Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

We developed a tool to perform intravital imaging on live mice using an inverted confocal microscope. This enables us to track skin cell dynamics throughout homeostasis and compare cell behaviors during disease onset and progression. Multi-photon microscopy is the primary approach applied to achieve intravital cell tracking.

However, the disadvantages of these microscopes include limited day-to-day imaging versatility, high cost, and technical complexity. Our new tool enables the use of versatile inverted confocal microscopes for in vivo intravital imaging. By modifying the design file, the stage insert dimensions can be customized to fit any make of inverted microscope.

The objective hole can be resized and repositioned to fit any animal model and tissue of choice. To begin installing the 3D printed live imaging insert on the inverted microscope stage, carefully place the insert into the large microscope stage groove. Using screws, secure each corner of the insert.

Slide the heat plate into the side opening of the insert in a plug side down orientation so the plate lays atop the lower insert grooves with the plug port underneath the stage. Align the grooved circular opening in the insert with a 40X silicone oil immersion objective and apply silicone oil to the center of the objective. Then using a syringe, apply a small amount of vacuum grease along the grooved circular opening.

Lay the coverslip disc on top of the opening to seal it onto the insert. Finally, raise the 40X objective until it barely touches the bottom of the glass coverslip. Before placing the anesthetized mouse on the live imaging insert installed on the inverted microscope stage, thread the isoflurane tubing with the attached nose cone through the corner tubing bracket on the insert.

After plugging the heat plate into the controller containing an attached anal thermometer probe and switching it on, transfer the anesthetized mouse from the induction chamber onto the heat plate. Then secure the nose cone onto the mouse. Insert the anal probe into the animal and adjust the controller to maintain a stable body temperature of about 36 degrees Celsius.

Cover the properly positioned mouse with a plastic cling wrap to trap body heat and elevate the body temperature. Position the mouse such that its head aligns with the coverslip disc and immobilize its ear onto the center of the glass coverslip using a metal ear clip or tape. After completing imaging, select the low flow option on the electronic vaporizer to cease isoflurane delivery.

Transfer the mouse to a heating pad until it becomes ambulatory. After removing the coverslip disc, wipe it clean with lens paper and lens solution. Store it properly to avoid scratches for reuse.

Next, detach the anesthetic tubing from the insert bracket and unscrew the insert from the microscope stage. The acquired Z-stack of fluorescently-labeled live mouse ear tissue showed minimal drift in the x, y, and z axes over time, validating the custom 3D printed live imaging inserts. The time points from a three-hour timelapse movie of the mCherry positive mouse ear epidermal cells of an adult male mouse revealed a single Z-plane remaining in focus over the extended time.

Similar observations were made from another three-hour timelapse movie of GFP positive ear dermal fibroblasts of an adult female mouse.