{"doi":"10.1117/1.jbo.30.s2.s23901","title":"Consensus guidelines for cellular label-free optical metabolic imaging: ensuring accuracy and reproducibility in metabolic profiling","abstract":"Significance: Cellular metabolism plays a central role in health and disease, making its study critical for advancing diagnostics and therapies. Label-free optical metabolic imaging using endogenous fluorescence from reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) provides nondestructive, high-resolution insights into metabolic function and heterogeneity from the sub-cellular to the tissue level. Standardized approaches are essential to ensure reproducibility and comparability across studies. Aim: We aim to establish a consensus framework for the acquisition, calibration, and reporting of microscopic imaging metabolic function assessments based on fluorescence intensity and lifetime measurements of NAD(P)H and FAD. Approach: We present best practices for calibrating, analyzing, and reporting fluorescence intensity-based optical redox ratios and fluorescence lifetime data using multiexponential fitting and phasor analysis. Guidelines for validation experiments and cross-system standardization are provided to improve accuracy and reproducibility. Results: We demonstrate the importance of calibration procedures and normalization strategies for intensity-based optical redox measurements. We highlight needed calibration, signal-to-noise ratio considerations, and the impact of distinct analytical approaches on fluorescence lifetime-based metabolic function metrics. Conclusion: We recommend a consistent, practical framework for reproducible, label-free, optical metabolic imaging, facilitating robust comparisons across studies and supporting the broader adoption of optical metabolic imaging technologies for biomedical research and clinical translation.","journal":"Journal of Biomedical Optics","year":2025,"id":514093,"datarank":0.37273599746820013,"base_score":2.4849066497880004,"endowment":2.4849066497880004,"self_citation_contribution":0.37273599746820013,"citation_network_contribution":0.0,"self_endowment_contribution":0.37273599746820013,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":11,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9479,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":108082,"name":"Melissa C. Skala","orcid":"0000-0002-6320-7637","position":1,"is_corresponding":false},{"id":426587,"name":"Kyle P. Quinn","orcid":"0000-0002-6876-3608","position":2,"is_corresponding":false},{"id":1376032,"name":"Chiara Stringari","orcid":"0000-0002-0550-7463","position":3,"is_corresponding":false},{"id":451761,"name":"Janet E. Sorrells","orcid":"0000-0003-0071-8509","position":4,"is_corresponding":false},{"id":1376033,"name":"Ahmed A. Heikal","orcid":"0000-0002-0370-4033","position":5,"is_corresponding":false},{"id":407040,"name":"Lin Z. Li","orcid":"0000-0001-9447-7982","position":6,"is_corresponding":false},{"id":407037,"name":"He N. Xu","orcid":"0000-0001-5127-2629","position":7,"is_corresponding":false},{"id":417933,"name":"Sixian You","orcid":"0000-0002-1243-1815","position":8,"is_corresponding":false},{"id":253453,"name":"Alex J. Walsh","orcid":"0000-0003-3832-8207","position":9,"is_corresponding":false},{"id":108078,"name":"Rupsa Datta","orcid":"0000-0002-6432-2389","position":10,"is_corresponding":false},{"id":744913,"name":"Kayvan Samimi","orcid":"0000-0002-2566-612X","position":11,"is_corresponding":false},{"id":108081,"name":"Amani A. Gillette","orcid":"0000-0002-1470-3300","position":12,"is_corresponding":false},{"id":72584,"name":"Kevin W. Eliceiri","orcid":"0000-0001-8678-670X","position":13,"is_corresponding":false},{"id":353419,"name":"Mihaela Balu","orcid":"0000-0002-8018-5134","position":14,"is_corresponding":false},{"id":366688,"name":"Stephen A. Boppart","orcid":"0000-0002-9386-5630","position":15,"is_corresponding":false},{"id":253616,"name":"Michelle A. Digman","orcid":"0000-0003-4611-7100","position":16,"is_corresponding":false},{"id":1376034,"name":"Kylie R. Dunning","orcid":"0000-0002-0462-6479","position":17,"is_corresponding":false},{"id":315666,"name":"Conor L. Evans","orcid":"0000-0003-2185-6505","position":18,"is_corresponding":false},{"id":525761,"name":"Alba Alfonso‐García","orcid":"0000-0002-6748-3751","position":19,"is_corresponding":false},{"id":690993,"name":"Jessica P. Houston","orcid":"0000-0001-8201-6396","position":20,"is_corresponding":false},{"id":1376035,"name":"Wonsang Hwang","orcid":"0000-0002-6156-9554","position":21,"is_corresponding":false},{"id":1376668,"name":"Matthew M. Lindley","orcid":null,"position":22,"is_corresponding":false},{"id":295612,"name":"Xingde Li","orcid":"0000-0002-4725-3297","position":23,"is_corresponding":false},{"id":443821,"name":"Zhiyi Liu","orcid":"0000-0002-8122-8474","position":24,"is_corresponding":false},{"id":289213,"name":"Laura Marcu","orcid":"0000-0003-2369-0748","position":25,"is_corresponding":false},{"id":1376036,"name":"Sangeeta Murugkar","orcid":"0000-0002-6350-4893","position":26,"is_corresponding":false},{"id":579611,"name":"Michael G. Nichols","orcid":"0000-0002-6565-6743","position":27,"is_corresponding":false},{"id":1028211,"name":"Raluca Niesner","orcid":"0000-0001-8969-5432","position":28,"is_corresponding":false},{"id":140050,"name":"Sapun H. Parekh","orcid":"0000-0001-8522-1854","position":29,"is_corresponding":false},{"id":507025,"name":"Narasimhan Rajaram","orcid":"0000-0003-1224-8567","position":30,"is_corresponding":false},{"id":288785,"name":"Suman Ranjit","orcid":"0000-0003-3058-6332","position":31,"is_corresponding":false},{"id":337581,"name":"K. Robert Shen","orcid":"0000-0001-9605-1635","position":32,"is_corresponding":false},{"id":454543,"name":"Lingyan Shi","orcid":"0000-0003-1373-3206","position":33,"is_corresponding":false},{"id":536321,"name":"Belén Torrado","orcid":"0000-0002-0679-3422","position":34,"is_corresponding":false},{"id":632686,"name":"Alexander Vallmitjana","orcid":"0000-0002-7149-9385","position":35,"is_corresponding":false},{"id":743491,"name":"Michael Evers","orcid":"0000-0002-6461-4891","position":36,"is_corresponding":false},{"id":937774,"name":"Roger J. Zemp","orcid":"0000-0001-6802-5624","position":37,"is_corresponding":false},{"id":308841,"name":"Irene Georgakoudi","orcid":"0000-0002-0049-6697","position":0,"is_corresponding":true}],"reference_count":3,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:48:23.284782Z","pmid":"41230436","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":[]}