Extended Data Fig. 2. Hemodynamic correction of 2P GRAB_NE signals.

(a) Left: Average projection of a single GRAB_NE recording with four regions-of-interest (ROI) in areas with clear GRAB_NE fluorescence (black squares, labeled 1–4) and one ROI in a blood vessel (red square, labeled BV). Middle: standard deviation projection. Right: average fluorescence in each ROI before hemodynamic correction. (b) Left: Estimated hemodynamic signal present in each pixel. Middle: recovered average projection after hemodynamic correction. Note average fluorescence after hemodynamic correction is broadly similar the uncorrected data in (a) with some blurring due to Gaussian smoothing in preprocessing. Right: average ROI fluorescence after hemodynamic correction. (c) ROI-free methodology for obtaining GRAB_NE signal. Left: Following hemodynamic correction, the bottom quartile of pixels with the lowest hemodynamic weights, excluding the bottom 1% which often had artefactual signals, were kept. Right: These pixels were averaged together to produce the corrected GRAB_NE signal (bottom) which had substantially less hemodynamic contamination than the uncorrected field fluorescence (top). (d) Correlation between the corrected GRAB_NE signal and the uncorrected ROIs in (a) and (b). (e) Left: Correlation between uncorrected GRAB_NE fluorescence and pupil diameter. Right: Example traces from the average of pixels that were positively correlated (top), negatively correlated (bottom), or showed little relationship (middle) with pupil diameter. (f) The corrected GRAB_NE signal is not similar to the uncorrected GRAB_NE signal (red dot) but instead reflects the GRAB_NE signal of binned pixels (black dots) correlated to pupil diameter (R² = 0.62, p = 1.55⁻⁵, two-sided t-test).