Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

The overall goal of this method is to perform long-term, high-resolution, single-cell imaging of the intact lung using a vacuum stabilized imaging window. This method can help answer key questions in the field of cancer research;such as, what interactions secure between stroma and tumor cells during the earliest steps of metastasis. The main advantage of this technique is that the live, intact lung can be theorically visualized at a single-cell resolution for up to 12 hours.

Begin by tying a three inch length of 2-0 suture to a tracheal catheter, one quarter inch above the bushing, with a double-knot. Then apply eye ointment to an anesthetized mouse's eyes and use depilatory cream to remove the hair from the left side of the animal from the mid-line of the chest to a quarter of the back and from the axilla to just under the rib cage. Clean any excess lotion and sterilize the exposed skin with 70%ethanol.

Next, intubate the animal. And tie the 2-0 suture around the snout, under the front teeth. Move the animal to the surgical area, taking care not to dislodge the catheters, and turn on the ventilator.

Connect the ventilator to the tracheal catheter. Then tape the catheter to the snout and the left forelimb to the catheter. After confirming a lack of response to toe-pinch, use sharp scissors to remove one square centimeter of skin above the left chest wall.

Lift the mammary fat pad and cauterize any exposed blood vessels. Next, resect the fat pad and remove the muscle layer down to the rib cage, taking care not to cut the axillary vein at the base of the forelimb. Use forceps to lift the sixth rib.

Then, holding the sharp scissors at a shallow five degree angle, cut the rib near the edge of the opening in the skin. Remove four consecutive ribs to widen the opening in the chest wall, exposing the entire lung lobe. Then carefully transfer the mouse onto the microscope imaging stage.

With the vacuum off, fill the inner chamber of the vacuum window with PBS. Invert the mouse and position the exposed lung over the vacuum imaging coverslip window. Slowly, turn on the vacuum.

Place a restraining harness, made of tissue paper folded in half, twice over the chest of the mouse and tape the harness to the stage plate. Then attach a pulse oximeter to the animal's paw. Then place the environmental chamber on the stage and turn on the heat to maintain the mouse at a physiological temperature.

When the animal is in position, bring the 25 by 95 numerical aperture objective lens near the cover slip. In the epifluorescence mode, view the fitzy channel and bring the lung tissue into focus. Next, load a sterile syringe with 100 microliters of the tumor cell suspension, remove the syringe of PBS from the tail vein catheter, and connect the tumor cell syringe.

Slowly inject the tumor cells into the tail vein. Then reconnect the PBS syringe to the tail vein catheter and locate the tumor cells throughout the lung tissue via the microscope ocular. Now replace the PBS syringe with a syringe loaded with 100 microliters of rhodamine dextran and slowly inject the dextran into the mouse via the tail catheter.

Follow the dextran administration with a 50 microliter, sterile, PBS flush. Then obtain images at each of the previously located tumor cell locations to visualize the flow and integrity of the vasculature. Twenty-four hours post injection, single tumor cells can be visualized interacting with macrophages and monocytes within the vasculature.

The stability of the imaging is such that sequential Z-stacks of the field can be acquired and a three dimensional reconstruction can be generated. Here, a five by five mosaic of imaging fields, covering an 890 micron field of view, over 200 and five minutes, at 15 microns below the surface of the lung of a single metastatic lesion, 12 days post injection is shown. Despite the large field of view, the high resolution of the underlying frames, composing the mosaic, enables the capture of subcellular events, such as the mitosis of a single cell, as evidenced by chromosomal separation.

Intravascular injection of a high molecular weight, fluorescently labeled dextran, results in the labeling of the vascular lumen;although, unlabeled circulating erythrocytes and leukocytes can occlude the dextran. In the small capillaries of the lung, this occlusion results in a flashing of the dextran signal and a loss of definition in the vascular boundaries. The high spatial stability, provided by this protocol, allows time-averaging of the blood channel without blurring, thus restoring the temporary occlusions.

The other signal channels can then be overlayed on the vasculature to provide a clear view of the vessel boundaries. Once mastered, this technique can be completed in about an hour if it's performed properly. While attempting this procedure, it's important to remember to take care not to cut any large blood vessels without first cauterizing them and to avoid touching the lung tissue at all.

Following this procedure, the earliest stages of tumor cell dissemination and proliferation can be visualized. After watching this video, you should have a good understanding of how to perform intravital imaging in the intact lung with a vacuum stabilized imaging window. Thanks for watching and good luck with your experiment.