Fig 1

(A) FRCaMPi sensor is created by topological inversion of FRCaMP. Upper panel, a diagram showing the design of FRCaMP and FRCaMPi; the lower panel predicted 3D protein structures of indicators75 and a table of amino acid sequences of the linkers. (B) Basal brightness and photobleaching curves of red GECIs characterized in transfect HeLa cells by side-by-side comparison. For the plasmids used to transfect HeLa cells, a nuclear exclusion sequence (NES) was added to each red GECI and mGreenLantern reference was attached via a P2A peptide to express red and green FP in near equimolar ratio. *NES was not added to XCaMP-R due to the presence of F2A sequence C-terminal reported to have nuclear exclusion. The basal fluorescence of red GECIs (F_red) was normalized against the fluorescence of mGreenLantern (F_green). Fluorescence trace of FRCaMPi, jRGECO1a, XCaMP-R, and K-GECO1 over 10-min photobleaching, normalized to the mean brightness of jRGECO1a in the first frame. (C) Representative images showing the expression of mGreenLantern and FRCaMPi in HeLa cells. (D) Representative fluorescence trace induced by histamine (50 μM) and ionomycin (5 μM) in HeLa cells expressing K-GECO1, XCaMP-R, jRGECO1a, and FRCaMPi. (E) ΔF_max/F_min of FRCaMPi compared to other red GECIs during the period of pharmaceutically induced Ca²⁺ response (K-GECO1: n = 53 cells from 4 wells; XCaMP-R: n = 71 cells from 5 wells; jRGECO1a: n = 50 cells from 4 wells; FRCaMPi: n = 50 cells from 4 wells, two independent cultures). Kruskal–Wallis multiple-comparison test: p < 1 × 10–15. Pairwise Dunn’s comparison test with FRCaMPi: p = 5.92e–5 (jRGECO1a), p = 9.73e–7 (XCaMP-R) and p < 1e–15 (K-GECO1). (F) Scatter plot of ΔF_max/F_min against basal brightness of individual cells. Statistics were the same as (E). The quantitative data presented in this figure can be found in S1 Data.