Two photon imaging Is useful for immunology because it allows us to follow the migration of a single cell as it encounters its environment. By following thymic site migration. Using two photon microscopy, we can characterize the mechanism of positive and Negative selection.
Hi, I'm Ina Lottie from the laboratory of Dr.Ellen Roby at the University of California in Berkeley. And I'm Paul Herzmark, also from Ellen Robbie's lab. Today we're going to show you how we do in situ imaging of thymus tissue, and I'm gonna show you how we can use our two photon microscope to image the thymus.
And I'll show you the steps leading up to the two photon imaging. This includes the following procedures, isolation of hematopoietic stem cells from bone marrow adoptive transfer of bone marrow cells into neonates, dissection, and bisection of the thymus, preparing the tissue for imaging by two photon laser scanning microscopy. So let's get started.
Before we can prep the thymus For imaging, we must first adoptively transfer bone marrow cells into a neonatal mouse. These bone marrow cells were isolated from the hind legs of transgenic mice that express GFP ubiquitously. This is an insulin syringe with 50 microliters of bone marrow.
We do two to four injections into hosts that are three to seven days old, using bone marrow from one donor for every pup injected. And here we have a five day old pup, pinch the skin back to keep the pup from moving during the injection and to keep the skin topped. Aim for an injection site that is below the lungs and ribcage, but above the gut and stomach.
As you inject, loosen your grip on the skin to allow it to expand. Lastly, slowly remove the syringe to limit the amount of bone marrow that is lost. These mice will be allowed to develop for four to six weeks, and then the thymus can be removed for Imaging.
After four to six weeks, the thymus can Be removed. For imaging, I'll be using a set of large forceps and scissors as well as a set of small forceps and scissors. So this mass has been sacrificed in accordance with our animal use protocol and sprayed down with ethanol.
And I'm gonna use the large forceps to pinch the skin and make an incision at the midline. Now I can either use the scissors to cut down the midline or I can tear the skin away from the peritoneum and I'm gonna pin down the skin to expose the chest cavity. And I'm gonna use the sternum to hold onto while I cut through the peritoneum and I go into the chest cavity through the diaphragm and then cut along the sides of the rib cage.
And you can see the the thymus lying right on top of the heart. I'm gonna put them back. So there's the thymus.
I'm gonna avoid touching it. You use the smaller forceps, still hold onto the tissue around the thymus. Okay, now I'm gonna spray it down with some PBS and start removing some of the excess tissue.
So I'm gonna use the tissue around the thymus to hold onto. When I'm cleaning up the thymus, I'm gonna take off the excess tissue, carefully snipping it away. Now there are two lobes in the thymus that we also have to separate while cleaning it up.
But first I'm gonna remove all of these extra blood vessels that lie on surface of the thymus. I'm very careful not to disturb the architecture of the thymus that we're gonna be imaging. So I'm only pulling tissue off that's right at the surface.
Now I'm gently gonna start separating the two lobes. This is the ventral lobe and the dorsal lobe that we're gonna separate and gently cutting the tissue, teasing them apart. We don't want the tissue to dry out, so we're gonna squirt a little bit of PBS onto it.
Now I've separated the two lobes. So this is the dorsal lobe, which has a characteristic guitar shape to it. And this is the ventral lobe right here.
I'm gonna clean off the excess tissue off of each of these lobes so that the imaging area is free of excess tissue. But I'm gonna leave just a little bit of tissue for us to hold onto the thys. When we're imaging, we're gonna be imaging through this excess tissue, which really isn't necessary, and it'll improve our image quality by removing it.
So now we've got our two bisected lobes that are ready for mounting onto a cover slip for imaging. In order to prep the bisected Thymus for imaging, you will need some veterinary tissue adhesive and 18 by 18 cover slips. I like to use a pipe petman to put a fraction of a microliter of glue onto the cover slip.
So I'm gonna put a drop of glue that's about the length of the thymus that we're gonna be imaging. And then I like to spread it around. So if it's the width of the thymus, again, I'm gonna put both lobes on the same cover slip.
So I'm gonna put another one right here. So I'm gonna pick up the lobe from the excess tissue and just lay it on the cover slip and then drag it to the area where the glue is. I'm gonna do the same for the other lobe, again, touching against the cover slip where there isn't glue to steady my hand and then laying the the other thymic globe on the cover slip.
Now that we've attached both thymic globes to The cover slip, we're going to pass it off to Paul for imaging. Thank you Ina. Let's take this sample and put it in the DME medium that we're going to use to image it.
So this is our two photon microscope setup. Two photon microscopy is very good for imaging immune cells because you can look deep into the thymus and see the fluorescent cells moving around. It uses a pulse infrared laser to get the cells to fluoresce.
And we have in our home belt microscope four different cameras which allow us to simultaneously visualize four different colors and uses a special objective lens, which is a 20 x magnification. And it is a special lens that dips into the DME medium that we've submerged this the thymus into, and that allows it to have a 0.95 na, which is very good, will make our cells very bright. In order to keep the tissue healthy, we bubble 95%oxygen and 5%CO2 through the DMEM medium.
From there, it's pumped over to the perfusion chamber where it's heated to 37 degrees, and that's where the thymus lobes are submerged. With this system, we can keep the cells in the thymus healthy for four hours of imaging. So I showed you how to set up the imaging for the two photon microscope.
Now let's look at some of our results in this movie that's looping. You can see the thymus that we imaged in four different colors here. The red are blood vessels from the thymus and the yellow are dendritic cells from the host that are expressing yellow fluorescent protein under the control of a dendritic cell specific promoter.
The blue and green thymocytes that you see here are from two different donors that we use to make a neonatal chimera using an adoptive transfer technique that Ena showed you earlier. The green cells are thymocytes that are expressing green fluorescent protein, and the blue cells are thymocytes from the donut that was expressing cyan fluorescent protein. This is the same looping movie that I showed you before, but I wanna show you the three dimensionality of the data sets that we collect to get these movies.
We focus on a plane and take an image and then focus a little bit deeper and take another image and a little deeper in another image. And then for this movie, we went down about 60 microns, and then we repeated that volume of images about once every 30 seconds to get the moving volume. And this movie was taken over about 20 minutes.
So we can track the cells through three dimensions over time. We've just shown you How we image the thymus using two photo microscopy. The most important aspects of this procedure are one, injecting the neonates with bone marrow from donors.
Two, dissecting and bi, dissecting the thymus without damaging its structure. Three, firmly attaching the thymus to a cover glass four imaging using a two photon microscope. Imaging in the thymus can answer many questions about the development of thymocytes and the generation of a diverse T-cell repertoire.
So that's it. Thank you for watching and good luck with your Experiments.
