Step 4: Starting the acquisition
Checking the bidirectional phase correction
We will scan bidirectionally for speed. If the bidirectional phase correction value is not set properly, the image will appear jagged. Below, the left image shows what things should look like and the right image shows an example of a bad setting.
The phase correction value is set in the CONFIGURATION window of ScanImage. To set this, do the following:
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.
Scroll in with the mouse wheel to get a detailed view. DO NOT change the "zoom" (scan amplitude) value.
If necessary, set the averaging to about 20x to get a better feel for what is going on.
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 until things look good.
Return the averaging to the desired value for imaging.
Once done, you can save the configuration file and the value will be reused. It should need little or no tweaking across samples.
At faster speeds with linear scanners (line periods shorter than about 800 us) the phase can vary 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
Now set Power/Depth Adjust in POWER CONTROLS. If you haven't done this before, try "Default Exponential" and a constant of about around 250 to 500:
Place the objective over a region containing mostly grey matter then hit "Grab" in the main ScanImage window. You should see images of the sample at each depth and hopefully it will look similarly bright at each depth. You can increase the ramp of the power with depth by making the depth constant smaller. Once you have a good value, it can be reused for similar samples. Likely 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.
Start!
If you are in auto-ROI mode you should now re-run the preview scan at the new, higher, laser power. This should be the laser power used for imaging. Under normal circumstances you do not want more than about 200 mW at the sample, as higher powers tend to result in fluorophore saturation. In other words, any further increase in brightness is due to the optical section becoming thicker and hence resolution decreasing. For bright samples 120 to 150 mW should be adequate. Then press "Auto-Thresh" to find the sample (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
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.
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