This method can help us to visualize the movements of blood vessels within the mouse liver along evolution of the dynamics of the liver in multiple dimensions. Using this method, we can visualize the dynamics of blood vessels within the liver in three dimensions. We have demonstrated the use vascular dye, but you can perform the vessel staining with your substance of interest before microscopic visualization.
After confirming a lack of righting reflex in an anesthetized eight week old male C 57 black six mouse. Wipe the mouse's tail with 75%alcohol and use a one 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 five 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 two 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 60 X 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.
Turn on the laser and press and hold the power button and shutter for three seconds. Then start the microscope operating software and set the laser to 800 nanometers. To set the image acquisition control click the fluorescent switch and turn off the room and equipment lights.
Open the light path shutter on the microscope and rotate the fluorescence filter to the fourth gear. Pull the two levers of the optical switch and looking through the eye piece, use the course and fine focusing quasi spirals to adjust the focal length. Then use the X Y axes to adjust the field of view to locate the target area.
For imaging, turn off the fluorescent switch in the software and switch the fluorescent filter to the second gear. Push the two levers of the optical switch and click focus times two to preview the target area adjust the acquisition settings and the image acquisition control parameters and press control plus C to adjust the high voltage gain and offset. Click stop to stop the preview, click the X Y button to scan in two dimensions and click save.
Select a region and click depth and preview to select the end and start sets in the microscope settings. Scan 3D images and save. After scanning, select a region click time and adjust the other acquisition settings and image acquisition controlled parameters.
Then scan through the different slices and save to obtain the resulting image movie. Here visualization of the distribution of blood vessels within the liver using multi photon microscopy as demonstrated, can be observed. This blood vessel is divided into a plurality of branches emanating from a trunk and distributed into the surrounding space.
The outer circumference of the blood vessel can be observed in red while the inner cavity is dark, as observed in the video non red objects moving within the vessels could be cells. The darkness of the tissue observed in this video is likely due to the liver not having been fixed well over time. It is important to remember that how scar-less liver is fixed, affects the stability of the image.
This methodology can also be applied to the position of other structures within the liver for the direct observation of the distribution or changes in specific labor structures of interest.
