Pm100a

We used a high-powered red LED (Red XP-E, 620–630 nm wavelength) and Buckpuck driver (RapidLED, Randolph, Vermont) to stimulate Chrimson expressing neurons. The LED was mounted directly underneath the preparation and light was presented at 0.238 mW/mm² as measured by a Thorlabs light meter PM100A with light sensor S130C. Flies expressing Chrimson were raised on food containing 0.2 mM all-trans-retinal. mM all-trans-retinal. All-trans retinal was prepared as a stock solution in ethanol (35 mM), and 28 μl of this stock was mixed into approximately 5 ml of rehydrated potato flakes and added to the top of a vial of conventional food. For Figures 6A, 7A and 8A, we varied the red light intensity as a 10 Hz sine wave from 0 to 0.238 mW/mm². This phasic stimulation should prevent adaptation of the optogenetic tool.

The leaves of Camelina sativa were placed on a cover slip that was immersed with water. Light from an LED with a green emission that was centered at 530 nm (LXZ1-PM01) was collimated and delivered to the sample to simulate the interference of sunlight in the wavelength range that is analyzed with our macroscope. To match the optical power that is sent to the sample with the real sunlight power, a photodiode power meter (PM100A, Thorlabs, DE) was used to measure the sunlight and the intensity of the LED on the sample. Since the recorded green fluorescent emission is detected after bandpass filtering (ET525/36m, Chroma, USA) in our setup, we applied the same filter to record the irradiance of the direct sunlight in the same green waveband (525 ± 18 nm). We measured 10 mW/cm² (λ = 525 nm, Δλ = 36 nm). We correspondingly adjusted the power of the green LED to deliver the same power in our experiments. The images of the leaves have been recorded under both darkroom and artificial sunlight conditions at a sampling rate of $f_{\mathrm{s}}$  = 200 Hz by modulating the excitation lights at 2.5 Hz for eight periods.

Worms expressing ChR2 first grew to L1-L2 stage on standard NGM plates. They were then transferred to new NGM plates either with or without (for negative control) all-trans retinal 2 days before experiments. The retinal plates were prepared by spotting each plate (60-mm diameter with 10-ml agar) with 200-µl OP50 containing 2-mM retinal (R2500, Sigma-Aldrich). In most experiments, blue light pulses (2 sec or 5 sec, 470 ± 20 nm) were generated by a Lambda XL light source (Sutter Instrument, Novato, CA, USA) with a 470 ± 20-nm excitation filter (59222, Chroma Technology Corp., Bellows Falls, VT, USA) and SmartShutter^® (Sutter Instrument). Light intensity was adjusted by applying the three standard neural density filters (ND4, ND8, ND16) of the Nikon FN1 microscope in various combinations, which resulted in eight different light intensities ranging from 0.01 to 4.38 mW/mm². The maximal light intensity was used in all experiments except those assessing the effect of light intensity-dependent activation of ASH neurons on AVA membrane potential. Light intensities were measured with an optical power meter (PM100A, Thorlabs, Newton, NJ, USA) equipped with a photodiode power sensor (S121C, Thorlabs). The on-and-off of light stimulation was controlled by NIS-Elements imaging software (version 4.51) through the SmartShutter in the Lambda XL light source.