Improved orange and red Ca²± indicators and photophysical considerations for optogenetic applications

Abstract

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca(2+)) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca(2+) indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca(2+) indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca(2+) imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Figure 1

Characterization of improved indicators. (a) Excitation spectra of O-GECO1 (orange), R-GECO1.2 (red) and CAR-GECO1 (dark red) with (solid line) and without (dashed line) Ca²⁺. Inset: 25× y-axis zoom. (b) Emission spectra represented as in (a). (c) Two-photon excitation spectra of O-GECO1, R-GECO1, and CAR-GECO1, colored as in (a, b). (d–f) Models of (d) CAR-GECO1; (e) R-GECO1.2; and (f) O-GECO1, showing location of substitutions relative to R-GECO1. The structural model used in (d–f) is based on PDB IDs 3EVR(36) and 2H5Q(28) and was previously described.

Figure 2

Imaging ChR2-induced Ca²⁺ transients in INS-1 cells. (a, d) Example fluorescence images of INS-1 cells expressing ChR2(T159C)-EGFP and either (a) R-GECO1.2 or (d) Lyn-R-GECO1.2. Scale bar = 2 μm. (b, e) ChR2-dependent fluorescence vs time traces for cells expressing ChR2(T159C)-EGFP and either (b) R-GECO1.2 or (e) Lyn-R-GECO1.2, during intervals of blue light irradiation (480–500 nm at 135 mW/cm²) for 50–200 ms. (c, f) ChR2-independent fluorescence vs time traces for cells expressing only (c) R-GECO1.2 or (f) Lyn-R-GECO1.2, during intervals of blue light irradiation as described for (b, e). (g, h) Average signal enhancements of R-GECO1.2 or Lyn-R-GECO1.2 upon increasing doses of blue light illumination in cells expressing (g) R-GECO1.2 only (n = 22) or R-GECO1.2 with ChR2(T159C)-EGFP (n = 33); or (h) Lyn-R-GECO1.2 (n = 18) or Lyn-R-GECO1.2 with ChR2(T159C)-EGFP (n = 24).

Figure 3

Confocal imaging of ChR2(T159C)-induced Ca²⁺ elevations in mouse neocortical slice culture. Fluorescence images and response to photoactivation light for neocortial neurons transfected with only CAR-GECO1 (a–c) or cotransfected with CAR-GECO1 and ChR2(T159C)-EGFP (d–f). Scale bar = 50 μm. (b, c) Fluorescence vs time traces for cells transfected with only CAR-GECO1, during intervals of illumination at region of interest 1 (ROI-1) with a 405 nm laser at either 100% (90 μJ/μm²) (b) or 5% (4.5 μJ/μm²) (c) power. Control cells at ROI-2 and 3 were not illuminated. (e, f) Identical experimental conditions to (b, c) using tissue that has been cotransfected with CAR-GECO1 and ChR2(T159C)-EGFP. Based on the colocalization of green and red fluorescence, the neuron being photoactivated (at ROI-1) is expressing both CAR-GECO1 and ChR2(T159C)-EGFP.

Figure 4

Reversible photoactivation of Ca²⁺ indicators during 405 nm illumination. Solutions of purified Ca²⁺ indicators were illuminated with a violet light laser (405 nm, 150 mW) for 5 s intervals. (a–c) Absorbance changes for the fluorescent (anionic) form of the Ca²⁺-free state during 5 s violet light pulses (black solid line) for (a) CAR-GECO1 at 560 nm; (b) R-GECO1.2 at 570 nm; (c) O-GECO1 at 545 nm. (d–f) Transient absorbance spectra (dashed lines) acquired immediately after the onset of illumination for (d) CAR-GECO1; (e) R-GECO1.2; (f) O-GECO1. (g–i) Transient absorbance spectra (dashed lines) acquired immediately after the end of illumination for (g) CAR-GECO1; (h) R-GECO1.2; (i) O-GECO1.

Figure 5

pH dependence of photoactivation of Ca²⁺ indicators. Change of absorbance in the Ca²⁺-free state (a–c) and Ca²⁺-bound state (d–f) with violet light (405 nm) illumination as a function of pH for: (a, d) CAR-GECO1; (b, e) R-GECO1.2; (c, f) O-GECO1.

Figure 6

Proposed mechanism of photoactivation in red fluorescent GECOs.