Fig. 1: Concept and photophysical characterization of rsGCaMP1.1 and rsGCaMP1.4-ER.

a, Concept (left) showing the dependence of photo-switching on ligand binding (Ca²⁺ in the case of rsGCaMPs). The engineering concept of rsGCaMPs is based on GCaMP architecture (right). Red dots symbolize regions of mutational screening, which eventually allowed photo-switching (bottom) in rsGCaMPs. US, ultrasound. b, Dependence of changes in absorption or fluorescence on Ca²⁺ concentration. Norm., normalized. c–e, Change in absorption spectra of GCaMP5G (c) and rsGCaMP1.1 (d), and the fluorescence spectrum of rsGCaMP1.4-ER (e), as Ca²⁺ concentration increases from 0 to 750 µM. f–h, 488- and 405-nm-dependent photo-switching in rsEGFP2 (f) and rsGCaMP1.1 (g) in the presence of 0 and 39 µM free Ca²⁺, and fluorescence of rsGCaMP1.4-ER (h) in the Ca²⁺-saturated (sat.; 750 µM total Ca²⁺) and free state (0 mM total Ca²⁺). i, Major rsGCaMP variants used in the key imaging experiments in this study. A full overview of all variants can be found in Extended Data Table 1b. All data were recorded in triplicate and are reported as mean and standard deviation. Error bars are omitted in the spectra for clarity. A side-by-side comparison of all binding curves, absorbance spectra and fluorescence spectra with errors can be found in Supplementary Figs. 1–3. Photo-switching was achieved using a 405/12.5- and a 490/26-nm light-emitting diode (LED), and a 5-mm cross-section liquid light guide delivering 1,528 and 270 mW cm^–2, at 405 and 488 nm, respectively. a.u., arbitrary units.

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