After preparing the mouse for surgery, identify the target area, seven millimeters to the left of the sternum and about seven millimeters above the subcostal margin. Lift the skin over the identified area using forceps and make a 10 millimeter circular incision using sharp micro dissecting scissors to cut only the skin. Then remove the soft tissue over the rib cage and cauterize any major blood vessels at both ends using a cautery pen.
Elevate the sixth or seventh rib with forceps and use a single blade of the blunt micro dissecting scissors to pierce the intercostal muscle. Now gently rotate the scissors and make an approximately 10 millimeter incision in the intercostal space while avoiding contact with the lung tissue and the ribs. Release compressed air at the wrist or hand delicately to determine the suitable flow strength and clear the nozzle of debris.
Then apply short bursts of air at the incision to avoid causing lung injury. With the forceps, lift the sixth or seventh rib and insert the retractor into the intercostal incision with blades parallel to the chest wall. Then release the handle carefully to let the retractor open and reveal the lung gradually.
Now apply two to three drops of sterile PBS warmed to 37 degrees onto the exposed lung tissue to prevent it from drying. Overlay a piece of aluminum foil with a small cutout over the mouse, positioning it directly over the exposed lung tissue. Ensure the cutout only shows the open thorax and the nearby tissue for targeted lung photo conversion.
Next position a box with a cutout over the mouse and aluminum foil, ensuring the cutout aligns with the exposed lung. And set the photo conversion lamp directly above the cutout on the box. Then activate the photo conversion lamp to illuminate the exposed lung tissue for a continuous span of six minutes.
After the photo conversion, deactivate the lamp and clear the lamp box and aluminum foil mask away from the mouse. Separate the lung from the chest wall by discharging air at the wrist, and then discharge air at the chest incision. Firmly grasp the retractor handle and squeeze it to close the blades.
Carefully maneuver the closed retractor out of the intercostal space, ensuring not to contact the lung tissue. Then use the 5-0 silk suture to close the intercostal incision with a simple continuous suture pattern, ensuring that the rib on both sides of the incision is incorporated. Pull the suture firmly to ensure the wound edges align, re-establishing the chest wall's integrity.
Secure the suture by knotting the end four times. Trim the suture tails as close to the knot as possible, and close the skin using surgical staples. To remove excess air from the thoracic cavity, insert a one milliliter syringe with a 28 gauge needle below the xiphoid process.
Draw the syringe back to approximately one milliliter. Remove the syringe from the mouse and discharge the air. Images of exposed Dendra2 expressing lung metastases prior to blue light exposure are shown.
Exposing Dendra2 expressing lung metastases to blue light showed that six minute exposure produced the brightest signal in the photo converted channel in both ex vivo and in vivo samples. Photo converted and non-photo converted cells were identified in all three tissue types from mice that underwent surgery with blue light exposure. Only non-photo converted tumor cells were found in the tissues of mice that underwent surgery without blue light exposure.
