Figure 2.

Imaging of a head-fixed mouse with LOTUS-V. (A) Venus fluorescence in the primary visual cortex (V1) of a paraformaldehyde-fixed brain. (B,C) Examples of two-photon fluorescence images of V1 area containing LOTUS-V-expressing neurons (B) and a single V1 neuron spatially well resolved by two-photon microscopy (C). (D) Schematic drawing of the prepared cranial window. The headplate and o-ring were glued over the cranial window. The furimazine solution was enclosed in the o-ring bath by a cover glass. (E; left) Schematic drawing of the imaging setup for a head-fixed mouse. A mouse was placed on a running disk while the head was fixed to a bar via the headplate. The velocity was recorded through a rotary encoder during imaging. LOTUS-V bioluminescence from the V1 was collected through a CCTV lens. NLuc and Venus emissions were separated by image splitting optics. These emissions were acquired using an image intensifier unit, which can amplify the signal up to 10⁶ times, and recorded with an EMCCD camera in the dark condition. (E; right) Overlaid images of bright-field and bioluminescence in NLuc and Venus channels acquired by this system. (F) Averaged time series of velocity and LOWESS-smoothed ΔR/R₀ at the locomotion onset (n = 12 sessions from N = 3 mice). The Granger causality test was performed to determine whether the velocity Granger-causes ΔR/R₀ statistically (p < 0.05). (G,H) Plots of z-normalized ΔR/R₀ in resting (<5 cm/s) and active (>5 cm/s) states of head-fixed mice, using (G) all time-points (p < 0.0001, Wilcoxon rank sum test; n = 707871; 31653 time-points), or (H) average values from each mouse (p < 0.05, Wilcoxon signed-rank test; N = 7 mice). Error bars indicate mean ± standard error; ^*p < 0.05; ^****p < 0.0001.