포유 동물 조직의 레이저 캡처 Microdissection

Hi, my name is Rob Edwards. I'm the director of the University of California Irvine Laser Capture microscopy facility. I'm going to be describing for you the use of the laser capture microscope that we have here.

Our instrument is a Leica A-S-L-M-D, which we purchased approximately three years ago. There are two different types of laser capture systems. One can be likened to a spot welder where one has a piece of tissue on a glass slide on top of a plastic membrane, which overlies the tissue.

A laser source is then used to melt the membrane, in effect spot welding the tissue onto the membrane and then that membrane is removed, taking the captured tissue with it.Okay. To start the LCM system, we power on a number of instruments in sequence here. We'll start with the A-S-L-M-D control box, which is powered on here.

We then turn on the computer simple enough. If you have a specimen that requires a fluorescence optics for identifying the cells to be captured, you would turn on the mercury lamp for the fluorescence illumination. We're not going to be using that for this demonstration.

If you want to capture high quality images of the before and after specimens, one can turn on the spot camera separately right here after the logic box and the computer are turned on, one next turns on the laser power control unit here, which is actually turned on by a key back here on the bottom of the instrument. And finally, one turns on the computer monitor. There are a number of controls that are present on the screen that you'll need to be familiar with.

You'll use them quite regularly. This is a classic Microsoft Windows based program and so there are a number of menus that we'll discuss. But first let me talk about the the desktop here.

There's a live window which will show you what the microscope is actually seeing. And up across the menu bar here, you can see a button for live images and there's also a freeze button which you can toggle and then use the file menu to capture still images of a portion of tissue, say before capture and then after capture to document the tissue that you've isolated. The microscope can run either in brightfield or fluorescence mode, depending on whether you're capturing hematin counter stain tissue or fluorescently stain tissue.

The next two buttons include the specimen mode, which focuses on the plane of the microscope stage, and then there's a collector mode which moves the glass slide out of the way and allows you to visualize the captured tissue in the top of the collection tube. This is a quality control type setting. Next in the menu bar are a number of drawing tools, which you will use to identify the portions of tissue or cells that you wish to capture.

And then down the right side of the screen there are some additional drawing and capture tools, including some drawing shape options, some cutting options, and then the cutting control buttons. Also on the control panel here at the bottom is the collector device, which is the portion of the microscope below the stage, which holds the einor tubes into which your tissue is captured. And these four buttons labeled A, B, C, and D control which tube is to be moved directly Under the specimen being captured.

This is an Example of a a typical frozen section tissue which has been processed for laser capture microdissection. This, these are coronal sections of a mouse embryonic head which have been captured on a pen foil slide. These were eight micron frozen sections which were then dried and counter stain with hematin and stored at minus 80 degrees Celsius until use.

So to mount these slides onto the microscope, the microscope comes with a special holder and remember that the tissue is going to be captured into an einor tube which lies beneath the plane of the stage. And so the tissue is on this side of the slide. It's actually going to be mounted in the slide holder tissue side down like so.

The slide fits there and is held into place, held in place by spring pressure. And then this intact, this hole. The slide holder is placed onto the microscope stage between these two guides.

You will note that there is a small detent which fits under a clip at the back of the stage holding the specimen in place. And as that is inserted, you feel a positive click. Okay, the next thing to set up is the einor tubes, which will actually capture the tissue.

One has the option of either using 0.2 or 0.5 milliliter einor tubes and they fit into these little holders which then fit in this tray, which goes in a special slot under the stage. And so the tissue will actually be captured within the top of the einor tube cap. And the einor tube is, I'm using a 0.5 milliliter tube for illustrative purposes here.

So the cap goes in like so slide it underneath through, and then the, the lips of the cap are held in place by those teeth and then the cap clips underneath like so, and then this entire cap goes into the holder. Now, if one is capturing nucleic acids, particularly for RNA isolation, one can put a several hundred microliter drop of a triol solution directly in the cap. It's important not to spill this into the instrument of course, because of the phenol and other caustic chemicals in the triol reagent.

This is the microscope controller as I stated before, the objectives and the illumination and focus are fully motorized on this instrument. So the focus knob is here on top. The Y translation control for the stage is this knob.

The X translation control is here. On the bottom to the left is the iris control, which varies the illumination and the, the microscope remembers what illumination level was set at each magnification. So when you change from objective to objective, the illumination is automatically adjusted.

And then finally on the right side of the controller is the objective turret control which moves between the objectives. This instrument has five objectives, a four x, a 10 x, a 20 x, a 40 x, and a 63 x objective. The first step In actually capturing tissue is to align the microscope so that what you see on the screen is actually what you will capture to align the microscope.

We move to the laser menu option and we select calibrate, and we accept we hit continue. Now I am on the four x setting and you can see there's a relatively large area of membrane here. Note there's no tissue.

We do the alignment off on one corner of the slide. The laser has cut a crosshatch in the upper right corner of the screen and I will use the mouse and center it in that screen and then click or in that crosshatch and click the computer cuts another cross hatch. I will align with that and click, and then it cuts a third cross hatch and we align there.

The calibration for the four x objective is now complete for microscope, is now aligned and ready to actually capture tissue. You may have noticed during the alignment process that the cross hatches that were being cut were rather thick. And that's actually because the laser control power was set to a very high setting.

We probably don't need that degree of cutting power. And so I will illustrate how one adjusts the laser power by cutting a a line. And if I draw a free hand, a diagonal line on the screen and then select a a cap and then come over and hit start cut, the computer will cut along the line that I have defined.

And you can see how wide that line is. I I need to back off the laser power. So do that.

I go to the laser menu, I go to control and you'll note that the intensity has been maxed out. I'm going to decrease the laser intensity down to approximately 30 or so and I'll accept that setting. Now if I draw a new line and cut it, you'll see that the width of the burned tissue or of the burned membrane is considerably less.

And so as a general principle, one wants to set the laser control power to as low a setting as possible to minimize the amount of Tissue that is getting vaporized. So I'll Now demonstrate the actual capture of some tissue using this mirroring eye. I am currently in the Forex objective lens and will make an attempt at capturing a relatively large portion of tissue.

We will isolate the entire lens out of the center of this eye. And so to do that I'm going to cut freehand. And so using the drawing control at the top of the screen, I will cut.

I will draw a line around the specimen to be captured. And I'm doing this very low magnification. If one is interested in more accurate cuts or smaller areas of tissue, one obviously can capture at a higher magnification.

So I have selected the area to be cut and I will over in the draw shapes menu, I usually keep the closed lines option on. What closed lines does is if one draws an irregular shape around something and doesn't completely enclose the object to be captured, it draws a straight line across the opened ends of the shape. So with with the area to be cut identified, we will then start the cutting process and you will see that the control box focuses the laser through the areas and it cut almost completely away.

We have a hanging Chad often when capturing large pieces of tissue. One does not get complete separation of the tissue. And so the first thing to try is to just cut it again and the laser will follow the tissue around.

Sometimes you can alternatively just where it's hanging, you can draw a small space and cut that and there it falls. And so we've now captured this portion of tissue. As I mentioned before, we can go to collector mode and verify that that piece of tissue is in fact in the collector or in the einor tube cap.

So I will come up here to our collector control button, push that, and you will see that the microscope will now change the view. And we're now looking at the inside surface of the einor tube and there is the captured piece of tissue in the lower right portion of the screen and actually focus and see the individual cells. So we verified that we did in fact capture the tissue that we selected.

Now, not the capture process does not occur with a hundred percent efficiency because these tissues that are being captured are essentially like falling leaves. A a certain small percentage of captured tissue fragments will not fall within the the einor tube lid, but the loss rate is very low once one is completed capturing the desired tissue. The next step, of course is to remove the einor tube with the captured tissue from the instrument and to securely cap it.

And to do this, we very gently pull straight out on the holder like so. And then keeping in mind that you want the tube cap to stay upright, remove the holder from the tray and then very gently pull the tube out. Slide like so, move out of the holder and then move the einor, not the lid, like so.

Now, if one has already placed a drop of triol in the lid, your tissue will already begin the solubilization process and you can return to your laboratory and complete your RNA isolation. There are a number of manufacturers that produce microscale, RNA isolation kits such as Amon is the manufacturer that we, we prefer. And that's all there is to it.

So today I took you through The laser capture microscope facility here at uc Irvine. We demonstrated how one captures frozen section tissues from pen foil coated slides in preparation for isolating RNA from said tissue. Thanks for your attention and best of luck with your own laser capture Microdissection.