Fig. 3: Adaptions allowing imaging in a lit environment.

a, Microscope schematic highlighting the detector system, showing light paths and on-board detectors for green (green) and third-harmonic generation signal (blue) channels (Ch.). Scanner and z-drive optics omitted. The direction of the excitation path is indicated by the red arrow. b, Photograph of one of the on-board detectors (SiPM). c, Quantification of image signal-to-noise ratio (SNR) using SiPM and remote PMTs coupled via a POF. d, Schematic of pixel-wise timing of excitation pulses (top) and emitted fluorescence integration window (bottom, green boxes) used for dark count rejection. e, Schematic illustration of the line-wise image acquisition and timing of the environmental lighting activation (LED, yellow) and acquisition of a line in the image (line acquisition, green). f, Example images (average of 50 frames) acquired with environmental lighting off (left) or on (right). Data for f and g from neurons in layer 6, laser power 27 mW. g, Example Ca²⁺-fluorescence traces from the neurons indicated in f during a transition from the environmental lighting being on to off. h, Average pixel intensity over the whole frame from green channel data one frame before (Light) and one frame after (Dark) transitions from light to dark (left) and fluorescence traces from each neuron (gray) from one example recording during a transition from dark to light. Mean of all neuronal traces shown in black. Traces have been normalized to the time point one frame before the time the environmental lights were activated and have been denoised. Open circles in the left panel show data from individual transitions, red diamond represents the mean (N = 22 transitions, P = 1, two-sample Kolmogorov–Smirnov test). Data in left panel presented as raw analog-to-digital units (ADu), data in the right panel as arbitrary units (a.u.) after denoising.