Extended Data Fig. 1: Development and characterization of QuasAr3.

a, Screening pipeline. Rationally designed constructs were cloned in the Optopatch configuration, expressed in primary neurons and tested for spike SNR using light-induced spikes. Constructs with improved SNR were then expressed in vivo using in utero electroporation and tested for spike SNR in acute slices. The process was repeated iteratively. b, Examples of SNR measurements in cultured neurons. Left, wide-field epifluorescence images of GFP (green) fused to CheRiff, an opsin with excellent membrane trafficking. Middle, fluorescence of QuasAr (red) mutants. Scale bar, 10 μm. Right, QuasAr fluorescence transients in response to optogenetically induced spikes (10 ms blue-light stimulation at 1 mW mm⁻²). Each construct was tested on at least five cultured neurons. c, Hierarchical screen for improved membrane trafficking of QuasAr variants (see Methods for details). Diagram, schematic of the FCK_DuEx1.0 construct and overview of the screening pipeline. E. coli colonies were transformed with libraries in FCK_DuEx1.0. The colonies with the brightest fluorescence were picked for lentivirus production and secondary screening in primary neuronal culture. Images, example images of the fluorescent protein (FP) channel of QuasAr2–FP fusions: (i) mOrange; (ii) mRuby2; (iii) mKate2; (iv) citrine. Scale bar, 10 μm. d, SNR of N-terminal modifications compared with QuasAr2. All constructs showed reduced SNR (see Methods for details). e, Replacing mOrange2 with Citrine as a fusion protein improved the trafficking only with two specific linkers. f, Adding additional TS sequences at the linker and C-terminal improved the spike SNR. g, The mutation K171R increased the expression level of QuasAr, quantified by normalizing QuasAr fluorescence by the fluorescence of the co-expressed CheRiff–GFP. d–g, Data are mean ± s.e.m., one-tailed Student’s t-test. h, Top, diagram of the QuasAr2 and QuasAr3 constructs. Bottom, confocal images of brain slices expressing QuasAr2 and QuasAr3. Scale bar, 500 μm. Insets, single-cell bodies; scale bar, 10 μm. Representative images from n = 2 mice (QuasAr2) and n = 3 mice (QuasAr3). i, Confocal images of brain slices expressing Cre-dependent QuasAr3 with sparsity controlled by co-expression of hSyn-Cre. Scale bar, 50 μm. j, Simultaneous fluorescence and patch-clamp recordings from two neurons expressing QuasAr3 using AAV virus in acute brain slices. Left, image of QuasAr3 fluorescence in the soma. Scale bar, 10 μm. Middle, spiking during ramp current injection. Right, mean spike, overlay of fluorescence and voltage. Inset, boxed regions showing correspondence of optical and electrical recordings of subthreshold voltage overlaid. See Extended Data Fig. 3 for statistics.