Step 3: Selecting the imaging area

It's now time to find the surface of the sample and decide which area to image.

Open the laser shutter

The first job is to open the laser shutter. Hit the "Open Shutter" button. Once opened, the button text will switch to "Close Shutter" and Shutter Open indicator will turn from red to green.

Finding the sample surface

Prepare the rig for imaging: turn off the lights then turn on the PMTs, etc. Press the green "Preview" button in the main BakingTray window to open the Acquisition GUI, which we will use for navigating the sample.

This opens the Acquisition View:

Acquisition View. The main axes show a schematic of the slide to which the samples are glued. The dark gray rectangle represents the frosted part of the slide. The blue square is the current position of the objective.

The stages can be moved by performing a middle-click (mousewheel-click) on the image. Middle-click to where you think the sample should be on the slide (use the representation of the frosted area to guide you). Press "Focus" in ScanImage to start Scanning. You should see the sample. If you do not see anything check the laser shutter is open, the PMTs are on, and the laser "Modelock" indicator in the GUI is green. If all that is good, you may need to hunt more in X/Y or search in Z. If you need to search in Z for the sample, the easiest thing is to place the objective a little too close to the sample and move it away whilst imaging.

The following image shows what a brain looks like 25 micron down from the sample surface. You should use a lower power (e.g. 50 to 100 mW) at this stage, as you'll be dwelling at a single FOV for a while and don't want to burn the sample. The image quality can be improved later by turning up the laser power.

Finding the correct imaging depth

Zero the Z position indicator and slice again. You should find that the surface of the sample remains at around the same depth (+/- about 5 microns). If the depth varies by more than about 10 microns, cut again and re-measure. Note that the surface may not be flat in some regions. e.g. if you're cutting from the cerebellum to the bulb, then you should reference from the pons as this area is physically more stable than the cerebellar hemispheres (at least around the transition region between the cerebellum and cortex). Usually the surface height stabilises within a 3 cuts. Sometimes it takes longer and rarely it alternates in a way that suggests the system is alternating between thick and thin sections. If this happens, switch to cutting thicker sections. If this is not an option, try cutting a couple of slightly thicker sections then switch back to your intended section thickness. Once the sample surface is stable, move the objective down to about 20 to 35 microns beneath the surface. This will be your first imaging depth.

MOTOR CONTROLS window in ScanImage Basic

Choosing an imaging area

BakingTray acquires either the same rectangular area in all sections (Manual ROI) or automatically finds the tissue to image in each section (Auto-ROI). The following instructions assume you are using the Auto-ROI, as this is suitable for most situations and is faster to run and set up.

The auto-ROI finds and tracks whole samples visible during the interactive preview scans you are about to take. If a sample is not visible at this stage it will not be imaged by the auto-ROI. Either trim down until the sample is visible or use the manual ROI mode. More details on the auto-ROI can be found here.

Hit the "ROI" button in the Acquisition View and draw a rectangle over the area you wish to do image. You can drag this box to translate it and pull on an edge to re-size it. If you resize, it will snap to the nearest whole tile. Once you move or re-size the box, its size in tiles is shown at its mid-point. Double-click to accept the ROI. Press "Preview Scan" (Until this bug is fixed, wait until the scanning starts before changing any GUI values in ScanImage). The sample will be imaged at low resolution using only one optical plane. The completed preview scan should look something like this:

Auto-ROI preview scan with three samples. The pixels in the green border are used to estimate background. In this case there is sample tissue in the green border so the image needs to be re-taken.

Don't worry about the tile illumination artifacts, those will be cleaned up during final image assembly by StitchIt.

After the preview scan is complete, you may zoom back out to see the whole slide by pressing "Slide".

Once you have run a preview scan in auto-ROI mode a green border will appear at the image edge. This region is used for determining the intensity threshold between sample and agar. It is important there is no sample in the green zone and that the green zone contains agar. If your agar is cropped too close to the sample, alter the look-up table to confirm no out-of-agar pixels exist here. The image above has tissue in the green border and has to be re-done:

The samples have been re-imaged so there is no tissue in the green border. The left brain was mounted so it overhung the slide slightly.

Once you have a good preview image you can move on to the steps in Step 4: Starting the acquisition. After these steps you will turn up the laser power and use the "Auto-Thresh" button to find the sample. Do not do this yet.