Extended Data Fig. 3: Protocol and validation of the microablation procedure.

a. Protocol for microablation. See also Methods for further details. b. Example trace of Calcium fluorescence during laser exposure on a target neuron when doing point scanning. Ca²⁺ fluorescence was monitored while exposing the laser, and the time-lapse imaging continued for a couple of seconds to a few tens of seconds until we observed abrupt elevation of the signal. In this example, an abrupt increase of fluorescence intensity was observed around 21 sec from the onset of laser exposure. c-d. Examples of targeted neurons imaged in vivo over the time course of up to 4 days. In c, the targeted neuron showed a permanently elevated fluorescence signal one hour after the microablation procedure. After 4 days, both the nucleus fluorescence and the calcium fluorescence were undetectable. In another example targeted neuron in d, after 4 days of the microablation procedure, filled calcium fluorescence remained while the nucleus fluorescence was almost eliminated. When target neurons are successfully microablated, in most cases both signals disappeared (c, 89.4 %), in rare cases nucleus signal was lost with filled Ca²⁺ fluorescence (d, 10.6 %). Scale bar: 20 μm. e-h. Evaluation of the effectiveness of the procedure by re-identifying individual targeted neurons in fixed brain sections counter stained by 4’, 6-diamidino-2-phenylindole (DAPI). Four days after the microablation procedure, neurons which lost the H2B::mCherry signal exhibited an absent DAPI signal (e) or an abnormally fragmented DAPI morphology (f-h). Scale bar: 20 μm. i. Distribution of collaterally damaged off-target neurons surrounding targeted neurons along xy-axis and z-axis. The red dot indicates the aligned positions of the targeted neurons (n = 111). The gray dots are off-target damaged neurons considering a cubic volume of 60 × 60 × 60 μm surrounding the targeted neuron. The majority of neurons suffering collateral damage fall within a 15 μm radius (dashed line) from the targeted neurons (64.3%), which corresponds to the radius that was applied by default to filter out neurons for further image analysis (see Methods). No statistical difference was observed in distributions between xy-distance and absolute z-difference (two-sided Kolmogorov-Smirnov test, p = 0.27). j. Summary bar plots of the number of co-damaged off-targeted neurons out of 111 ablation experiments targeting a single neuron. k. Summary bar plots of nucleus fluorescence signals and post-hoc DAPI signals in targeted neurons categorized based on in vivo images 4 days after the microablation procedure.