Step 4: Starting the acquisition

Checking the bidirectional phase correction

The software scans bidirectionally for speed. This means that it acquires image data as the fast scan axis moves left to right and also when it returns, moving right to left. Data from odd and even lines will be out of phase if the bidirectional phase correction value is not set properly. An improper values will result in images that appear jagged. Below, the image on the right shows what things should look like and the left image shows an example of a bad phase setting.

Consequence of a bad bidirectional phase correction value. Left shows a bad image and right a good one.

The phase correction value is set in the CONFIGURATION window of ScanImage:

ScanImage CONFIGURATION window. The Scan Phase correction slider is highlighted by the orange box.

To tweak the phase correction value:

• Identify an area of the tissue which has bright features. Ideally thin vertical features that cross many scan lines. You can middle-click on the Preview image to move the stages to that location in the sample.

• Hover the mouse over the live image window of ScanImage and scroll in with the mouse wheel to get a detailed view.

• If only autofluorescence is visible, set the averaging to about 10 frames to get a clearer view. In areas with bright signal averaging is not necessary.

• Ensure that the image is not clipping too much. i.e. if you have the red clipping setting enabled then you shouldn't be seeing much red. Too much clipping will mask out the bidi artifacts.

• Tweak the bidi value by clicking on the slider arrows. Allow a couple of seconds for your changes to have an effect due to the averaging.

• You can also try the "Auto Adjust" button, but be aware that this often fails.

• Return the averaging to the desired value for imaging.

If you have galvos only (not a resonant scanner) then line periods shorter than about 800 us will lead to variable phase offsets across the scan line. So either don't use very fast values, or switch to resonant scanning, or apply a correction post-hoc.

Confirming the beam intensity with z-depth

Deeper optical planes will yield less signal. To compensate for this, ScanImage can increase laser power with depth. We will no confirm that this laser power ramping is reasonable.

Find the BEAM CONTROLS window and if necessary press the "Power Z-Profile" button to expand it. Ensure the exponential option is chosen in the P/Z Adjust drop-down menu. The length constant (Lz) associated with this will determine the rate at which power should increase with depth. A value of about Lz=200 to Lz=500 is normally sufficient.

Checking power as a function of depth. "Exponential" has been selected with Lz=249. Laser power ramps up with depth over the 8 optical planes shown here. Images look very similar across depth, indicating that the correction is working well.

• Place the objective over a region containing mostly grey matter.

• Hit "Grab" in the main ScanImage window. You will now see a grid of images, one from each depth.

• If you have the "High saturated" (bright pixels are red) look-up table, adjust the histogram settings such that only a small number of pixels appear red in the first image (the top plane).

• Adjust Lz. e.g. If deeper images look dimmer, you can increase the slope of the power curve by making the depth constant smaller.

No single value will be perfect throughout the sample. For example, in brains the signal is attenuated much faster with depth when imaging white matter compared to grey matter (see below). The attenuation due to white matter can not be corrected by increasing power, so chose areas with mainly grey matter for testing the length constant.

Areas with a lot of white matter should not be used to assess power/depth

Start!

If you are in auto-ROI mode you must re-run the preview scan if you have changed laser power or PMT gain since it was originally made. Once you are ready, press "Auto-Thresh" to have BakingTray find the sample. Finding the samples will take about 5 seconds. Once the samples have been found, proposed ROIs are overlaid on top of the image.

The Auto-Thresh button (highlighted in red) will identify the samples in the FOV. The proposed tile scan is overlaid on top of the brains after a threshold has been found.

More details on the auto-ROI can be found here.

In both manual and auto-ROI modes you can now hit "Bake" in the Acquisition View to start acquiring the sample

Press Bake to start the acquisition

There will be a confirmation dialog box before the acquisition starts and there is a short delay after accepting this before the acquisition begins.

After the acquisition has started

You can now start syncAndCrunch on the StitchIt analysis machine to begin pre-processing data and sending preview stitched images to the web.

Once the acquisition has started you can pause and resume at any time from the preview window. The following settings can be changed during acquisition:

• Laser power: any time

• Laser power depth ramp: during slicing only

• PMT gain: any time

• Frame averaging: during cutting only

• To alter the number of sections you need to need to stop the acquisition, resume it, then modify the remaining number of sections to image.

Make sure you also read through Step 5: Concluding the acquisition, which sumarises post-acquisition tasks and explains how to resume a stopped acquisition.