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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. Move the sample so the objective is over the middle of it and hit "Focus" in ScanImage. In this case the objective is on a motorized stage and can be controlled using both a hand-pad and ScanImage. 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 the very top of the sample surface. The image looks noisy as we are only just imaging the top of the sample and are using a relatively low laser power with a 12 kHz scanner. 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 will 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.

Choosing an imaging area

BakingTray images either the same rectangular area in all sections (Manual ROI) or automatically finds the tissue to image in each section (Auto-ROI). In both cases you initially need to acquire a preview tiled image of the sample. The rectangular area begins at the front/left coordinates defined in Prepare GUI. If your sample is not a brain, roughly position the objective at this location and hit the "Set front/left" button. If your sample is a brain and you're imaging from cerebellum to the bulb, find the ventral midline (you may see the basilar artery if you're at the right point) and press "Set ventral midline". This will automatically set the front/left position. For this option to work, the ventral surface will need to need to be facing towards the right as you look at the sample standing in front of the microscope.
The instructions below are for imaging one sample only. If you have multiple samples in the same agar block, refer to "Imaging multiple brains at once" once you have read through rest of this page.
You will now open the Preview GUI, from which you will select the XY area to image. Press the green "Preview" button in the main BakingTray window to open the Acquisition GUI.
Press the "Preview Scan" button on the right (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:
Don't worry about the tile illumination artifacts, those will be cleaned up during final image assembly by StitchIt.
Once the preview scan is taken, you can middle-click on it to move the X/Y stages to that location.
After the preview scan completes the resonant scanner (if present) will remain on. This is to allow it to achieve a steady temperature and reduces drift in the bidirectional phase delay.

Manual-ROI acquisitions

These instructions are for manual ROI acquisitions. For Auto-ROI skip to the next section. Use the manual ROI if you want to image a sub-region of the sample or if you have reason to believe the auto-ROI might struggle with your sample (e.g. it has very low SNR or tends to be extensively occluded by membranes)
These instructions for the manual ROI pick up from the previous image where we are too close to the ventral surface of the brain. The easiest way to correct this is to use the box-draw button (the small B to the left of the Preview button). In this case we zoom out first (the - button in the GUI) then click the B. A box is drawn around the original imaged area.
You can now 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.
Note that once the preview scan is complete, the text on the top left of the Acquisition View will report the stage coordinates as the mouse is moved over the preview image. You can use these values directly to set a new front/left position in the Prepare GUI edit boxes for the front/left position.
Reading the stage coordinates
If the area to be imaged needs to be a different size, you can also manually edit the area to image in the main view (use the tiles as a scale). Re-take the preview scan to confirm you selection.
When the sample is framed to your satisfaction, you can move on. Remember to take into account the changing size of the sample at later sections and also any tilt in the mounting of the sample that might cause it to appear to translate across the imaged field as cutting progresses.

Auto-ROI acquisitions

The auto-ROI has been extensively tested and is unlikely to result in data loss through mis-placing the ROI. Auto-ROI acquisitions are quicker to set up and generally run faster because fewer tiles are imaged. Once you have run a preview scan in auto-ROI mode a green border will appear at the image edge. This region is for determining the 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.
Once you have checked this (and if necessary re-drawn the ROI and re-generated the preview), perform the next steps in 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.
More details on the auto-ROI can be found here.
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