Red-shifted excitation enhances the sensitivity of red genetically encoded Ca2+ indicator and enables crosstalk-free two-photon holographic optophysiology.

Significance: Two-photon (2P) holographic optophysiology, which combines optogenetic actuators and genetically encoded Ca 2 + indicators (GECIs), enables precise in vivo interrogation of neuronal networks. However, this approach is hindered by crosstalk, which is unintentional activation of actuators by imaging light, especially when using blue-light-activated GECIs (e.g., GCaMP) with red-light-activated actuators (e.g., ChRmine). Aim: To eliminate crosstalk in 2P holographic optophysiology, we employed the inverse combination, namely red GECIs and blue-light-activated actuators and optimized the excitation wavelength, as conventional 2P excitation failed to detect optogenetically induced GECI responses. Approach: PC12h cells expressing various combinations of GECIs and optogenetic actuators were subjected to simultaneous Ca 2 + imaging and optogenetic stimulation under both single-photon (1P) and 2P excitation. Results: Under 1P excitation, crosstalk was evident in the GCaMP6m (blue)/ChRmine (red) pair, but negligible in the R-CaMP1.07 (red)/eTsChR (blue) pair. Under 2P excitation, R-CaMP1.07 showed significantly enhanced sensitivity at a red-shifted wavelength ( ∼ 1200    nm ) compared with the expected 2P excitation wavelength (1125 nm). Conclusion: Red-shifted excitation was essential for detecting the small Ca 2 + elevation following optogenetic stimulation. This optimized condition improves the sensitivity of red GECIs and enables a more robust 2P optophysiology free from crosstalk.