Two photon microscopy allows Image acquisition in areas deep inside the organs. The excitation of fluorescent proteins in two photon microscopy is achieved when two photons of about equal energy interact with the molecule, producing an excitation equivalent to the absorption of a single photon possessing twice the energy. Therefore, in a two photon microscope, infrared lasers are used, allowing imaging acquisition in thick tissues, minimizing photobleaching and photo damage.
Also, since the excitation is restricted to the focal plane, there's no out-of-focus fluorescence. Together with new stereotactic tools and surgical procedures, it becomes a powerful technique to document cellular behaviors in live animals. In this paper, two photon microscopy was used to acquire intra vital images of the thymus.
In this organ, T-cell maturation occurs and therefore this technique can be useful in studies involving thy cyte development. Now let's start the protocols First to prepare the animals irradiate Eight week old nude animals with 900 rads. After irradiation, put the animals back into the cage until bone marrow transfer.
To prepare the bone marrow suspension After euthanasia with carbon dioxide of donor mice and removal of hind limbs, skin and muscle are removed from the bones and reserved in PBS. Transfer the bones to a new Petri dish containing PBS. Cut the ends of the bones.
Fill a syringe with PBS and flush the bones using a 25 gauge needle. Alternatively, transfer the bones to a mortar and smash them with a pestle, homogenized bone marrow with vigorous pipetting centrifuge. Suspension for five minutes at 400 G.Resus.
Suspend cells in PBS and inject intravenously into your irradiated mouse. A certain soft bone marrow occurs during the first days after transfer. Since non reconstituted by 10 pl, mice start to die 10 days after radiation.
However, we wait for six to eight weeks to consider our animals fully reconstituted. To continue the animal preparation. Four to six weeks after bone marrow transfer, embryonic thymus transplantation will be performed to allow observation of thymocytes in vivo.
First, after euthanizing day 15, pregnant females with carbon dioxide and harvesting the embryos, remove the thymus of each embryo and place them in a plate with cold PBS until use anesthetize the recipient animals with ketamine, xylazine, and keep them on top of a heating pad. When the animal is deeply anesthetize, wash the region with ethanol. Cut the skin, cut the peritoneal membrane, localize and expose the kidney.
Once the kidney is exposed, make a small cut in the kidney capsule and a pocket underneath it. The embryonic thymus are added inside this pocket. Finally put the kidney back to its original position and close the peritoneal cavity and the skin.
With stitches, the animal must receive analgesics and be placed on a heating pad until they start to recover from the anesthetics. The ratio between CD four positive and CD eight positive cells can be followed weekly by blood sample analysis. Animals with a ratio around 1.521 accepted the thymus.
The percentages of thy cyte populations in transplanted thymus are similar to the percentages found in endogenous thymus of control.Animals. Intravital imaging of the transplanted thymus will require a new animal surgery and the use of our animal holder device. First, inject ketamine, xylazine, and aesthetics.
Once the animal is unconscious, put it on top of a heating pad intravenously. Inject Rodin b isothiocyanate dextran to allow future observation of blood flow in your tissue. Open the skin and peritoneal cavity to expose the kidney containing the transplanted thymus to prevent the kidney from going back to its original position.
Close the lateral side of the skin incision with stitches. Transfer the animal to the heating pad on top of the animal holder stage. Pinch both sides of the kidney.
Using a clamp made of aluminum foil. Close the animal holder and place the top heater. Probe as close as possible to your tissue.
Add low melting aros on top of your preparation. Cover the whole preparation with the top heater. Monitor the anesthesia and give anesthetic Boosts every 40 minutes.
After finishing the surgery, the whole setup is now Transferred to the two photon microscope for further imaging acquisition. In our case, we used a Prairie Ultima two photon microscope equipped with a tie sapphire laser four top PMTs, and a 20 x water immersion objective. First turn on the laser and the microscope.
Then add RIC mirrors and filter sets according to the wavelengths desired. Put the animal in the microscope. Connect all the heater pads to their controllers.
Add water on top of the top heater aperture, and carefully lower the objective into the water. Focus on blood vessels or GFPT regs using the XY, Z external controller. Quickly find the region of interest.
Adjust the gain on the PMTs to optimize color separation and minimize the laser required to achieve sufficient signal over background. Once the region of interest is located, begin the time-lapse imaging acquisition by acquiring sequential images of 50 micrometer depth tissue volume in our basic setup. Each volume takes 30 seconds to be acquired.
Therefore, a 30 minute image acquisition is reached after 60 sequential Repetitions of this volume. This video shows GFP positive Cells inside the thymus. Note, the cell movement and the blood flow inside the vessels.
However, if the preparation is not good, in this case, the organ temperature is low. The cell stop Moving even as blood flow continues. The method here describe it allows in ViiV observation of thymocytes.
These methods should be very useful to any immunologist that is interest to observe Thymocytes distribution and interactions during T-cell development, despite quite simple one, should take care of few steps to avoid any experimental artifact. For example, you should take care to not damage any blood vessel because this will decrease the oxygenation inside the tissue. Also, the temperature should be kept at 37 Celsius degree all the time.
Finally, the results here acquired should be very useful to validate any other results or obtained by x vivo techniques.
