Extended Data Fig. 2: Characterization of two-photon optogenetic stimulation and evoked response.
From: Neural signal propagation atlas of Caenorhabditis elegans
a, Two-photon (2p) stimulation spot size (point-spread function). b, Imaging excitation wavelength and intensity were chosen to avoid GUR-3/PRDX-2 activation. GCaMP response to 500 nm activation of GUR-3/PRDX-2 expressing neuron as reported in24. Vertical grey line indicates light intensity typically used for calcium imaging in present work. Inset: GCaMP6 excitation spectra from26. Vertical cyan line indicates 505-nm imaging excitation wavelength used in present work. c,d, A neuron near (c) and a neuron far (d) from the objective are photobleached to demonstrate targeted illumination. tagRFP-T is photobleached by 2p stim (20 s illumination, 200 μW, 500 kHz repetition rate, 3.1 μm diameter FWHM spot). Difference image shows tagRFP-T fluorescence merged with a false-colour blue-green image to reveal change in intensity after targeted illumination. Only targeted neuron and not nearby neurons appear photobleached. Insets shows zoomed-in image of the targeted neuron’s original tagRFP-T intensity (left) and difference image (right). Laser power was chosen to avoid saturated bleaching. For a sense of scale, C. elegans interneuron cell somas are roughly 4 microns in diameter. e, In vivo demonstration of 2p effective spot size. Activity from a neuron expressing GUR-3/PRDX-2 and GCaMP6s is shown in response to a 300-ms 2p stimulation delivered at t = 11 s,4 μm beyond the ≈ 3.5 μm diameter soma on the optical axis (z), and at t = 35 s, centred on the soma (t = 35 s). Only on-target soma stimulation evokes a transient, called an “autoresponse”. A stimulus artefact at t = 35 s is visible because no smoothing or filtering is applied to this trace. Schematic via BioRender. f, Distribution of autoresponses under typical stimulus conditions (1.2 mW, 500 kHz; 0.5 s for WT, 0.3 s for unc-31). Autoresponses are required for inclusion. g, Measured calcium response of neuron AIY to optogenetic stimulation of AFD. Compare to figure 4b in ref. 48. A variety of stimulus durations was used to generate autoresponses of different amplitudes (n = 1 (0.1 s), n = 2 (0.15 s), n = 1 (0.2 s), n = 3 (0.25 s), n = 3 (0.3 s), n = 3 (0.35 s), n = 6 (0.4 s), n = 2 (0.45 s), n = 4 (0.5 s) cross-hairs indicate s.d. h, Blue light evoked more reversals in animals expressing GUR-3/PRDX-2 in AVA (n = 11 animals) than WT (n = 8 animals). ~480 nm peaked light was delivered to freely moving animals. Unpaired t-test, p = 0.025. Bars show mean and s.d. i,j, Probability density (i) and CDF (j) of evoked calcium responses in a 30-s post-stimulus window for the targeted neuron (0 μm) or for neurons different distances away. Autoresponses are required. Cross-hairs in j, 75% cumulative distribution at ΔF/F0 = 0.1.
