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Summary:

In this study, the authors report the development of a genetically encoded voltage indicator (GEVI) based on a rhodopsin protein combined with a red-fluorescent dye, enabling fast two-photon (2P) imaging of cell membrane potentials called 2Photron. While other existing GEVIs can perform 2P voltage imaging, they cannot be multiplexed with green fluorescent protein–based indicators. This novel red-shifted GEVI addresses that limitation, offering sub-millisecond, high–spatial-resolution voltage imaging with sufficient sensitivity for experiments in awake, behaving animals, while simultaneously detecting calcium signals with a green-fluorescent calcium indicator. This work therefore presents an important and useful tool for studying diverse electrical events in neurons.

Major Concerns:

1.      The authors performed the initial rhodopsin screening using the JF525 dye rather than the JF552 dye ultimately used for multiplexing. Could this choice explain why the fluorescence sensitivity (ΔF/F₀) in Fig. 1 differs from that observed in cultured neurons with JF552? Since the fluorescence change relies on FRET which is highly sensitive to the spectral overlap between the protein pair, the authors need to explain the rationale for using different dyes.

2.      A more thorough study of the photophysics of this GEVI is lacking. The authors should present the 2P absorption spectrum, which would be helpful for reference for future efforts or improvements. Similarly, they state in the discussion that, because of FRET mediation, illumination details can influence fluorescence amplitude and kinetics. This claim should be supported by experimental data showing how illumination conditions affected the voltage sensitivity since they performed experiments under different conditions.

3.      The authors compare 2Photron to another GEVI, JEDI-2P-Kv, by co-expressing them in cultured cells to extract performance metrics. While they acknowledge that co-expression can hinder the performance of both probes, a more rigorous comparison with single expression would clarify the true performance of each indicator.

4.      The manuscript does not explain why the authors did not include a comparison with GCaMP6f. The authors should indicate whether the discrepancy between spike number and amplitude was greater with GCaMP6f than with GCaMP8, as this would be relevant information for the neuroscience community, since GCaMP6 is still widely used.

Minor Concerns:

·         In the introduction, when noting that 1030–1080 nm lasers are convenient for excitation, it would be useful to explicitly state that JF552 is a suitable dye for this wavelength range to improve reader understanding[AH1] .

·         The supplementary materials indicate that different setups were used for the experiments, but this is not clearly described in either the main text or supplementary text. The authors should explicitly state which setup was used for each measurement, using consistent naming or numbering (e.g., SLAP microscope = Setup 1, Bergamo microscope = Setup 2, uLOVe microscope = Setup 3). Supplementary Table 3 should list intensity instead of power to allow a consistent comparison between setups.

·         Fig. 1c’s bit plot needs an explanation for non-expert readers [AH2] or should be omitted if it is not essential.

·         In Fig. 1d–e, the 1P vs. 2P comparisons would be easier to read if the fluorescence bars for the same sensors were placed side-by-side. The current ordering makes direct comparison difficult.

·         To convey the relevance of positive-going sensors in the context of Fig 1., the authors should explain why they included 1P signals from positive-going sensors in the text.

·         The discussion of Fig. 2c and Supplementary Figs. 7–8 does not mention the differences in illumination conditions. A brief description (e.g., “X times higher intensity,” “higher repetition rate”) would clarify the intended message. The discussion of the second-best GEVI, PaR+, could be moved to the supplementary material, as it is not used in in vivo experiments and its performance metrics are not detailed in the main text. This would free space to describe more relevant experimental details about how the illumination conditions affect the performance.

·         For Fig. 3f, the authors mention that they analyzed 16 cells co-expressing GCaMP. They should clarify whether GCaMP co-expression affects 2Photron performance or include a comparison in the absence of co-expression for a fairer evaluation.

·         The discussion of normalized amplitude could be moved to the supplementary material, and the main text could instead expand on correlations between GCaMP responses and other spike properties.

·         The definition of spike onset is not clear in the supplementary text. A more thorough explanation is needed for better comprehension.

 [AH1]Can you let the authors know why it would be useful? For example, Is it to improve reader understanding?

 [AH2]Can you briefly explain why more explanation would be helpful?

Competing interests

H. Schroff has collaborated with E. R. Schreiter