Step 3: Selecting the imaging area

It's now time to find the surface of the sample, make sure the system is cutting consistently, and decide which area to image.

Open the laser shutter

If not done already, 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. Double check the wavelength is correct.

Set the laser power

Set the laser power to a suitable value. Typically about 100 to 150 mW is good, but it will vary based on your labelling and the laser itself (e.g. does it have a pre-chirper?). Higher powers are not necessarily better, as the brighter signal comes from activating fluorophores from a larger volume. In other words, increase in brightness is due to the optical section becoming thicker and hence resolution decreasing.

The BEAM CONTROLS (laser power) window in ScanImage. Note the power is calibrated here, so we have a value in mW as well as the percent power value.

Finding the sample surface

Prepare the rig for imaging: turn off the lights, shut the enclosure, then turn on the PMTs.

The PMTs can be damaged by room or enclosure lights when powered on. Do not turn on the PMTs if the lights are on.

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 following:

• Is the laser shutter is open?

• Are the PMTs are on?

• Is the laser "Modelock" indicator in the GUI is green?

• Are you definitely over the sample?

• Is the laser power enough?

• Could the image look-up table (right click on image and open the histogram) too stretched out?

• Is the objective Z wrong? 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 series of images shows what a brain might look like at a range of depths. Once you have found the brain, focus up and down until you identify the brain surface. In the image series below, this would be somewhere between the -30 and -20 micron depths. The brain surface is very salient: there is a sudden and obvious transition from "nothing" to "brain." Make a note of the surface depth. If you have a motor connected via ScanImage, you can zero the depth reading to mark the surface (see also below).

A series of depths spaced 10 microns apart. That labeled "0 microns" is suitable depth for the first imaging plane.

Ensuring slicing is reproducible

The system does not automatically find the brain surface during imaging. Instead, it assumes that the surface of the tissue is a constant distance with respect to the objective for the whole acquisition. This assumption will be correct if the cut thickness is consistent. Now we will check if that is so.

You have already found and noted the depth of the brain surface. Press "Slice once" in the Prepare GUI. This will take a slice: moving the sample up the thickness of one section, slicing the section, then returning the X/Y stage to its original location. If slicing is consistent, the sample surface will be located within 5 or 10 microns of the previous measurement for the last section. If the depth is off by more than about 10 microns, cut again and re-measure. Usually the surface height stabilises within 3 cuts. Sometimes it takes longer and rarely it alternates between thick and thin sections. If this happens, see the cutting troubleshooting guide.

Setting the imaging depth

You will likely be imaging multiple optical planes. The first optical plane is that which is visible currently in the ScanImage live preview window. Currently this is the sample surface, which is not evenly illuminated and will probably contain cutting artifacts. We want the first depth(s) to look nicer. Move the objective down about 20 microns. If you have coarse Z control via ScanImage, you can simply enter "20" in the edit box (see below).

MOTOR CONTROLS window in ScanImage Basic. The objective has been moved down 20 microns with respect to the sample surface at zero microns.

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 (see image below). 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.

The preview window initially presents with just a schematic of the slide (left). Press the "ROI" button and draw a ROI around the area you anticipate the brains to be. Then press return.

Press "Preview Scan" (Until this bug is fixed, wait until the scanning starts before changing any GUI values in ScanImage). This will initiate a tile scan of the sample at low resolution using only one optical plane.

"Preview Scan" initiates a tile scan of the sample at low resolution and with one optical plane.

An example of a completed preview scan with two brains is shown below. Don't worry about the tile illumination artifacts, those will be cleaned up during final image assembly by StitchIt.

Completed preview scan.

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

Is the green border clear?

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 below shows a preview scan with brain tissue in the green border. You would re-do this preview scan.

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.

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.

The surface of all samples roughly the same?

All samples should look roughly the same and should not have gradients across them in the left/right direction. Such gradients are an indication that the blade is tilted. You can verify this if you suspect it by panning to different brain locations and measuring the brain surface there. See the Acquisition Problems section of the troubleshooting guide if the tilt is severe and you can't fix it by just moving the objective down another 10 or 20 microns.

Samples of different ages or that have had different degrees of fixation can exhibit very different levels of autofluorescence. This is normal and not a defect in the imaging or cutting.