This article describes a method for visualizing the vasculature in living tissue using multiphoton microscopy. The technique involves injecting a fluorescent dye into an anesthetized mouse to label blood vessels, followed by high-resolution imaging of the liver.
Multiphoton microscopy enables the capture of high-resolution, three-dimensional images of the fluorescently-labeled vasculature within the living tissue. Begin with an anesthetized mouse in the supine position.
Carefully inject a suspension of fluorophore conjugated-dextran into its tail vein. The injected molecules flow through the circulation and reach organs like the liver. The fluorescent dye labels the vascular serum, aiding blood vessels' visualization, while the attached high-molecular-weight dextran prevents their leakage from the vasculature.
Prep the mouse's abdominal area. Transfer it onto the base of the organ imaging frame equipped with an imaging lens-containing suction ring connected to a vacuum pump. Incise along its lower sternal border to expose the liver.
Guiding the suction ring to the liver, apply vacuum or negative pressure which facilitates the adsorption of the liver to the lens in the suction ring. This step reduces the motion artifacts during imaging. Transfer the mouse-containing imaging frame onto the multiphoton laser scanning microscope stage.
Cover the lens with saline which acts as immersion medium for high-resolution imaging. During imaging, the focused long-wavelength lower-energy photons allow deeper tissue penetration and excite fluorophores in the blood vessels at the focal point, causing the emission and detection of fluorescent signals.
A series of scans from the selected optical sections at different depths recreates a complete three-dimensional image of the blood vessels within the tissue.
After confirming a lack of righting reflex in an anesthetized 8-week-old male C57BL/6 mouse, wipe the mouse's tail with 75% alcohol and use a 1-microliter syringe equipped with a 30-gauge needle to inject 10 milligrams per milliliter of freshly prepared Rhodamine B isothiocyanate-dextran in 100 microliters of Rhodamine B isothiocyanate-dextran into the caudal tail vein.
When all of the solution has been delivered, use a cotton swab to apply pressure to the puncture site and use gauze wet with sterile water to soak the abdominal fur. Use a razor to shave the abdomen, making strokes in the direction of the fur, and place the mouse onto a 37 degrees Celsius, alcohol-disinfected heating pad.
To fix the mouse liver with a body organ imaging frame, first, place a clean 5-millimeter diameter suction cup in a fixed position and wipe the heating pad and suction cup with 75% alcohol. Connect the suction cup to the vacuum pump hose and turn on the pump.
On a 75% alcohol-sterilized table, use surgical scissors to cut 2 centimeters of skin from the lower sternal border of the mouse and expose the liver. Place the mouse and the heating pad onto the holder base of the body frame and adjust the organ imaging fixture so that the suction cup can hold the liver. Then, use a negative pressure of 30 to 35 kilopascals for suction so that the liver attaches to the cup.
To set up the multiphoton laser scanning microscope, turn on the microscope and select the 60x objective. Fix the frame and the mouse under the objective and add a drop of normal saline to the cup large enough to cover the lens. Adjust the objective so the lens just touches the normal saline and turn on the laser software.
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