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Resolving this organisation requires techniques capable of probing dynamics at the single-molecule level with high spatial and temporal resolution. In this study, we employ the complementary fluorescence fluctuation spectroscopy approaches, Fluorescence Correlation Spectroscopy (FCS), Photon Counting Histogram Analysis (PCH), Raster Image Correlation Spectroscopy (RICS) and Number and Brightness Analysis (N&amp;B), in conjunction with Fluorescence Recovery After Photobleaching (FRAP), to investigate the membrane organisation of the C-X-C chemokine receptor 4 (CXCR4), a GPCR known to undergo ligand-induced reorganisation. At the nanoscale, FCS highlighted opposing effects on diffusion after agonist (CXCL12) and inverse agonist (IT1t) treatment, whilst RICS also showed ligand-mediated changes in particle number. Both single-point and image-based brightness analyses (PCH and N&amp;B) showed increased brightness after CXCL12 treatment, consistent with the pre-internalisation clustering of CXCR4. At the microscale, FRAP showed an increase in immobile CXCR4, not visible to FFS approaches, following CXCL12 stimulation. This integrated approach, performed on a single commercial confocal microscope, provides valuable insight into the reorganisation of CXCR4 in the plasma membrane over a range of temporal and spatial scales, which are not detectable using standard imaging.<\/jats:p>","DOI":"10.3390\/biom16081107","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T10:13:47Z","timestamp":1785320027000},"page":"1107","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Characterising C-X-C Chemokine Receptor 4 Dynamics in the Cell Membrane Using Fluorescence Fluctuation Spectroscopy"],"prefix":"10.3390","volume":"16","author":[{"given":"Noemi","family":"Karsai","sequence":"first","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen\u2019s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK"},{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6227-4483","authenticated-orcid":false,"given":"Jo\u00eblle","family":"Goulding","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen\u2019s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK"},{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leigh A.","family":"Stoddart","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen\u2019s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK"},{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6331-5606","authenticated-orcid":false,"given":"Laura E.","family":"Kilpatrick","sequence":"additional","affiliation":[{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"},{"name":"School of Pharmacy, Biomedical Discovery Institute 3, University of Nottingham, Nottingham NG7 2RD, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4424-239X","authenticated-orcid":false,"given":"Stephen J.","family":"Hill","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen\u2019s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK"},{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meritxell","family":"Canals","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen\u2019s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK"},{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8514-0827","authenticated-orcid":false,"given":"Stephen J.","family":"Briddon","sequence":"additional","affiliation":[{"name":"Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, Queen\u2019s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK"},{"name":"Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/j.tcb.2020.11.004","article-title":"van Dynamic Plasma Membrane Organization: A Complex Symphony","volume":"31","author":"Arp","year":"2021","journal-title":"Trends Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1111\/tra.12628","article-title":"Regulation of G Protein-Coupled Receptor Signaling by Plasma Membrane Organization and Endocytosis","volume":"20","author":"Weinberg","year":"2019","journal-title":"Traffic"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1042\/BST20150236","article-title":"Compartmentalization of GPCR Signalling Controls Unique Cellular Responses","volume":"44","author":"Ellisdon","year":"2016","journal-title":"Biochem. 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