Scanbox makes sure the data acquisition of the signals from the photo-multipliers are synchronized to the laser pulse. Let me explain why. Suppose you run an asynchronous data acquisition clock at 25Mhz while the laser has a pulse rate of 80Mhz. Then you may obtain images of single cells that look like this:
This is a zoomed in view of a single cell labeled with GCamp6s. Notice something funny about this picture? If you look along individual lines you will see that bright pixels are often next to black pixels. If you look at another frame you will see the same pattern, except that the location of bright/dark pixels would have shifted.
What’s going on?
What is happening is that the phase of the 25Mhz clock relative to the laser pulses changes over time, producing a beat pattern. For these choice of frequencies, the beating pattern has a period of 5 pixels. This is because 5 pixels @ 25Mhz is the same as 16 pulses @ 80Mhz, both of which are equal to 200ns. The pattern is obvious when you compute the average auto-correlation of the fluctuations of the lines:
Yuck… Two pixels away there is a 40% anti-correlation in the signal! How do you get rid of this problem? Running the sampling clock at 20Mhz does not solve it. Why? Because the laser does not run exactly at 80Mhz so the relative phase will still drift. Moreover, the actual frequency of the laser changes with your selection of the wavelength and we want a solution that will work for all wavelengths. Another partial solution is to temporally filter your signal by selecting PMT amplifiers with a relatively low high-cut frequency. This, however, will decrease the spatial resolution along the resonant axis and will not solve the problem if your selection of frequencies causes the phase to drift slowly in time. In this case, the overall brightness of your images will drift slowly back and forth between bright and dark levels. The simplest solution to this problem is to synchronize the data acquisition with your laser. Indeed, then your single frames look nicer (note image is from a different cell) and you get sharp bright/dark edges with single-pixel resolution.
In Scanbox this synchronization is simply achieved by ‘cleaning-up’ the sync out output of the Chameleon with a BBP-70+ band pass filter from Mini-circuits. Then, the level and slope of the trigger in the Alazartech board is fine-tuned to find the relative phase that achieves a maximal signal. The resulting settings will depend on the PMT filters that you use, but will generate the crispiest, noise-free images you optics can support.
So, if you see the sync-out of your laser not connected to anything, and your single images look like the one at the top, it is time to get this fixed. Your data will thank you.