Some time ago we talked about the advantages of synchronizing the digitizer sample clock to the laser. Our new system incorporates an on-board PLL multi-phase clock that allows Scanbox to automatically measure the contrast in a test image as a function of sample phase delay and select the optimal value for your setup.
A typical measurement showing the change in normalized contrast of a target image as a function of phase delay (here the range from -8 to 8 covers the entire 12.5 ns period of the laser).
Scanbox automatically shows the resulting images, on the same scale, for each phase delay value as well:
Scanbox will also plot the normalized images obtained for the settings that yield the lowest and and highest contrasts:
One can clearly see from the images that phase does not simply scale the contrasts of the images, but has an obvious effect on their SNR. (Why this occurs exactly is still a matter of debate here, but data rule and the results are clear.)
This option will be automatically available to those who adopt the new tower system. What can you do if you have one of the older systems? You can simply change the sample clock phase by extending the length of the cable running from the laser SYNC OUT to the external clock of the digitizer. Extensions of 50cm in length can be connected together to yield phase steps of 1/8th of the laser period. So if you want to optimize the contrast and SNR of your images take a day off to find your optimal delay and improve the quality of your data.
A new checkbox within the Laser panel (labeled AGC) allows you to turn an automatic control of laser power on and off.
When AGC is on, Scanbox checks the distribution of pixel values on the image every T seconds, and increases or decreases the laser power by a certain factor if the fraction of pixels above a threshold is outside the desired range.
The values of these parameters are found in a new section of the sbconfig.m file:
% Laser AGC
sbconfig.agc_period = 1; % adjust power every T seconds
sbconfig.agc_factor = [0.93 1.08]; % factor to change laser power down or up if outside prctile bounds
sbconfig.agc_prctile = [1e-5 1e-3]; % bounds on percent pixels saturated wanted
sbconfig.agc_threshold = 250; % threshold above which is considered saturated (255 is max value)
Below is a video showing AGC in action.
At the beginning of the video, I focus on pollen grains using low power. When the AGC is turned on, it brings the power up. Then, if I increase the PMT gain, the laser power is decreased in response. If the laser power is changed manually, AGC will re-adjust it to bring the pixel distribution within the desired limits.
AGC of laser power is useful when running a z-stack with a range that is larger than 100um or so. In that case, engaging AGC will make Scanbox adjust laser power as a function of depth. Another situation where AGC may be useful is while running very long experiments/sessions where water may evaporate slowly leading to a reduction of the signal. In that case, turning AGC would compensate and could render the data usable.