{"doi":"10.1117/12.2608888","title":"Fluorescence lifetime imaging of metMyoglobin formation due to nitric oxide stress","abstract":"Myoglobin is a protein that is expressed quite unevenly among different cell types. Nevertheless, it has been widely acknowledged that the Fe<sup>3+</sup> state of myoglobin, <i>met</i>myoglobin (<i>met</i>Mb) has a broad functional role in metabolism, oxidative/nitrative regulation and gene networks. Accordingly, real-time monitoring of oxygenated, deoxygenated and <i>met</i>Mb proportions- or, more broadly, of the mechanisms by which <i>met</i>Mb is formed, presents a promising line of research. We had previously introduced a Förster resonance energy transfer (FRET) method to read out the deoxygenation/oxygenation states of myoglobin, by creating the targetable oxygen (O<sub>2</sub>) sensor Myoglobin-mCherry. In this sensor, changes in myoglobin absorbance features that occur with lost O<sub>2</sub> occupancy -or upon <i>met</i>Mb production control the FRET rate from the fluorescent protein to myoglobin. When O<sub>2</sub> is bound, mCherry fluorescence is only slightly quenched, but if either O<sub>2</sub> is released or met is produced, FRET will increase- and this rate competing with emission reduces both emission yield and lifetime. Nitric oxide (NO) is an important signal (but also a toxic molecule) that can oxidize myoglobin to <i>met</i>Mb with absorbance increases in the red visible range. mCherry thus senses both <i>met</i> and deoxygenated myoglobin, which cannot be easily separated at hypoxia. In order to dissect this, we treat cells with NO and investigate how the Myoglobin-mCherry lifetime is affected by generating <i>met</i>Mb. More discriminatory power is then achieved when the fluorescent protein EYFP is added to Myoglobin-mCherry, creating a sandwich probe whose lifetime can selectively respond to <i>met</i>Mb while being indifferent to O<sub>2</sub> occupancy.","journal":"PubMed","year":2022,"id":300792,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9528,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":669638,"name":"Mateus Prates Mori","orcid":"0000-0003-1349-6378","position":1,"is_corresponding":false},{"id":247630,"name":"Paul M. Hwang","orcid":"0000-0002-5976-7834","position":2,"is_corresponding":false},{"id":401702,"name":"Dan L. Sackett","orcid":"0000-0002-3492-1866","position":3,"is_corresponding":false},{"id":438071,"name":"Jay R. Knutson","orcid":"0000-0001-9990-1738","position":4,"is_corresponding":false},{"id":458258,"name":"Rozhin Penjweini","orcid":null,"position":0,"is_corresponding":true}],"reference_count":20,"raw_metadata":null,"created_at":"2026-07-19T00:31:57.812980Z","pmid":"35463920","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":[]}