Optical constraints on two-photon voltage imaging.
| Autor: | Brooks FP 3rd; Department of Chemistry and Chemical Biology, Harvard University., Davis HC; Department of Chemistry and Chemical Biology, Harvard University., Wong-Campos JD; Department of Chemistry and Chemical Biology, Harvard University., Cohen AE; Department of Chemistry and Chemical Biology, Harvard University. |
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| Jazyk: | angličtina |
| Zdroj: | BioRxiv : the preprint server for biology [bioRxiv] 2024 May 17. Date of Electronic Publication: 2024 May 17. |
| DOI: | 10.1101/2023.11.18.567441 |
| Abstrakt: | Significance: Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo , but the relative merits and limitations of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. Aim: We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. Approach: We measure brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. Results: Compared to 1P excitation, 2P excitation requires ~10 4 -fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in mouse cortex with a target SNR of 10 (spike height:baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of > 300 μm, 2P voltage imaging using an 80 MHz source can record from no more 12 cells simultaneously. Conclusions: Due to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at depth > 300 μm will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging. Competing Interests: Disclosures. The authors have no competing interests to declare. |
| Databáze: | MEDLINE |
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