Abstract
All-optical interrogation, based on high-resolution two-photon stimulation and imaging, has emerged as a potentially transformative approach in neuroscience, allowing for the simultaneous precise manipulation and monitoring of neuronal activity across various model organisms. However, the unintended excitation of light-gated ion channels such as channelrhodopsin (ChR) during two-photon calcium imaging with genetically encoded calcium indicators (GECIs) introduces artifactual neuronal perturbation and contaminates neural activity measurements. In this study, we propose an active pixel power control (APPC) approach, which dynamically adjusts the imaging laser power at each scanning pixel, to address the challenge. We aim to achieve simultaneous two-photon optogenetic manipulation and calcium imaging with a single femtosecond laser, while minimizing the cross-talk between manipulation and imaging. To study this technology’s capabilities, we applied it to the larval zebrafish brain in vivo. Our results demonstrate that the APPC approach preserves GECI signal quality while suppressing optogenetic artifacts significantly. This enhances the accuracy of neural circuit dissection and advances the precision of all-optical interrogation, offering a robust framework for probing neural circuit dynamics and causality in vivo with high fidelity, potentially across various model organisms. Importantly, this technology can be seamlessly integrated with commonly used two-photon microscope systems in laboratories worldwide.
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Data availability
The main data supporting the findings of this study are available within the paper and Supplementary Information files. The source data files for all data presented within the figures can be found at: https://github.com/QuLab1234/APPC.
Code availability
The custom codes for image processing are available online at: https://github.com/QuLab1234/APPC.
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Acknowledgements
This study was supported in part by the Research Grants Council of Hong Kong to J.Y.Q. (16102825, 16102123, 16102122, 16102421, 16102920, 16102518); Collaborative Research Fund (C6001-19E); the Innovation and Technology Commission (ITCPD/17-9); the Theme-Based Research Scheme (T12-611/25-N) and the Hong Kong University of Science & Technology through grant 30 for 30 Research Initiative Scheme, and Research Grants Council of Hong Kong to J.L.S. (16101221, 16103224, 16103522, and 16103625).
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G.Y., G.T., and J.Y.Q. conceived the research idea and designed the experiments. G.Y. built the imaging systems and Y.F. and Z.S. provided technical support; G.T. and K.Y.C. prepared animals; G.Y. performed imaging experiments under the supervision of J.Y.Q. and J.L.S.; G.Y. and G.T. analyzed the data with assistance from Y.H.; G.Y., G.T., Y.H., J.L.S., and J.Y.Q. wrote the paper with input from all other authors.
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Yan, G., Tian, G., Fu, Y. et al. Active pixel power control for crosstalk-free All-optical neural interrogation. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69419-8
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DOI: https://doi.org/10.1038/s41467-026-69419-8
