{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T19:40:25Z","timestamp":1786995625656,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,13]]},"group-title":"Neuroscience","reference-count":41,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T00:00:00Z","timestamp":1786579200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["CP2-1-0000000704"],"award-info":[{"award-number":["CP2-1-0000000704"]}],"id":[{"id":"https:\/\/ror.org\/02qenvm24","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders and Stroke","doi-asserted-by":"crossref","award":["DP2NS136990"],"award-info":[{"award-number":["DP2NS136990"]}],"id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000070","name":"National Institute of Biomedical Imaging and Bioengineering","doi-asserted-by":"crossref","award":["R01EB037653"],"award-info":[{"award-number":["R01EB037653"]}],"id":[{"id":"10.13039\/100000070","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000025","name":"National Institute of Mental Health","doi-asserted-by":"crossref","award":["F30MH138009"],"award-info":[{"award-number":["F30MH138009"]}],"id":[{"id":"10.13039\/100000025","id-type":"DOI","asserted-by":"crossref"}]},{"award":["T32GM136577"],"award-info":[{"award-number":["T32GM136577"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/02320dz84","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,13]]},"abstract":"<jats:p>\n                  Recent advances in fluorescent indicators and optical microscopy now enable\n                  <jats:italic>in vivo<\/jats:italic>\n                  synaptic imaging of glutamate, which transmits the majority of signals between neurons in the brain. Extracting fluorescence signals from these recordings is complicated by the minuscule scale and dense clustering of synapses on dendrites, as well as brain motion in behaving animals. Here we present the Glutamate Imaging Analysis Toolbox (GIAnT), a set of automated tools for glutamate imaging data that corrects sample motion, identifies active synapses with super-resolution precision, and extracts synaptic fluorescence signals. Compared to methods designed for cellular imaging, GIAnT reduces motion artifacts, more accurately identifies active synapses, and improves extracted signal quality by reducing contamination from overlapping synapses. By pairing\n                  <jats:italic>in vivo<\/jats:italic>\n                  glutamate imaging with\n                  <jats:italic>post hoc<\/jats:italic>\n                  expansion microscopy, we find that &gt;70% of the putative synapses extracted using GIAnT matched one-to-one with glutamatergic synapses onto the postsynaptic cell. Our results establish GIAnT as an automated and validated pipeline for processing synaptic glutamate imaging data at scale.\n                <\/jats:p>","DOI":"10.64898\/2026.08.07.743580","type":"posted-content","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T04:50:14Z","timestamp":1786683014000},"source":"Crossref","is-referenced-by-count":0,"title":["GIAnT: a Glutamate Imaging Analysis Toolbox"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4026-9181","authenticated-orcid":false,"given":"Michael E.","family":"Xie","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD"},{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1321-5866","authenticated-orcid":false,"given":"Johannes","family":"Friedrich","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"Wirsching","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caleb Jones","family":"Shibu","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3742-394X","authenticated-orcid":false,"given":"Maedeh","family":"Seyedolmohadesin","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naveen","family":"Ouellette","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tim","family":"Wang","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6670-7362","authenticated-orcid":false,"given":"Karel","family":"Svoboda","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9045-3489","authenticated-orcid":false,"given":"Adam S.","family":"Charles","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0374-2005","authenticated-orcid":false,"given":"Kaspar","family":"Podgorski","sequence":"additional","affiliation":[{"name":"Allen Institute, Neural Dynamics, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081712200556000_2026.08.07.743580v1.1","doi-asserted-by":"crossref","first-page":"3558","DOI":"10.1038\/s41467-021-23829-y","article-title":"The functional organization of excitatory synaptic input to place cells","volume":"12","year":"2021","journal-title":"Nature Communications"},{"key":"2026081712200556000_2026.08.07.743580v1.2","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1038\/s41592-023-01863-6","article-title":"Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission","volume":"20","year":"2023","journal-title":"Nature Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.3","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1038\/s41592-025-02965-z","article-title":"Glutamate indicators with increased sensitivity and tailored deactivation rates","volume":"23","year":"2026","journal-title":"Nature Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuroscience.2005.11.038"},{"key":"2026081712200556000_2026.08.07.743580v1.5","first-page":"041402","article-title":"Review of data processing of functional optical microscopy for neuroscience","volume":"9","year":"2022","journal-title":"Neurophotonics"},{"key":"2026081712200556000_2026.08.07.743580v1.6","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1038\/s41592-019-0582-9","article-title":"ilastik: interactive machine learning for (bio)image analysis","volume":"16","year":"2019","journal-title":"Nature Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.7","doi-asserted-by":"crossref","unstructured":"Berlanga, A. E. , G. Kang , A. Kwok , T. Broggini , J. Lawlor , K. Kuchibhotla , D. Kleinfeld , G. Mishne , & A. S. Charles . 2025. Fast and accessible morphology-free functional fluorescence imaging analysis. bioRxiv.","DOI":"10.1101\/2025.04.15.648462"},{"key":"2026081712200556000_2026.08.07.743580v1.8","doi-asserted-by":"crossref","first-page":"101179","DOI":"10.1016\/j.crmeth.2025.101179","article-title":"A deep learning pipeline for accurate and automated restoration, segmentation, and quantification of dendritic spines","volume":"5","year":"2025","journal-title":"Cell Reports Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.9","doi-asserted-by":"publisher","DOI":"10.1109\/tip.2022.3171414"},{"key":"2026081712200556000_2026.08.07.743580v1.10","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1146\/annurev-neuro-100119-110036","article-title":"Toward Community-Driven Big Open Brain Science: Open Big Data and Tools for Structure, Function, and Genetics","volume":"43","year":"2020","journal-title":"Annual Reviewof Neuroscience"},{"key":"2026081712200556000_2026.08.07.743580v1.11","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1126\/science.1260088","article-title":"Optical imaging. Expansion microscopy","volume":"347","year":"2015","journal-title":"Science (New York, N.Y"},{"key":"2026081712200556000_2026.08.07.743580v1.12","doi-asserted-by":"crossref","unstructured":"Chen, T.-W. , X.-B. Huang , S. E. Plutkis , K. L. Holland , L. D. Lavis , & B.-J. Lin . 2025. \u201cImaging neuronal voltage beyond the scattering limit.\u201d Nature Methods, 1\u201310.","DOI":"10.1038\/s41592-025-02692-5"},{"key":"2026081712200556000_2026.08.07.743580v1.13","doi-asserted-by":"crossref","first-page":"1298","DOI":"10.1038\/s41592-024-02309-3","article-title":"High-throughput volumetric mapping of synaptic transmission","volume":"21","year":"2024","journal-title":"Nature Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.14","doi-asserted-by":"publisher","DOI":"10.1126\/science.aec9923"},{"key":"2026081712200556000_2026.08.07.743580v1.15","doi-asserted-by":"crossref","unstructured":"Chen, Z. , G. I. Coste , E. Li , R. L. Huganir , A. R. Graves , & A. S. Charles . 2025. Automatic detection of fluorescently labeled synapses in volumetric in vivo imaging data. bioRxiv.","DOI":"10.1101\/2025.01.22.634278"},{"key":"2026081712200556000_2026.08.07.743580v1.16","unstructured":"Collman, F. C. 2010. High resolution imaging in awake behaving mice: Motion correction and virtual reality. Ph.D., Princeton University."},{"key":"2026081712200556000_2026.08.07.743580v1.17","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.73775"},{"key":"2026081712200556000_2026.08.07.743580v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(00)81237-4"},{"key":"2026081712200556000_2026.08.07.743580v1.19","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.1984.tb09689.x"},{"key":"2026081712200556000_2026.08.07.743580v1.20","doi-asserted-by":"crossref","unstructured":"Frazier, P. I. 2018. Bayesian optimization. In Recent advances in optimization and modeling of contemporary problems, 255\u2013278. Informs.","DOI":"10.1287\/educ.2018.0188"},{"key":"2026081712200556000_2026.08.07.743580v1.21","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1038\/s41592-022-01422-5","article-title":"Detecting and correcting false transients in calcium imaging","volume":"19","year":"2022","journal-title":"Nature Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.22","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.38173"},{"key":"2026081712200556000_2026.08.07.743580v1.23","doi-asserted-by":"publisher","DOI":"10.1364\/OL.33.000156"},{"key":"2026081712200556000_2026.08.07.743580v1.24","doi-asserted-by":"crossref","first-page":"100205","DOI":"10.1016\/j.crmeth.2022.100205","article-title":"PatchWarp: Corrections of non-uniform image distortions in two-photon calcium imaging data by patchwork affine transformations","volume":"2","year":"2022","journal-title":"Cell Reports Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.25","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1038\/s43586-021-00038-x","article-title":"Single-molecule localization microscopy","volume":"1","year":"2021","journal-title":"Nature Reviews Methods Primers"},{"key":"2026081712200556000_2026.08.07.743580v1.26","doi-asserted-by":"crossref","first-page":"3850","DOI":"10.1038\/s41467-020-17523-8","article-title":"Light microscopy of proteins in their ultrastructural context","volume":"11","year":"2020","journal-title":"Nature Communications"},{"key":"2026081712200556000_2026.08.07.743580v1.27","doi-asserted-by":"crossref","unstructured":"Mishne, G. , R. R. Coifman , M. Lavzin , & J. Schiller . 2018. Automated cellular structure extraction in biological images with applications to calcium imaging data. bioRxiv.","DOI":"10.1101\/313981"},{"key":"2026081712200556000_2026.08.07.743580v1.28","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0623-08.2008"},{"key":"2026081712200556000_2026.08.07.743580v1.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2010.08.026"},{"key":"2026081712200556000_2026.08.07.743580v1.30","doi-asserted-by":"crossref","unstructured":"Ouellette, N. , E. Turschak , & J. Zhuang . 2022. \u201cCo-registration of mouse cortical structures between in vivo and ex vivo images using tangential sectioning.\u201d STAR Protocols 3.","DOI":"10.1016\/j.xpro.2022.101405"},{"key":"2026081712200556000_2026.08.07.743580v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/j.jneumeth.2017.07.031"},{"key":"2026081712200556000_2026.08.07.743580v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2015.11.037"},{"key":"2026081712200556000_2026.08.07.743580v1.33","unstructured":"Podgorski, K. , A. Charles , D. A. Flickinger , G. Jaindl , A. Negrean , J. Rohde , & M. E. Xie . 2023. Second-generation scanned line projection microscopy (SLAP2) for in vivo imaging of synaptic input. In 2023 Neuroscience Meeting Planner, PSTR241.30. Washington, D.C.: Society for Neuroscience."},{"key":"2026081712200556000_2026.08.07.743580v1.34","doi-asserted-by":"crossref","first-page":"e1007791","DOI":"10.1371\/journal.pcbi.1007791","article-title":"Localized semi-nonnegative matrix factorization (LocaNMF) of widefield calcium imaging data","volume":"16","year":"2020","journal-title":"PLOS Computational Biology"},{"key":"2026081712200556000_2026.08.07.743580v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jneumeth.2021.109173"},{"key":"2026081712200556000_2026.08.07.743580v1.36","doi-asserted-by":"crossref","unstructured":"Stringer, C. , C. Ki , N. DelGrosso , P. LaFosse , Q. Zhang , & M. Pachitariu . 2026. Extracting large-scale neural activity with Suite2p. bioRxiv.","DOI":"10.64898\/2026.02.04.703741"},{"key":"2026081712200556000_2026.08.07.743580v1.37","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1038\/s41586-025-08985-1","article-title":"Light-microscopy-based connectomic reconstruction of mammalian brain tissue","volume":"642","year":"2025","journal-title":"Nature"},{"key":"2026081712200556000_2026.08.07.743580v1.38","doi-asserted-by":"crossref","unstructured":"Varol, E. , J. Boussard , N. Dethe , O. Winter , A. Urai , T. I. Brain Laboratory , A. Churchland , N. Steinmetz , & L. Paninski . 2021. Decentralized Motion Inference and Registration of Neuropixel Data. In ICASSP 2021 - 2021 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), 1085\u20131089.","DOI":"10.1109\/ICASSP39728.2021.9414145"},{"key":"2026081712200556000_2026.08.07.743580v1.39","doi-asserted-by":"crossref","first-page":"788","DOI":"10.1038\/s41592-025-02614-5","article-title":"DREDge: robust motion correction for high-density extracellular recordings across species","volume":"22","year":"2025","journal-title":"Nature Methods"},{"key":"2026081712200556000_2026.08.07.743580v1.40","doi-asserted-by":"publisher","DOI":"10.1126\/science.ads4706"},{"key":"2026081712200556000_2026.08.07.743580v1.41","first-page":"024307","article-title":"Comprehensive software suite for functional analysis and synaptic input mapping of dendritic spines imaged in vivo","volume":"11","year":"2024","journal-title":"Neurophotonics"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.08.07.743580","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T19:20:19Z","timestamp":1786994419000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.08.07.743580"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,13]]},"references-count":41,"URL":"https:\/\/doi.org\/10.64898\/2026.08.07.743580","relation":{},"subject":[],"published":{"date-parts":[[2026,8,13]]},"subtype":"preprint"}}