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. 2020 Apr 30;9:e54441. doi: 10.7554/eLife.54441

Figure 5. Simulations: iGluSnFR signal time course and SNR depends on the imaging volume.

Figure 5.

(A) iGluSnFR (300 μM) signals measured across different spherical regions of interest (ROIs). (B) Summary data shows that the dependence on ROI volume is greater at higher iGluSnFR concentrations. (C) Peak iGluSnFR response to the release of 5000 glutamate molecules as a function of iGluSnFR concentration. Dashed line indicates maximal signal based on maximal occupancy (see Figure 1B). (D) iGluSnFR signal variability decreases with indicator concentration. Inset, individual responses at three different iGluSnFR concentrations (gray, 1 μM; blue, 10 μM; orange, 100 μM). (E-F) iGluSnFR single-synapse response signal (ΔF/F0) depends on indicator concentration (E) and ROI dimensions (F). (G) iGluSnFR compound ΔF/F0 responses depend on ROI dimensions and the density of activated synapses. (H) When iGluSnFR was evenly sampled throughout even a small volume, only a small fraction of the indicator was activated by glutamate. Laser line scanning, by contrast, yields higher ΔF/F0 values (Helassa et al., 2018).

Figure 5—source data 1. iGluSnFR signals depend on imaging volume.
Simulated iGluSnFR signal decay time constants measured across different ROI radii and at different iGluSnFR concentrations (Figure 5B). Peak iGluSnFR signals vs. iGluSnFR concentration (Figure 5C). iGluSnFR response variability (CV) vs. iGluSnFR concentration (Figure 5D). Simulated peak iGluSnFR ΔF/F values (single synapse) across different ROI radii and at different iGluSnFR concentrations (Figure 5F). Simulated peak iGluSnFR ΔF/F values (multiple synapses) across different ROI radii and at different iGluSnFR concentrations (Figure 5G). Fraction of iGluSnFR bound within a 0.5 μm radius ROI vs. iGluSnFR concentration (Figure 5H).