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Fig. 1. | BMC Biology

Fig. 1.

From: Colored visual stimuli evoke spectrally tuned neuronal responses across the central nervous system of zebrafish larvae

Fig. 1.

Experimental setup, visual stimulation protocol, and regression-based identification of responsive neurons. a H2B-GCaMP6s fluorescence maximum intensity projection of the CNS of a 5-dpf larva. Scale bar, 200 μm. b Schematic of two-photon microscope used for imaging zebrafish larva brain combined with visual stimulation. Stimuli are presented dorso-frontally using LEDs of four different wavelengths. GM, galvanometric mirror; FF, fluorescence filter; DM, dichroic mirrors. Obj, Olympus × 20, 0.95NA, water immersion; L1, L2, L3, and L4 are the LEDs used for visual stimulation. c Normalized absorption spectra of zebrafish cones (solid lines, modified from Guggiana-Nilo and Engert [10]) overlapped with the LEDs emission spectra (dashed lines) used for visual stimulation. d Protocol of visual stimulation. e GCaMP fluorescence of a selected plane of a 5-dpf larva viewed dorsally (left image) and corresponding segmentation image (right image). Each identified neuron is labeled with a distinct arbitrary color. The dotted orange squares show the area magnified in the right panels, displaying cellular resolution of imaging (top) and segmented neurons (bottom). The inset in the bottom panel shows a neuron selected for the examples in fh. Scale bar, 100 μm. f Fluorescence intensity F(t) measured in the 13-pixel cluster attributed to the selected cell highlighted in e (F0 is shown with the red line; see the “Methods” section for the detailed description of F0 and ΔF/F0 calculation). Stimulus time points are shown at the top. g ΔF/F0 trace. h Fitted regression data (colored trace) and corresponding T values. ik Examples of ΔF/F0 traces and calculated T values obtained from the regression analysis on other neurons demonstrating the responsiveness to the different stimuli

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