{"doi":"10.1117/1.nph.11.3.035007","title":"Optical constraints on two-photon voltage imaging","abstract":"SignificanceGenetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of one-photon (1P) versus two-photon (2P) voltage imaging are not well characterized.AimWe 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.ApproachWe measure the 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 the 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.ResultsCompared with 1P excitation, 2P excitation requires ∼104-fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in the mouse cortex with a target SNR of 10 (spike height to 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 than ∼12 neurons simultaneously.ConclusionsDue 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 a depth of >300 μm will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging.","journal":"Neurophotonics","year":2024,"id":440954,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9613,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":806835,"name":"Hunter C. Davis","orcid":"0000-0003-1655-692X","position":1,"is_corresponding":false},{"id":807433,"name":"J. David Wong-Campos","orcid":null,"position":2,"is_corresponding":false},{"id":253698,"name":"Adam E. Cohen","orcid":"0000-0002-8699-2404","position":3,"is_corresponding":false},{"id":656788,"name":"F. Phil Brooks","orcid":"0000-0002-4334-8192","position":0,"is_corresponding":true}],"reference_count":73,"raw_metadata":null,"created_at":"2026-07-19T02:01:01.842286Z","pmid":"39139631","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}