Extended Data Fig. 1: dLight validation, photometry setup and motion artifact removal.

a) Maximum projection epifluorescence images of HEK cells transfected with the dLight plasmid, and excited with either 480 nm blue (top) or 400 nm UV (bottom) light. Green emission (527 nm) was collected for both excitation wavelengths. Scale bar indicates 20 µm. Three separate experiments were performed with n = 28, n = 45, n = 42 regions of interest (ROIs) from the first, second, and third experiment respectively. b) Scatter plot of pixel fluorescence for each pixel location under both 400 nm and 480 nm excitation. A regression line was fit to the scatter plot (blue). The strength of the correlation demonstrates that both excitation wavelengths cause similar spatial patterns of dLight emission at 527 nm. c) Single cell dLight responses to perfused dopamine at 480 nm (left) or 400 nm (right) wavelengths. White dashed lines indicate time points where dopamine is washed into and out of the sample. Each row is an individual cell region of interest (ROI). d) Correlation in single cell dLight responses to perfused dopamine imaged with blue (x-axis) or UV light (y-axis). Each dot is an individual cell. Blue line indicates linear fit. The near-zero slope indicates that the UV-light-dependent green emission is almost entirely independent of dopamine concentration. e) Validation of dLight1.1 response to optogenetic stimulation. To assess how quickly DLS dLight reports dopamine transients in vivo, SNc axons were optogenetically stimulated using ChrimsonR (whose excitation spectrum is separated from that of dLight) while dLight fluorescence was recorded. Left: schematic illustrating viral injection and implant procedure to simultaneously record dLight transients and optogenetically stimulate dopamine axons in DAT-Cre animals. dLight was injected into the right dorsolateral striatum, Cre-dependent ChrimsonR was injected into the right SNc, and an optical fiber was placed above the dLight injection site in the DLS (see Methods). Middle: coronal slice depicting the expression of both dLight and ChrimsonR in the striatum. Right: mean stimulation-evoked dLight fluorescence using ChrimsonR in dopaminergic axons originating from SNc. The green shaded region indicates bootstrap SEM. The gray shaded area indicates the 95% bootstrap confidence interval of the mean trace pre-stimulation. Red shading indicates the duration of optogenetic stimulation. dLight transients are resolvable starting 67 ms from stimulation onset, suggesting that dLight can report rapid dopamine dynamics via photometry in DLS (p = 0.005, U = 2610, f = 0.32, one-sided Mann-Whitney U test). f) Schematic describing the photometry recording setup. A blue and UV LED were modulated at different frequencies, and light delivered to the mouse via a single fiber optic cable. Green fluorescence was acquired using a photodetector and the blue (dLight) and UV (isosbestic) components were separated using lock-in amplification. g) Example data depicting a dLight trace along with the simultaneously recorded reference signal acquired during free behaviour. The UV, or reference signal, represents the contributions of motion and mechanical artifacts and other non-ligand dependent changes in sensor fluorescence. Since UV and blue excitation of dLight cannot be perfectly matched, a gain and bias term were fit to match the UV-excited emission to the blue-excited emission per previously published papers⁵⁴; this fitting process maximizes the correlation between the UV and blue signals, enabling us to effectively subtract the reference signal from the dLight signal. Left: example fit showing the UV trace scaled to match the dLight trace (bottom) aligned to syllables (top). Right: histogram of r² values between the photometry reference (UV component) and signal (dLight component), prior to fitting and subtracting the reference signal (top) and after fitting and subtracting the reference signal (bottom). Note that the baseline correlation between the reference and signal channels prior to subtraction is low. h) Top: the probability, on average, of observing a dopamine transient across all mice and experiments – defined as the ΔF/F0 trace crossing 1 standard deviation (computed per experiment) – at a given timepoint in the experiment. The probability was estimated in one minute bins. Shading indicates the 95% bootstrap confidence interval across per-mouse averages. Bottom: the maximum ΔF/F0 per 1 min time bin across all mice and experiments. As in the top plot, the average across all mice and experiments is shown, with shading indicating the 95% bootstrap confidence interval across per-mouse averages.