Extended Data Fig. 3: Data acquisition and post-processing for two-photon imaging with 16 time-multiplexed excitation beams.

a, Block diagram of the electronics for data acquisition and instrument control. PMT, photomultiplier tube; Pre-amp, pre-amplifier; ADC, analogue-to-digital converter; FPGA, field-programmable gate array; EOM, electro-optic modulator. b, Computer simulation of signal sampling in different stages of the pipeline in a. The ADC samples the analogue, pre-amplified and low-pass filtered signals (blue) from one of the PMTs at a rate of 5 × 10⁷ samples per second. In each of the four temporal phases, the FPGA sums the digitized signals (red) from the ADC to yield the fluorescence intensity values of each image pixel (grey). c, Raw fluorescence images for each of the four excitation phases, acquired in an awake mouse expressing GCaMP6f in layer 2/3 cortical pyramidal cells and averaged over 100 frames (7.23 Hz acquisition rate). In each of the four phases, a distinct set of four PMTs detects most of the fluorescence emissions, creating four active image tiles within the 4 × 4 array. (Each of the four PMTs corresponds to one of the four laser beams that is active in that phase.) To illustrate, the four active tiles within the phase I image are shaded with a different colour (shaded large square regions). However, close to the boundaries of each active tile, some fluorescence photons are detected by the other 12 PMTs. During signal unmixing these photons are reassigned to corresponding pixels in the correct adjacent active image tile. For instance, within the phase I image photons detected in the areas outlined in colour (rectangles and small squares) are reassigned to the colour-corresponding active tiles. d, An image compiling the four sets of four active image tiles from the panels in c. e, During signal un-mixing, we re-assign scattered fluorescence photons to their correct pixels of origin, using the method shown in c, by reassigning the boundary regions of 128 pixels width. The resulting image is displayed with the mean contrast equalized across tiles. Scale bars: c, e, 500 μm.