Supplementary Figure 7: Maintenance of brain temperature within 2 °C of normal body temperature during high-speed two-photon imaging in awake mice.

Studies of the brain temperature variations that arose during high-speed two-photon imaging and imaging protocols that kept the brain in the range of normal body temperature.(a) Inset: An image showing a thermocouple implanted beneath a 4.5-mm-diameter glass window in the cranium of a GCaMP6f-tTA-dCre mouse, proximal to the site of two-photon imaging. Red dashed box encloses the field-of-view for high-speed two-photon imaging. Main panel: Measurements of peak brain temperature, determined via 10-Hz-sampling of brain temperature within 20-s-periods of two-photon imaging (400 beamlets; 2 mW illumination power per beamlet; 100 Hz image frame acquisition rate; 200 kHz laser repetition rate), plotted as a function of the lateral distance between the tip of the thermocouple (100-200 µm beneath the dura) and the center of the field-of-view. Peak temperature declined linearly with this distance and the beamlet power with a proportionality constant of –1.07 ± 0.03 °C · mm^-1 · mW^-1. Error bars denote s.d. across N = 4 bouts of imaging (each 20 s in duration) per data point. (b) Example traces of brain temperature measured with a thermocouple located within ~0.5 mm of the same field-of-view before, during and after 20-s-periods of two-photon imaging (gray shading) at one of six different illumination power levels (0.125–3 mW per beamlet; 400 beamlets; 100 Hz image frame acquisition rate; 200 kHz laser repetition rate). Due to the cranial window, in the absence of laser illumination the brain temperature was ≥10 °C below normal body temperature in mice, as previously reported²³. The example traces are representative of data taken at 6 different fields-of-view studied in a total of 3 mice. (c) Time-course of the temperature measurements in panel b, normalized by illumination power. The blue trace and shading denote, respectively, the mean and s.d. of the same 6 time-courses shown in b, divided by total illumination power. Red trace: Parametric fits to the rising and declining phases of the temperature time course using two different exponential functions with distinct time constants (τ = 0.092 ± 0.001 s^–1 and 0.071 ± 0.0002 s^–1 for the rising and declining phases, respectively). Inset: Peak temperatures, attained at the end of each 20-s-period of illumination, plotted as a function of the laser beamlet power (linear proportionality constant: 5.5 ± 0.6 °C · mW^-1). Each curve shows the data from one of 6 different fields-of-view, studied in a total of 3 mice. (d) Validation of three different imaging protocols to keep the brain within 2 °C of normal body temperature (37 ± 2 °C; shaded regions of the graphs). Solid traces are empirical measurements. Dotted lines are the predicted temperature values based on the kinetic parameters determined as in panel c. At 1 mW power per beamlet (400 beamlets in total), the illumination was continuous and the brain temperature reached ~39 °C after ~190 s of illumination. At 2 mW and 3 mW per beamlet, periods of imaging and rest were interleaved, mimicking the repetitive trial structure of a rodent behavioral task that might normally be performed in the context of a brain-imaging experiment. Uncertainty values stated here for all fit parameters are the 95% confidence intervals.