit a small quantity of surgical adhesive on the edges of the window that are now under the skin, and push down on the skin with forceps to secure the skin to the window. Apply the glue around the edges of the window to create a rim that will prevent the skin from growing back over the window. Before beginning the imaging session, clean the window from any residual debris by gently wiping it with a cotton swab dipped in 70%ethanol.

When utilizing a water immersion lens, apply the ultrasound gel to the window avoiding bubbles. To start the imaging, locate and place the grid in focus through the oculars. Then determine the approximate tissue imaging depth by setting the bottom of the grid to zero.

The information will help in locating the corresponding Z-plane in subsequent imaging sessions. To identify the same location over multiple imaging sessions, utilize the squares on the grid as a reference point. During the imaging session, note the grid orientation and the location of each field of view imaged within the grid.

The grid can be used to navigate back to specific grid areas and imaging fields in the success of imaging sessions. Following each imaging session remove any residual lens immersion medium and debris by gently wiping the window with a cotton swab dipped in 70%ethanol. The silicone window allows capturing the dynamic immune cell infiltration over time.

The representative analysis illustrates the neutrophil infiltration within the remodeling mammary gland tissue at days two and four. The silicone window allows tumors to be visualized at multiple time points over weeks after window insertion. The windows cast with 200 milligrams of PDMS are generally better for surgical implantation as they exhibited significantly greater toughness than the windows cast with 150 milligrams.

Of note, the PDMS windows were not as rigid as standard glass, represented by the Young's modulus of the glass windows being significantly greater than PDMS. The window insertion in different organs was well-tolerated as most mice continued to gain weight over time. The most important thing to remember when attempting this procedure is to fully seal the window using glue.

This prevents infection, the tissue from drying out, and the displacement of the window. Following the procedure, orthotopic injections can be done through the silicone window, allowing researchers to study a wide variety of topics, such as cell proliferation, cell-cell interactions, and therapeutic efficacy.