Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The mammary gland is highly dynamic and undergoes multiple rounds of remodeling throughout life. Our protocol allows capturing the cellular dynamics driving tissue rearrangements in the healthy and diseased mammary gland. Our mammary imaging window with a replaceable lid enables to perform longitudinal intravital imaging of the mammary gland over long time periods, up to several weeks.

The replaceable lid of the mammary imaging window ensures optimal imaging conditions at all times and allows tissue manipulation or drug administration in between imaging sessions. Our method not only provides insights into healthy mammary gland biology, but also allows us to study mammary tumor initiation and progression at the single-cell level. Begin by placing the lid of the mammary imaging window on a sterile surface and applying cyanoacrylate adhesive glue around the entire rim lid perimeter.

Carefully place a 10-millimeter round glass cover slip on top of the lid. Use a sterile wooden stick to position the glass cover slip and apply some force to push it down to ensure an airtight seal between the glass cover slip and the lid's frame. Make sure the glass does not cover up the holes in the arms of the lid.

Disinfect the lids and the mammary imaging window by submerging them in 80%ethanol for at least 30 minutes in a closed Petri dish. Use a cotton tip soaked in acetone to remove the excess cyanoacrylate adhesive glue on the glass cover slip. To prepare the mouse for surgery, shave the area around the fourth mammary gland using a razor blade.

Use the fourth nipple as a landmark and remove the loose hairs using sticky tape. Perform a paw withdraw test to verify if the animal is adequately anesthetized. Gently lift the skin using fine Graefe forceps and use small spring scissors to make a 10-to 15-millimeter diagonal incision in the skin on top of the fourth mammary fat pad Use the fourth nipple as an orientation point and make sure not to cut or damage the peritoneum or the mammary gland.

Separate the skin from the mammary fat pad and underlying tissue layers using blunt dissection of up to five to eight millimeters all around the incision line to create a pocket to fit the mammary imaging window frame. Define the region-of-interest, or ROI, based on the macroscopic features of the mammary tissue, including the location of the lymph node or a tumor lesion. Try to keep the ROI central with regard to the incision.

Pick up the mammary imaging window using sterile forceps and test whether the incision is large enough to insert the window. If needed, slightly enlarge the incision until the mammary imaging window fits in. Remove the window and use a 5-0 silk suture to place a purse string suture one to two millimeters away from the edge of the incision starting from the caudal end and from outside to inside of the skin.

Move approximately five millimeters up along the incision and pass the suture thread through the skin from inside to outside. Repeat this procedure along the edge of the incision, thereby creating a circular suture consisting of five to six loops. The final exit of the suture thread should be located approximately two to five millimeters away from the first entrance.

Fit the mammary imaging window inside the incision and use the Graefe forceps to carefully place the skin in the groove of the window. Make sure to leave the loops of the suture outside. Pull the loops of the purse string suture and gently pull both ends of the suture to tighten it in the groove of the mammary imaging window.

Tie off with surgical knots and cut away the excess thread. Use a toothpick to apply petroleum jelly to the inner rim of the window, avoiding the underlying tissue. Gently push the window frame downwards with sterile forceps and position the lid into the window frame.

This will avoid a volume of air in between the mammary gland tissue and the mammary imaging window lid. Place the tips of the thin forceps through the holes in the arms of the lid and tighten the lid by twisting it clockwise. If some air remains after tightening the lid, introduce a sterile insulin needle through the skin into the cavity between the mammary tissue and the lid and slowly pull out the plunger, thereby creating a vacuum between the mammary gland tissue and the mammary imaging window lid.

Now the lymph node can be easily observed after this procedure. Inspect the condition of the mammary imaging window frame and the lid, including the stability of the sutures or any potential damage to the lid. Position the mouse on the inlay in such a way that the mammary imaging window falls in the hole of the imaging box inlay.

Apply semi-transparent and paper tape over the back of the mouse to stabilize the mouse and reduce tissue motion due to respiration. For imaging sessions greater than three hours, place a flexible needle subcutaneously in the neck of the mouse and secure it with paper tape. Attach a 10-millimeter syringe with a nutrient mix to a flexible silicone tube and push the nutrient mix until it reaches the end of the tube.

Connect the silicone tube to the flexible needle and inject 50 microliters of the infused solution every 30 minutes. Place the lid over the imaging box to close it. Then, secure the imaging box in the stage of the microscope.

Verify that the mouse is stable inside the imaging box. Define the area of interest using the epifluorescence mode of the microscope and apply the microscope settings. Use the Spiral function in the imaging software navigator mode to generate a large overview tile scan and create a reference map of the tissue.

Define the region-of-interests within the Spiral overview and then define the upper and lower Z planes to determine the Z stack. Press Start to obtain the detailed three-dimensional or four-dimensional Z stacks of the tissue. Place the tips of the thin forceps through the mammary imaging window holes in the arms of the lid and loosen the lid by twisting in anti-clockwise.

Alternatively, the tissue can be manipulated or repositioned using sterile blunt forceps to optimize the imaging conditions for the specific region-of-interest. Apply petroleum jelly to the inner rim of the mammary imaging window, avoiding the underlying tissue. Gently push the window frame downwards with sterile forceps and position the lid into the window frame.

Finally, place the tips of the thin forceps through the holes of the arms of the lid and lighten the lid by twisting it clockwise. Pubertal Rosa26 Cre/ERT2 Rosa26-membrane tomato-membrane GFP mice were injected with a low dose of tamoxifen, leading to Cre-mediated recombination of the Rosa26-membrane tomato-membrane GFP allele. The mice were imaged on days one, three, and five to follow the dynamic changes in the developing mammary gland.

The representative images show the 3D rendering of an elongated and branching mammary duct over multiple days imaged through a mammary imaging window. Single Z-plane images of the branching tip and the elongating branch are shown here. Single cells in the stroma are highlighted by white asterisks.

The representative images show the inguinal lymph node of the mouse imaged through a mammary imaging window. The second harmonic generation is depicted in green. 3D-rendered images of a mammary ductal structure of an adult female E-cadherin membrane CFP mouse over multiple consecutive days are shown here.

The collagen I pattern was used to retrace the same ROI and the E-cadherin membrane CFP signal was used to mark the ductal cells. Kikume green-red mice were imaged at day zero before and after exposure to violet light. The imaging sessions were repeated on days two, six, and 10 after conversion.

The same area of the mammary gland was imaged through a mammary imaging window for 10 days. Smaller regions were photo-converted on day zero and showed a similar dilution rate of the Kikume red signal over time, indicating an equal turnover rate throughout the epithelium. Zoom images of the indicated regions show proliferative heterogeneity at the cellular level six and 10 days after photo-conversion.

Quantification of the red to green ratio of different cells 10 days after photo-conversion revealed highly variable dilution rates of some cells within the same ductal microenvironment. A crucial step of this procedure is opening the lid in between the imaging sessions. This unique feature of the window improves imaging conditions and allows for longitudinal imaging of the tissue.

The mammary imaging window with a replaceable lid will facilitate novel questions regarding in vivo, fast acting drug testing and screening in the field of tumor initiation and progression in the mouse mammary gland.