Hi, I am Melanie Matthew. I'm a graduate student in Micah Hollins lab at the University of California Irvine in the Department of Physiology and Biophysics. And today, along with Dr.Christine Beaton, we're going to show you how we image the responding effect or memory T-cell population in the rat ear.
Dr.Christine Beaton will first induce delayed type hypersensitivity DTH in the rat ear. And then we'll move on to in situ imaging of the rat ear by two photo microscopy. Two days ago I injected GFP labeled of of albumin specific T cells in hyper to a Lewis.
And today I'm going to challenge the same rat in the ear. So in the right ear I'm going to inject of albumin. And in the left ear I'm just going to inject some PBS.
The dose of albumin I'm injecting is 20 micrograms in 20 microliter. And the albumin I'm using is a mixture one to one of Texas Red labeled of albumin and unlabeled of albumin. So this is the to right on which I'm going to show you how I'm going to inject.
So I have my left hand, I'm right-handed. So my left hand I took the glove off to be able to put one finger under the ear and the other one above to stretch the skin so that I can see exactly where I am with the needle and go in between the two layers of skin in the air, which is very thin. So for that I use 27 gauge needles to have something extremely thin to go in there.
So if I turn it around, you definitely see where the Texas red went and where the PBS went. So we are now 42 hours after challenge in here and I'm going to use a spring loaded micrometer to measure ear thickness. So I'm going to measure both ears, the right ear that got challenged with the albumin and the left ear that wasn't challenged and the difference in your thickness will give us an idea of the intensity of the inflammation.
Oh, 62 0 60 0 61, 0 60 or 58 or 57 or 57. And the other ear 4 49 4 55 4 48.4 47 4 46, 4 50. Hi.So Christine Beaton has just given us the rat that she removed after measuring for DTH response.
We've transported them over here on ice. This is the left ear from the rat. And we're here now in Ian Parker's laboratory doing two photon microscopy.
So I'm going to prep these ears for in situ two photon imaging. So I'm going to prep one of the rat ears here at the base of the ear. The tissue's a lot thicker and we have some hair sticking up that's gonna be a little messy if I just start cutting into it.
So first thing I'm going to do is just cut some of that hair off. I'm just gently holding the ear. My finger makes it a lot easier to actually control the tissue when you hold it between your thumb and your forefinger.
I prefer it to using forceps because forceps, I just, I kind of just can't feel what the tissue's doing as easily and we're always worried about damaging the tissue. So we wanna be able to feel what we're doing with it. So I just like using my fingers.
This is the dermal layer peeling away from here in the rat ear. So as I was trimming away some of the hair, I was able to gently trim some of those dermis just kind of peels away. There's some very small dissecting scissors to do so just some nice curved scissors.
You don't wanna jab the tissue and you can work your way up from the base of the ear to the top, just gently cutting away some dermal layer. So we don't need a very big window for imaging. This area just right here will be enough to get some, some nice images of the cells infiltrating the tissue.
So you can see we have some intact blood vessels still here at the base of the ear and pretty much all the way up to the tip of the ear. This whole area will be really good for imaging DTH. So I'm going to go ahead and cut that piece of tissue that I've exposed out to make a little piece of tissue we can glue down to our imaging stage.
So just make a cut here, turn it around. I can cut here. We have a nice little piece of tissue we can prep on our two photon scope for imaging.
These are unbreakable plastic cover slips that we cut to the size of our tissue. And then we mount the tissue onto these using the bet bond. And then we secure these unbreakable plastic cover slips using silicone grease to our insitu imaging stage.
I'm gonna take this small piece of air tissue, it has a little curvature to it, so I'm gonna have to flatten it out as I glue it down. Not ideal, but it'll work. So you just take one of these cover slips and you cut it, kind of estimate the size of your ear and just cut a little larger than you think it's gonna be.
To mount our tissue onto the cover slip. We use a tissue safe super glue called bet long. We get it from 3M and just put a couple drops of that onto our cover slip.
I don't wanna have too much. And you also wanna make sure that it's spread around equally. So I'm just gonna take a little piece of lens paper and use the corner of it to wick the glue across the cover slip here.
Gonna so up a little at the extra glue and as soon as this glue touches water, it will cure. So you wanna be very careful with your tissue sample. So I'm gonna grab the tissue corner of it here that we're probably not gonna image cause it's on the curved part.
I'm gonna dry it off just a little bit. The bottom of it, just on the lens paper so it's all dried off. And I'm going to touch the corner of it, this rat ear tissue to the cover slip.
And it should immediately cure where I've touched it. The rest of, I'm just gonna flatten out just a little bit by gently pressing on it, the edges of it. So hopefully we can have a nice imaging frame.
So now what I'm going to do is cure the glue by dipping it in media. So to prevent the glue from getting on top of the tissue, we flip it upside down and slowly dip it one corner first in our reservoir of media and it should be stuck down nicely and we can image it in our in tissue imaging chamber. So now we're going to mount the piece of tissue that we prepped for in situ imaging, the piece of beer tissue onto the stage.
So here's our piece of tissue with the silicone grease on the bottom of it, and we just place it gently in the flow of media. Now it's stuck to the bottom of the stage is the silicone grease. Brighten the flow of media with our temperature probe right next to it.
And now we're going to focus. So I'm going to turn on our focus, new light, our bright peel. So I'm gonna take our 20 x objective and lower it down into the chamber.
Make sure it doesn't touch the tissue. Look through the eyepiece and focus in on our ear tissue. And there it is.
Here you see autofluorescent green hair follicles in the dermal layer of the rat ear. Surrounding this hair follicles is a dense layer of collagen. This is a layer that we remove for the T photon imaging of the T fector memory cells.
The autofluorescent hair follicles will obscure the image as as the bright collagen of the upper dermal layer. Okay, so here you can see that we have APCs that have taken up Texas red conjugated volin, and they're nestled along these blue collagen fibers. The collagen fibers are blue due to second harmonic generation that we get into photon imaging along the collagen fibers.
We have highly motile tcell. They crawl up and down the collagen fibers and interact with the APCs. So in this video you saw Dr.Christine Beaton, induce adoptive DTH in the Lewis rat.
And then we've showed you our ear prep for imaging, the responsive T effector memory cells, and the rat ear and our two photon techniques to do so.
