{"doi":"10.1101/2020.09.04.283572","title":"High throughput instrument to screen fluorescent proteins under two-photon excitation","abstract":"Abstract Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":127321,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9532,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":576014,"name":"Jonathan King","orcid":"0000-0002-3679-5488","position":1,"is_corresponding":false},{"id":576629,"name":"Jacob Franklin","orcid":null,"position":2,"is_corresponding":false},{"id":576015,"name":"Nathan Clack","orcid":"0000-0001-6236-9282","position":3,"is_corresponding":false},{"id":555029,"name":"Christopher McRaven","orcid":null,"position":4,"is_corresponding":false},{"id":576630,"name":"В. А. Гончаров","orcid":null,"position":5,"is_corresponding":false},{"id":576016,"name":"Daniel Flickinger","orcid":"0000-0002-8361-3122","position":6,"is_corresponding":false},{"id":250506,"name":"Karel Svoboda","orcid":"0000-0002-6670-7362","position":7,"is_corresponding":false},{"id":275972,"name":"Mikhail Drobizhev","orcid":"0000-0001-9072-1300","position":8,"is_corresponding":false},{"id":275973,"name":"Thomas E. Hughes","orcid":"0000-0002-8430-070X","position":9,"is_corresponding":false},{"id":275971,"name":"Rosana S. Molina","orcid":"0000-0001-7891-2462","position":0,"is_corresponding":true}],"reference_count":33,"raw_metadata":null,"created_at":"2026-07-18T23:15:30.930746Z","pmid":null,"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":[]}