{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T13:57:52Z","timestamp":1791467872088,"version":"4.3.4"},"reference-count":38,"publisher":"American Chemical Society (ACS)","issue":"40","license":[{"start":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T00:00:00Z","timestamp":1790208000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/na","name":"National Science Foundation Major Research Instrumentation program","doi-asserted-by":"publisher","award":["1126386"],"award-info":[{"award-number":["1126386"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"National Science Foundation Major Research Instrumentation program","doi-asserted-by":"publisher","award":["1919670"],"award-info":[{"award-number":["1919670"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Infrastructure Innovation for Biological Research program","doi-asserted-by":"publisher","award":["2327468"],"award-info":[{"award-number":["2327468"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R35GM151033"],"award-info":[{"award-number":["R35GM151033"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Investigations of G protein-coupled receptors (GPCRs) interactions with nonvisual arrestins in living cells are essential to understanding the complex molecular mechanisms of GPCR-based signaling. Quantitative analysis of these interactions remains challenging in live cells, particularly when attempting to repeatedly image distinct cellular regions with high precision. Here, we describe the implementation of an optical imaging stabilization approach that integrates the recently developed Focal Readjustment for Enhanced Vertical Resolution (FREVR) technology into a multiphoton microscope, enabling high-precision alternation between image-forming sample planes with &amp;lt;20 nanometer repeatability and stability over time. Using this setup, we monitored the dynamic recruitment of arrestin-2 (Arr2) to the plasma membrane of HEK-293 cells expressing muscarinic acetylcholine M2 receptors (M2R) by alternately imaging distinct planes of interest, the basolateral membrane and a membrane cross-section. Following stimulation of M2R by agonist ligand, we observed a pronounced redistribution of cytoplasmic Arr2 toward the plasma membrane in both cellular cross sections and at the basolateral membrane. This method enables direct comparison of receptor and arrestin dynamics across regions of individual cells with very high precision, eliminating the need for averaging over numerous cells in order to denoise biologically relevant signals, and thereby capturing physiological cell-to-cell variability with accuracy.<\/jats:p>","DOI":"10.1021\/acs.jpcb.6c02313","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T16:02:50Z","timestamp":1790265770000},"page":"10371-10382","source":"Crossref","is-referenced-by-count":0,"title":["Precise Alternation\nbetween Image-Forming Sample Planes\nEnables Quantitative Monitoring of Receptor-Arrestin Interaction Dynamics\nat the Plasma Membrane of Live Cells"],"prefix":"10.1021","volume":"130","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-3997-4648","authenticated-orcid":true,"given":"Thomas D.","family":"Killeen","sequence":"first","affiliation":[{"name":"University of Wisconsin-Milwaukee , , , ,","place":["Milwaukee, Wisconsin, United States, 53211"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael R.","family":"Stoneman","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Milwaukee , , , ,","place":["Milwaukee, Wisconsin, United States, 53211"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3111-4716","authenticated-orcid":true,"given":"Ionel","family":"Popa","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Milwaukee , , , ,","place":["Milwaukee, Wisconsin, United States, 53211"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiuyan","family":"Chen","sequence":"additional","affiliation":[{"name":"Molecular Biology and Pharmacology, Indiana University School of Medicine , , , ,","place":["Indianapolis, Indiana, United States, 46202"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3427-8516","authenticated-orcid":true,"given":"Valerica\u0306","family":"Raicu","sequence":"additional","affiliation":[{"name":"University of Wisconsin-Milwaukee , , , ,","place":["Milwaukee, Wisconsin, United States, 53211"]},{"name":"University of Wisconsin-Milwaukee , , , ,","place":["Milwaukee, Wisconsin, United States, 53211"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"issue":"12","key":"2026100809093177700_cit1","doi-asserted-by":"publisher","first-page":"2519","DOI":"10.3390\/ijms18122519","article-title":"Molecular Mechanisms of GPCR Signaling: A Structural\nPerspective","volume":"18","author":"Gurevich","year":"2017","journal-title":"Int. J. Mol. Sci."},{"issue":"2","key":"2026100809093177700_cit2","doi-asserted-by":"publisher","first-page":"413","DOI":"10.1124\/pr.113.008052","article-title":"G protein-coupled\nreceptor oligomerization revisited: functional and pharmacological\nperspectives","volume":"66","author":"Ferre","year":"2014","journal-title":"Pharmacol. Rev."},{"issue":"7","key":"2026100809093177700_cit3","doi-asserted-by":"publisher","first-page":"672","DOI":"10.1016\/j.tips.2018.04.002","article-title":"GPCRs and Signal\nTransducers: Interaction Stoichiometry","volume":"39","author":"Gurevich","year":"2018","journal-title":"Trends\nPharmacol. Sci."},{"issue":"1","key":"2026100809093177700_cit4","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1038\/s41392-020-00435-w","article-title":"G protein-coupled\nreceptors: structure- and function-based drug discovery","volume":"6","author":"Yang","year":"2021","journal-title":"Sig. Transduct. Target. Ther."},{"issue":"4","key":"2026100809093177700_cit5","doi-asserted-by":"publisher","first-page":"251","DOI":"10.1124\/mol.117.111062","article-title":"GPCRs as targets\nfor approved drugs: How many targets and how many drugs?","volume":"93","author":"Krishna","year":"2018","journal-title":"Mol. Pharmacol."},{"key":"2026100809093177700_cit6","doi-asserted-by":"publisher","first-page":"897","DOI":"10.1146\/annurev-biochem-060614-033910","article-title":"The Molecular Basis of G Protein-Coupled Receptor Activation","volume":"87","author":"Weis","year":"2018","journal-title":"Annu. Rev. Biochem."},{"issue":"14","key":"2026100809093177700_cit7","doi-asserted-by":"publisher","first-page":"3090","DOI":"10.1111\/bph.16297","article-title":"Microscopy and spectroscopy approaches\nto study GPCR structure and function","volume":"182","author":"Fessl","year":"2025","journal-title":"Br. J.\nPharmacol."},{"issue":"4","key":"2026100809093177700_cit8","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1038\/nrd.2017.229","article-title":"Biased signalling: from simple switches\nto allosteric microprocessors","volume":"17","author":"Smith","year":"2018","journal-title":"Nat. Rev. Drug\nDiscovery"},{"issue":"9","key":"2026100809093177700_cit9","doi-asserted-by":"publisher","first-page":"102279","DOI":"10.1016\/j.jbc.2022.102279","article-title":"G protein-coupled\nreceptor interactions with arrestins\nand GPCR kinases: The unresolved issue of signal bias","volume":"298","author":"Chen","year":"2022","journal-title":"J. Biol. Chem."},{"issue":"11","key":"2026100809093177700_cit10","doi-asserted-by":"publisher","DOI":"10.15252\/embr.202050437","article-title":"Exploring GPCR-arrestin\ninterfaces with genetically encoded crosslinkers","volume":"21","author":"Bottke","year":"2020","journal-title":"EMBO Rep."},{"issue":"3","key":"2026100809093177700_cit11","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1242\/jcs.115.3.455","article-title":"The role\nof beta-arrestins in the termination and transduction\nof G-protein-coupled receptor signals","volume":"115","author":"Luttrell","year":"2002","journal-title":"J. Cell\nSci."},{"key":"2026100809093177700_cit12","doi-asserted-by":"publisher","first-page":"125","DOI":"10.3389\/fphar.2019.00125","article-title":"GPCR Signaling Regulation:\nThe Role of GRKs and Arrestins","volume":"10","author":"Gurevich","year":"2019","journal-title":"Front. Pharmacol."},{"key":"2026100809093177700_cit13","doi-asserted-by":"publisher","first-page":"1369","DOI":"10.3389\/fphar.2018.01369","article-title":"\u03b2-Arrestin Based\nReceptor Signaling Paradigms: Potential Therapeutic Targets for Complex\nAge-Related Disorders","volume":"9","author":"van Gastel","year":"2018","journal-title":"Front. Pharmacol."},{"issue":"5","key":"2026100809093177700_cit14","doi-asserted-by":"publisher","first-page":"854","DOI":"10.1124\/pharmrev.121.000302","article-title":"\u03b2-Arrestins:\nStructure, Function, Physiology, and Pharmacological Perspectives","volume":"75","author":"Wess","year":"2023","journal-title":"Pharmacol. Rev."},{"key":"2026100809093177700_cit15","doi-asserted-by":"publisher","first-page":"4708","DOI":"10.1021\/acs.jpcb.3c01419","article-title":"Fluorescence-Based Detection of Proteins\nand Their Interactions in\nLive Cells","volume":"127","author":"Stoneman","year":"2023","journal-title":"J. Phys. Chem. 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Biol."},{"issue":"24","key":"2026100809093177700_cit19","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2201103119","article-title":"The M(1) muscarinic\nreceptor is present\nin situ as a ligand-regulated mixture of monomers and oligomeric complexes","volume":"119","author":"Marsango","year":"2022","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"12","key":"2026100809093177700_cit20","doi-asserted-by":"publisher","first-page":"6503","DOI":"10.1002\/anie.202010049","article-title":"Probing Membrane\nProtein Association Using Concentration-Dependent Number and Brightness","volume":"60","author":"Paul","year":"2021","journal-title":"Angew. Chem., Int. 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Biol."},{"key":"2026100809093177700_cit24","unstructured":"Silfies, J. S.; Lieser, E.; Schwartz, S. A.; Davidson, M. W.\n          Correcting\nFocus Drift in Live-Cell Microscopy. https:\/\/www.microscopyu.com\/applications\/live-cell-imaging\/correcting-focus-drift-in-live-cell-microscopy (accessed October 13, 2025)."},{"issue":"6","key":"2026100809093177700_cit25","doi-asserted-by":"publisher","first-page":"2195","DOI":"10.1021\/acs.nanolett.4c05234","article-title":"Enabling Hours-Long Drift Correction with Nanometer Resolution in\nOptical Microscopy through Reflection Interferometry of Fiducial Beads","volume":"25","author":"Phan","year":"2025","journal-title":"Nano Lett."},{"issue":"11","key":"2026100809093177700_cit26","doi-asserted-by":"publisher","first-page":"12177","DOI":"10.1364\/OE.20.012177","article-title":"Using fixed fiduciary\nmarkers for stage drift correction","volume":"20","author":"Lee","year":"2012","journal-title":"Opt. 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Biol.\nChem."},{"issue":"22","key":"2026100809093177700_cit29","doi-asserted-by":"publisher","first-page":"16723","DOI":"10.1074\/jbc.M109.069443","article-title":"Oligomeric size\nof the M2 muscarinic receptor in live cells as determined by quantitative\nfluorescence resonance energy transfer (FRET)","volume":"285","author":"Pisterzi","year":"2010","journal-title":"J. Biol. Chem."},{"issue":"12","key":"2026100809093177700_cit30","doi-asserted-by":"publisher","first-page":"905","DOI":"10.1038\/nmeth819","article-title":"A guide to choosing fluorescent proteins","volume":"2","author":"Shaner","year":"2005","journal-title":"Nat. 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Methods"},{"key":"2026100809093177700_cit33","volume-title":"Software for Spectral\nUnmixing of Fluorescence Microscopy Data; Raicu Lab","author":"OptiMiS","year":"2026"},{"issue":"7","key":"2026100809093177700_cit34","doi-asserted-by":"publisher","first-page":"1613","DOI":"10.1016\/j.bpj.2015.02.021","article-title":"Experimental verification of the\nkinetic theory of FRET using optical microspectroscopy and obligate\noligomers","volume":"108","author":"Patowary","year":"2015","journal-title":"Biophys. J."},{"issue":"9","key":"2026100809093177700_cit35","doi-asserted-by":"publisher","first-page":"1456","DOI":"10.1016\/j.bbamem.2016.12.008","article-title":"Quaternary\nstructure of the yeast\npheromone receptor Ste2 in living cells","volume":"1859","author":"Stoneman","year":"2017","journal-title":"Biochimica\nEt Biophysica Acta-Biomembranes"},{"issue":"23","key":"2026100809093177700_cit36","doi-asserted-by":"publisher","first-page":"11678","DOI":"10.3390\/ijms262311678","article-title":"Intensity-Based\nEstimation of Monomeric Brightness for Fluorescent Proteins","volume":"26","author":"Stoneman","year":"2025","journal-title":"Int. J. Mol. Sci."},{"issue":"2","key":"2026100809093177700_cit37","doi-asserted-by":"publisher","first-page":"319","DOI":"10.1085\/jgp.95.2.319","article-title":"Effects of extracellular ATP on ion transport systems\nand [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic\nagonist carbachol","volume":"95","author":"Soltoff","year":"1990","journal-title":"J. Gen. Physiol."},{"key":"2026100809093177700_cit38","doi-asserted-by":"publisher","first-page":"450","DOI":"10.3389\/fncel.2018.00450","article-title":"Endocytosis of Activated\nMuscarinic m2 Receptor (m2R) in Live Mouse Hippocampal Neurons Occurs\nvia a Clathrin-Dependent Pathway","volume":"12","author":"Lambert","year":"2018","journal-title":"Front. Cell.\nNeurosci."}],"container-title":["The Journal of Physical\nChemistry B"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jpcb.6c02313","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/130\/40\/10371\/68642100\/acs.jpcb.6c02313.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article-pdf\/130\/40\/10371\/68642100\/acs.jpcb.6c02313.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T13:12:59Z","timestamp":1791465179000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jpcbfk\/article\/130\/40\/10371\/5437867\/Precise-Alternation-between-Image-Forming-Sample"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,24]]},"references-count":38,"journal-issue":{"issue":"40","published-online":{"date-parts":[[2026,9,24]]},"published-print":{"date-parts":[[2026,10,8]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jpcb.6c02313","relation":{},"ISSN":["1520-6106","1520-5207"],"issn-type":[{"value":"1520-6106","type":"print"},{"value":"1520-5207","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,24]]}}}