{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T13:12:01Z","timestamp":1788613921201,"version":"build-2803163510"},"reference-count":49,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T00:00:00Z","timestamp":1788566400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T00:00:00Z","timestamp":1788566400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["22K07379"],"award-info":[{"award-number":["22K07379"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["25K10758"],"award-info":[{"award-number":["25K10758"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Microscopy Res &amp; Technique"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>Liquid\u2013liquid phase separation (LLPS) plays a central role in intracellular compartmentalization and gene regulation. Although optogenetic optoDroplet systems are widely used to study LLPS in living cells, the mechanisms underlying the transition from dynamic liquid\u2010like condensates to operationally defined gel\u2010like assemblies remain poorly understood because intermediate assembly states are difficult to monitor in\u00a0situ. Here, we developed a confocal laser microspectroscopy\u2010based approach to characterize the assembly dynamics of the fused sarcomas (FUS)n\u2010mCherry\u2010CRY2 optoDroplet system in living NIH3T3 cells. Quantitative fluorescence analysis enabled the identification and discrimination of three distinct assembly states: monomeric, dimer\/oligomer intermediate, and gel\u2010like. Passive micro\u2010rheology, fluorescence recovery after photobleaching (FRAP), and automated morphological analyses further demonstrated progressive reductions in molecular mobility and increasing structural rigidity during condensate maturation. Three\u2010dimensional spatial analysis revealed that mature condensates exhibit a distinct core\u2013shell organization consisting of an operationally defined gel\u2010like core, a dimer\/oligomer\u2010rich intermediate layer, and an outer monomer\u2010rich boundary layer with liquid\u2010like properties. Upon cessation of blue\u2010light stimulation, the outer layer rapidly dissolved, whereas the central gel\u2010like core remained intact, indicating the acquisition of physical irreversibility. These findings establish fluorescence microspectroscopy as a quantitative approach for resolving molecular assembly states during condensate maturation and demonstrate that gelation proceeds from the condensate center toward the periphery. This framework provides new insights into pathological liquid\u2010to\u2010gel phase transitions associated with neurodegenerative diseases.<\/jats:p>","DOI":"10.1002\/jemt.70172","type":"journal-article","created":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T12:19:31Z","timestamp":1788610771000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Innovative Method for Evaluating the Optical Properties of Liquid\u2013Liquid Phase Separations Using Laser Scanning Microscopic Spectroscopy"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-3983-3165","authenticated-orcid":false,"given":"Johbu","family":"Itoh","sequence":"first","affiliation":[{"name":"Department of Neurology Tokai University School of Medicine  Isehara Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natsuko","family":"Fujii","sequence":"additional","affiliation":[{"name":"Department of Neurology Tokai University School of Medicine  Isehara Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saori","family":"Kohara","sequence":"additional","affiliation":[{"name":"Department of Neurology Tokai University School of Medicine  Isehara Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masatoshi","family":"Ito","sequence":"additional","affiliation":[{"name":"Department of Legal Medicine St. Marianna University School of Medicine  Kawasaki Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoto","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Department of Neurology Tokai University School of Medicine  Isehara Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6902-8309","authenticated-orcid":false,"given":"Eiichiro","family":"Nagata","sequence":"additional","affiliation":[{"name":"Department of Neurology Tokai University School of Medicine  Isehara Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,5]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580\u2010020\u201000326\u20106"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2694"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018\u2010022\u201004276\u20104"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.7"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M700616200"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1364\/OL.28.000695"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tcb.2018.02.004"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.201308087"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1017150108"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3891"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.9b05047"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/d41586\u2010018\u201003070\u20102"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010018\u201028858\u20100"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.REV119.007895"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2016.04.047"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ceb.2021.01.002"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2025.06.014"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E20\u201009\u20100568"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.1369\/jhc.6A7110.2007"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1385\/1\u201059745\u2010369\u20102:29"},{"key":"e_1_2_11_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.12.038"},{"key":"e_1_2_11_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41392\u2010024\u201002070\u20101"},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2012.04.017"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2940"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1155\/2022\/7165387"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature10879"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.08.018"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jpcb.8b08903"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.09.015"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.10.030"},{"key":"e_1_2_11_32_1","doi-asserted-by":"publisher","DOI":"10.1002\/mco2.223"},{"key":"e_1_2_11_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.01.013"},{"key":"e_1_2_11_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580\u2010018\u20100085\u2010z"},{"key":"e_1_2_11_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.07.047"},{"key":"e_1_2_11_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xplc.2024.101225"},{"key":"e_1_2_11_37_1","doi-asserted-by":"publisher","DOI":"10.1021\/ar900291y"},{"key":"e_1_2_11_38_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1037"},{"key":"e_1_2_11_39_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2016.11.054"},{"key":"e_1_2_11_40_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaf4382"},{"key":"e_1_2_11_41_1","doi-asserted-by":"publisher","DOI":"10.2144\/000113765"},{"key":"e_1_2_11_42_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms5925"},{"key":"e_1_2_11_43_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpc.2011.05.016"},{"key":"e_1_2_11_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2015.01.012"},{"key":"e_1_2_11_45_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2008.12.3902"},{"key":"e_1_2_11_46_1","doi-asserted-by":"publisher","DOI":"10.3389\/fphy.2021.644450"},{"key":"e_1_2_11_47_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.09.017"},{"key":"e_1_2_11_48_1","doi-asserted-by":"publisher","DOI":"10.1155\/2013\/890203"},{"key":"e_1_2_11_49_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2024.117448"},{"key":"e_1_2_11_50_1","doi-asserted-by":"publisher","DOI":"10.1002\/adma.202510115"}],"container-title":["Microscopy Research and Technique"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jemt.70172","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/jemt.70172","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jemt.70172","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T12:19:32Z","timestamp":1788610772000},"score":1,"resource":{"primary":{"URL":"https:\/\/analyticalsciencejournals.onlinelibrary.wiley.com\/doi\/10.1002\/jemt.70172"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,5]]},"references-count":49,"alternative-id":["10.1002\/jemt.70172"],"URL":"https:\/\/doi.org\/10.1002\/jemt.70172","archive":["Portico"],"relation":{},"ISSN":["1059-910X","1097-0029"],"issn-type":[{"value":"1059-910X","type":"print"},{"value":"1097-0029","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,5]]},"assertion":[{"value":"2025-12-16","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-05","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-05","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"jemt.70172"}}