Ocf 401

We calibrated the performance of the system using 0.2 µm fluorescent microspheres (TetraSpeck^TM, ThermoFisher Scientific, UK). The microspheres were excited by scanning an 8 × 8 beamlet array at 435 nm through a 100× NA1.45 objective (CFI Plan Apochromat Lambda 100× Oil, Nikon, UK) on an inverted microscope (Nikon Ti Eclipse, Nikon, UK) equipped with a perfect focus system. The effective pixel size was 62.5 nm in the x-y plane. The lateral and axial resolution was determined to be 302 ± 29 nm (s.d.) and 600 ± 96 nm (s.d.), respectively, from Gaussian fits to the data (Supplementary Fig. 1). Photon arrival times at the MF32 detector were calculated relative to a trigger (sync) signal from laser pulses incident on an optical constant fraction discriminator (OCF-401, Becker & Hickl, GmbH)) at the repetition rate of the laser, 80 MHz, which is sent to the MF32 detector. The temporal characterisation of the system was evaluated by adding a fixed delay of 5 ns to the photon arrival times by decreasing the sync cable (RG-174 type) length by 1 m. From measurements of the peak position and the decay curve from the sodium fluorescein solution we calculated the temporal bin size to be 57 ps. Representative intensity images of cells expressing empty vector mTurq2 and mTurq2-Epac1-td^dVenus and fluorescence decays from 5 × 5 pixel regions of the images are shown in Supplementary Fig. 2.

The TCSPC timer of the SPAD array is operated in reverse mode, that is, it is started by the detection of a single photon, and stopped by the arrival of a periodic trigger. The trigger is derived from detection of a reflection of the laser pulse with an optical constant fraction discriminator (Becker & Hickl OCF-401). The time range of the histogram acquired in TCSCP mode is 68.6 ns with a bin-width of 67 ps (determined by the 10 bit acquisition electronics), and the median dark count rate of the pixels is 50 counts per second. In the experiment, the SPAD camera detects an average of 0.0005 photon per pulse per pixel, thus operating in the photon-starved regime as required for TCSPC.