{"doi":"10.1016/bs.mie.2025.08.029","title":"Quantitative single-cell imaging of efflux pump heterogeneity and antibiotic response dynamics","abstract":null,"journal":"Methods in Enzymology","year":2025,"id":670773,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"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":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":625940,"name":"Somenath Bakshi","orcid":"0000-0003-3864-2546","position":1,"is_corresponding":false},{"id":1162610,"name":"Ruizhe Li","orcid":"0000-0003-2512-845X","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Quantitative single-cell imaging of efflux pump heterogeneity and antibiotic response dynamics","abstract":"Understanding how bacterial cells survive antibiotic treatment requires tools capable of resolving dynamic physiological heterogeneity at single-cell resolution. Here, we present a high-throughput, lineage-resolved microfluidic imaging platform for quantifying the expression and spatial distribution of the AcrAB-TolC efflux pump in Escherichia coli during antibiotic exposure. The system is built around a custom-designed multilayer microfluidic device, fabricated using a direct-write photolithography protocol, which ensures uniform delivery of nutrients and antibiotics while confining thousands of individual cells in dead-end trenches. Brightfield imaging provides a label-free, high-temporal-resolution method for tracking cell growth and division, enabling accurate reconstruction of lineages over time. Efflux pump abundance is quantified using GFP-tagged AcrB, while a red fluorescent reporter driven by a constitutive promoter serves as a control to account for growth-dependent fluorescence artefacts. To analyse the sources of heterogeneity in efflux abundance and its consequences for antibiotic response dynamics, we developed a lineage-resolved analytical framework that compares cells of similar replicative age but differing spatial positions, and vice versa. This analysis was made possible by a novel machine-learning-based image processing pipeline, which enables robust cell segmentation, tracking, and lineage reconstruction across multiple fields of view. By extracting time-resolved single-cell data, this platform allows precise dissection of non-genetic variability in efflux activity and its role in determining survival outcomes, offering a powerful foundation for future quantitative studies of bacterial physiology under antibiotic stress.","is_dataset_classified":null,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"41309184","pmcid":null,"openalex_id":"https://openalex.org/W4415531636","authors":[],"funders":[{"funder_name":"Engineering and Physical Sciences Research Council","grant_id":"EP/W032813/1","title":"Engineering robust, stable, and safe synthetic genetic circuits for smart therapeutics"},{"funder_name":"Wellcome Trust","grant_id":"RG89305","title":null},{"funder_name":"Wellcome Trust","grant_id":"NKXY ISSF3/46","title":null},{"funder_name":"University of Cambridge","grant_id":"","title":null}],"total_grants":4,"fwci":3.5606,"citation_percentile":0.92736532,"influential_citations":0,"citation_trend":[{"year":2026,"count":1}],"oa_status":"closed","license":"Elsevier TDM","oa_locations":[{"url":"https://api.elsevier.com/content/article/PII:S0076687925003453?httpAccept=text/xml","host_type":"publisher"},{"url":"https://api.elsevier.com/content/article/PII:S0076687925003453?httpAccept=text/plain","host_type":"publisher"},{"url":"https://doi.org/10.1016/bs.mie.2025.08.029","host_type":"book series"},{"url":"https://pubmed.ncbi.nlm.nih.gov/41309184","host_type":"repository"},{"url":"https://doi.org/10.1101/2025.08.06.668869","host_type":""}],"fields_of_study":["Innovative Microfluidic and Catalytic Techniques Innovation","Microfluidic and Capillary Electrophoresis Applications","Biosensors and Analytical Detection","0301 basic medicine","03 medical and health sciences","Single-Cell Analysis","Escherichia coli","Anti-Bacterial Agents","Escherichia coli Proteins","ATP-Binding Cassette, Sub-Family C Proteins","Lab-On-A-Chip Devices","Microfluidic Analytical Techniques","Bacterial Outer Membrane Proteins","Carrier Proteins","Green Fluorescent Proteins","Red Fluorescent Protein"],"mesh_terms":["Red Fluorescent Protein","Anti-Bacterial Agents","Bacterial Outer Membrane Proteins","Carrier Proteins","Escherichia coli","ATP-Binding Cassette, Sub-Family C Proteins","Multidrug Resistance-Associated Proteins","Escherichia coli Proteins","Microfluidic Analytical Techniques","Green Fluorescent Proteins","Lab-On-A-Chip Devices","Single-Cell Analysis"],"keywords":["Efflux","Microfluidics","Antibiotics","Fluorescence-lifetime imaging microscopy","Live cell imaging","Cell","Single-cell analysis","Chemostat","Machine Learning","Efflux pumps","Physiological Variability","Antibiotic Tolerance","Time-lapse Microscopy","Single-cell Imaging","Acrab-tolc","Fluorescent Reporters","Lineage Tracking","Bacterial Heterogeneity","Non-genetic Resistance","Direct-write Photolithography","Image Segmentation And Tracking","ATP-Binding Cassette, Sub-Family C Proteins","Escherichia coli Proteins","Lab-On-A-Chip Devices","Green Fluorescent Proteins","Escherichia coli","Microfluidic Analytical Techniques","Carrier Proteins","Anti-Bacterial Agents","Bacterial Outer Membrane Proteins","Red Fluorescent Protein"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-15T23:47:41.368516Z","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":[]}