Introduction: LMD Software Tutorial
The Leica Laser Microdissector (LMD) is both a microscope and a laser controlled by computer software. This enables users to move the laser - not the specimen - for optimal cutting. This allows high precision, real-time cutting, and the best view of your specimen.
The LMD can be used for genomics, transcriptomics, proteomics, metabolomics, and next generation sequencing.
This guide outlines the basics of how to use the LMD software, from setting it up to using it to cut.
Step 1: Opening the Software
Upon opening the LMD software, you should see a screen similar to that which appears above. The largest box on the screen should show the specimen you have placed in the LMD. On the far right is a list of controls for using the laser to cut your specimen, and a list of the cuts you have made, which should be blank.
Step 2: Viewing Collector Tubes
At the bottom left of the screen, you can see a section labeled “Collector Device: Tube Caps.” This section allows you to view the specimen you have collected in the tubes placed underneath the microscope. The tube that is currently selected, which the specimen the laser cuts will fall into, is highlighted in green. The other tubes which are not selected are shown in red. The middle button, which reads “No Cap” above it, will move the microscope so that no tube is underneath the specimen. If you are having difficulty visualizing your specimen, select “No Cap” to make it easier to see, but remember to switch to a tube to begin cutting.
To switch between tubes, simply click on the circle underneath the tube’s name, and the microscope will switch them for you.
Step 3: Viewing Your Slides
Next to the tube caps section is the “Slide Holder” section. This section shows 3 different rectangles for the 3 different slides that can be loaded into the microscope. Red crosshairs will appear on the slide you have selected. To switch between slides, similarly to the tube caps, simply click on the rectangle representing the slide you would like to switch to.
The intersection of the crosshairs represents where the microscope and laser are currently pointed on the slide. If you want to move the laser over a particular part of the slide, simply click on where you want them to move on the slide image, and the microscope will move for you.
Step 4: Creating Fast Overview of Slide
If you would like a small image of the slide in its rectangle in the “Slide Holder” section. Click the button to the bottom right of the section labeled “Create Fast Overview.” The microscope will take a few minutes to image the slides, and the image it takes will appear on the screen when completed.
Step 5: The Microscope Control Window
The Microscope Control Window is important to have open for laser use.
Click the square “MicCtrl” button along the top of the screen to open up the “Microscope Control” panel on the right. At the top, there is the “Contrast Methods” section, which allows you to change the type of light used to visualize the slide. Below that is the “Magnification” setting, which will automatically change the objectives on the microscope.
NOTE: Cutting with the laser is optimized at a magnification setting of 10x or higher.
There are also sliders to adjust the Focus and Light Intensity as well as a 4-arrow control pad at the bottom to move the stage in small increments.
Step 6: The Laser Window
Press the “Laser” button along the top bar. This opens up the Laser Control panel, which allows you to adjust various properties of the laser, such as
- Power - the strength of the laser
- Aperture - the thickness of the laser
- Speed - how fast the laser cuts (only applicable for Draw + Cut setting)
- Specimen balance - increases aperture at end of cut
- Heat guard - the level of protection between the laser and specimen
- Pulse frequency - how often the laser sends pulses at the specimen
It is recommended to make practice cuts on an unneeded part of the specimen whenever the laser settings are changed to make sure that the laser still cuts properly.
Step 7: Calibrating the Laser
This window also features the “Calibrate” button, which allows you to calibrate the laser. Clicking on the button will prompt a message asking if the area of the slide the microscope is on is ok to cut. If your microscope is currently pointed at an important part of your specimen, hit no! The laser will cut 4 crosses into the 4 corners of the image, so make sure the section you have selected is not crucial to your procedure.
Once you are ready to calibrate, press “Yes.” The laser will cut a calibration mark into the slide, and you will be prompted to click on the center of that cut.
This process will repeat 3 more times, and the program will tell you when calibration is complete.
Step 8: Cutting the Specimen: Draw + Cut
Draw + Cut allows you to draw your shapes before the laser actually cuts them, and you can draw as many as you want if you’d like the laser to cut many at a time. To do this, select the bullet that says “Draw + Cut.” Next, look over at the left toolbar on the page.
There are a few different shape cutting options such as Ellipse, Rec, and PtoP. Click on the shape you desire, then draw the shape around the part of the specimen you would like to cut.
A red outline represents where the laser will make its cut. Once you are satisfied with your shapes, select the “Start Cut” button on the far-right window.
Step 9: Cutting the Specimen: Move + Cut
The option Move + Cut allows you to cut the specimen in real-time, the laser moving as your hand moves on the software. Simply select the “Move + Cut” bullet and draw on the screen of where you would like the laser to cut. The laser will move as your hand moves.