The laser microdissection microscope is routinely used for widefield fluorescence imaging of large (normally brain slices) samples. The acquisition of large samples is performed by sequential imaging of several fields of view that are then stitched. Stitching is normally performed by the LAS X software, although other approaches exist (e.g. FIJI).
The microscope is also capable of using a UV laser to brand or cut through thin tissue (5-10 μm) for subsequent analysis of specific sections.
Instructions for fluorescence imaging
1. Switch on the fluorescence lamp and microscope control unit.
2. Start your session and start the LAS X (for fluorescence).
Choose the "DefaultDynamicsWidefieldTree" and the "Standard Configuration". Click "ok".
3. The LAS X software window is divided into three “panes”.
• On the left is the Projects/Acquisition pane. At the top of this pane you can see:
> The Open Projects tab - which is the data management area.
> The Acquisition tab – which controls the microscope (illumination, stage), the cameras, and any other parameters that you may want to configure to acquire the images.
• In the center is the filters/lenses/camera pane. More about this below.
• On the right is the image page. If you would like this pane to use a larger part of the screen, you can grab the divider in the middle of the left side of the image pane with the mouse (see arrow) and make this pane larger.
4. Image acquisition settings.
• Check that the camera being used is the DFC345 FX
• Set the exposure time of the camera. A rule of thumb, while finding field of view and focus, is 200 ms. Slower than that will delay display which will make focusing more difficult. The exposure time can be increased for image acquisition.
• Set the exposure and intensity (by adjusting the FIM – fluorescence
intensity manager) for each image channel that you intend to capture.
Note: If the camera is not the DFC345 FX, click "Preferences" under camera of the center pane. Select the correct camera and click "load".
5. Multi-field of view and stitching.
• Press the square grid next to the xyzt acquisition mode.
• To start defining the region you want to image, check the trash button is inactive (so there are no previous areas marked).
• Press "Tilescan". You can zoom in into the region to see how the software is registering marked positions.
• Add positions by pressing the button at the lower left of the x- y- grid (black/yellow arrow).
• To define a field you need at least 3 positions. It will appear the size of the field as e.g. 5 x 7.
• Check the "merge Images" and "auto stitching" are active. This will result in the software creating a single stitched image at the end of the acquisition.
• If everything is fine, press "start". You will see the current stage position change in the x- y- grid as the stage moves through the defined field.
Instructions for laser cutting
Frequently Encountered problems
Frequently Encountered problems...
... and their solution.
The dissection laser does not appear to work
The independent laser control has to be on and the key to 'on' position. Once the key is 'on' the laser controller box lights an 'error' red LED, which only indicates that the laser is on but not emitting. This is normal, although Leica labeled it with an 'error' sign.
Check on Settings > that the correct laser is being selected (LMD7000)
I don't see anything on the screen although I can see my sample on the eyepiece
Light is not getting to the camera. If you are using the microdissector software, then you need the color camera and two sliders have to be correctly positioned. If you are using the fluorescence only software for large field of view and stichting (LAS X) then you need the black and white camera. For this black and white camera only one slider has to be correctly positioned, in the "in" position.