In the scanning approach, the laser spot is spirally scanned over the cell body; in the scanless approach, a disk pattern (~12 μm in diameter) is generated by the SLM, covering the entire cell body at once. (A) Photostimulation triggered calcium response of a targeted neuron in vivo at mouse layer 2/3 of V1, for different stimulation modalities. For each modality, the multiplication of stimulation duration and the power squared was kept constant over four different stimulation durations. The average response traces are plotted over those from the individual trials. (B) ΔF/F response of neurons on different photostimulation conditions [10 cells over two mice in vivo (photostimulated one at a time), layer 2/3 of V1, over a depth of 100 ~ 270 μm from pial surface; one-way ANOVA test show significant different response between spiral scan and scanless approach at the same power for stimulation duration of 20 ms, 10 ms and 5 ms. At 1 ms, the p value is 0.17]. For each neuron and each stimulation duration, the power used in the scanless disk modality is 1 and 1.8 times relative to that in the spiral scan. For each neuron and each modality, the multiplication of the stimulation duration and the power squared was kept constant over four different stimulation durations. The power used in the spiral scan with 20 ms duration varies from 2.2 mW to 5 mW for different cells. (C) Boxplot summarizing the statistics in (B). The central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points (99.3% coverage if the data are normal distributed) not considered outliers, and the outliers are plotted individually using the '+' symbol. In this experiment, the mice are transfected with GCaMP6f and C1V1-mCherry. Repetition rate of the photostimulation laser is 1 MHz. The spiral scan consists of 50 rotations with progressively shrinking radius, and the scanning speed is adjusted to make different stimulation durations.