Encyclopedia of Experiments: Biology
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Transcript
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.
