Fig 3

(A, B) Schematics of expression cassette and neurons showing the engineering of soma-localized red GECIs by tethering red GECI to ribosome via rpl10a peptide. (C) Representative images of a neuron co-expressing SomaFRCaMPi (TRITC) and emiRFP670 (NIR) (n = 5 from two independent neuronal cultures). Scale bar, 50 µm. (D–G) Plot of fluorescence intensity along neurites, normalized to the intensity at the soma, from neurons expressing either (D) K-GECO1 or SomaK-GECO1, (E) XCaMP-R or SomaXCaMP-R, (F) jRGECO1a or SomajRGECO1a, (G) FRCaMPi or SomaFRCaMPi (K-GECO1: n = 6 neurites from 5 cells; SomaK-GECO1: n = 7 neurites from 5 cells; XCaMP-R: n = 6 neurites from 5 cells; SomaXCaMP-R: n = 6 neurites from 4 cells; jRGECO1a: n = 6 neurites from 5 cells; SomajRGECO1a: n = 6 neurites from 5 cells; FRCaMPi: n = 7 neurites from 6 cells; SomaFRCaMPi: n = 6 neurites from 6 cells). An unpaired student t-test was used. Dots, individual neurites; solid line, exponential fitting curve. The data of the non-targeted indicator was colored in gray to aid visualization. (H) Baseline brightness of soma-localized red GECIs characterized in primary hippocampal neurons by side-by-side comparison (SomaK-GECO1: n = 90 cells from 5 wells; SomaXCaMP-R: n = 84 cells from 4 wells; SomajRGECO1a: n = 85 cells from 5 wells; SomaFRCaMPi: n = 101 cells from 5 wells, two independent cultures). Kruskal–Wallis ANOVA was used followed by a post hoc Dunn’s multiple comparison test. Dot plot is used here and throughout the paper (dots, individual neurons; horizontal line, median; whiskers, interquartile range). Data is normalized to the median of the SomajRGECO1a values. (I–K) Averaged ΔF/F Ca²⁺ transients to one pulse (I), 10 pulses (J), and 160 pulses (K) by GECIs. All averaged traces are shown as mean ± s.e.m, indicated by solid lines and shaded area, respectively. Graph inset displays boxplot of peak ΔF/F₀ of each GECI to applied action potentials. Statistics in C–E are the same as in F. (L) Peak ΔF/F₀ of SomaFRCaMPi compared to other red GECIs as a function of pulses. (M) SNR of FRCaMPi compared to other red GECIs as a function of pulses. (N) Peak ΔF/F₀ of SomaFRCaMPi compared to that of FRCaMPi as a function of pulses. Statistics in o for SomaFRCaMPi and FRCaMPi are the same as in J and Fig 1F, respectively. (O) Half the rise time of red GECIs as a function of pulses. (P) Half decay time of red GECIs as a function of pulses (SomaK-GECO1: n = 61 cells from 4 coverslips, 3 independent cultures; SomaXCaMP-R: n = 64 cells from 4 coverslips, 3 independent cultures; SomajRGECO1a: n = 100 cells from 6 coverslips, 4 independent cultures; SomaFRCaMPi: n = 148 cells from 7 coverslips, 4 independent cultures). Graph inset displays boxplot of half rise/decay time of each GECI after 10 action potentials. Box indicates the median and 25–75th percentile range, and the whiskers represent 1.5 times the interquartile range. See S5 Table for detailed statistics and exact p values. The quantitative data presented in this figure can be found in S1 Data. Created in BioRender, https://BioRender.com/e01g235.