{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:57:37Z","timestamp":1766710657302,"version":"3.48.0"},"reference-count":69,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,25]],"date-time":"2025-12-25T00:00:00Z","timestamp":1766620800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"\u201cFlanders Resilience\u201d subsidy from the Flemish Government, originating from the \u201cEuropean Recovery and Resilience Facility\u201d","award":["VV021\/13"],"award-info":[{"award-number":["VV021\/13"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Recombinant adeno-associated viral vectors (rAAVs) are the leading gene delivery vehicles in clinical development, yet efficient nuclear delivery remains a major barrier to effective transduction. This limitation is partly due to the incomplete understanding of rAAV\u2019s complex subcellular trafficking dynamics. Here, we establish a longitudinal confocal live-cell imaging workflow that tracks rAAV2 from 4 to 12 h post-transduction, paired with an automated 3D analysis pipeline that quantifies spatiotemporal vector distribution, cytoplasmic trafficking, nuclear accumulation, and transgene expression at single-cell resolution. We use this platform to evaluate the effects of vector dose, cell cycle progression, and the behavior of empty particles. We identify previously undescribed trafficking features associated with high transgene expression. Higher rAAV2 doses enhanced cytoplasmic trafficking and nuclear delivery, while cell cycle progression facilitated both trafficking efficiency and transgene expression. We also characterize empty rAAV2 particles, revealing distinct trafficking patterns and markedly reduced nuclear accumulation compared to genome-containing vectors. By uncovering new bottlenecks in rAAV transduction, this platform provides mechanistic insights and potential strategies to improve AAV-based gene therapy. Its generalizable design further supports broad applicability to other non-enveloped viruses.<\/jats:p>","DOI":"10.3390\/ijms27010236","type":"journal-article","created":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:50:21Z","timestamp":1766710221000},"page":"236","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Longitudinal 3D Live-Cell Imaging Platform to Uncover AAV Vector\u2013Host Dynamics at Single-Cell Resolution"],"prefix":"10.3390","volume":"27","author":[{"given":"Marlies","family":"Leysen","sequence":"first","affiliation":[{"name":"Trellis Research Group, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4670-3700","authenticated-orcid":false,"given":"Nicolas","family":"Peredo","sequence":"additional","affiliation":[{"name":"VIB BioImaging Core Leuven, VIB Technologies, Center for Brain and Disease Research, B-3000 Leuven, Belgium"},{"name":"VIB BioImaging Core Leuven, Department of Neurosciences, KU Leuven, B-3000 Leuven, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benjamin","family":"Pavie","sequence":"additional","affiliation":[{"name":"VIB BioImaging Core Leuven, VIB Technologies, Center for Brain and Disease Research, B-3000 Leuven, Belgium"},{"name":"VIB BioImaging Core Leuven, Department of Neurosciences, KU Leuven, B-3000 Leuven, Belgium"},{"name":"VIB BioImaging Core Ghent, VIB Center for Inflammation Research, B-9052 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Benjamien","family":"Moeyaert","sequence":"additional","affiliation":[{"name":"Trellis Research Group, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Els","family":"Henckaerts","sequence":"additional","affiliation":[{"name":"Trellis Research Group, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium"},{"name":"Virus\u2013Host Interactions & Therapeutic Approaches Research Group, Department of Microbiology, Immunology and Transplantation, KU Leuven, B-3000 Leuven, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1467","DOI":"10.1073\/pnas.55.6.1467","article-title":"Studies of Small Dna Viruses Found in Various Adenovirus Preparations","volume":"55","author":"Hoggan","year":"1966","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1128\/jb.90.1.235-242.1965","article-title":"Structure and Composition of a Small Particle Prepared from a Simian Adenovirus","volume":"90","author":"Mayor","year":"1965","journal-title":"J. Bacteriol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1128\/jvi.8.5.766-770.1971","article-title":"Structural Proteins of Adenovirus-Associated Viruses","volume":"8","author":"Rose","year":"1971","journal-title":"J. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1128\/jvi.45.2.555-564.1983","article-title":"Nucleotide Sequence and Organization of the Adeno-Associated Virus 2 Genome","volume":"45","author":"Srivastava","year":"1983","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"7295","DOI":"10.1021\/ja502616y","article-title":"Defining the Stoichiometry and Cargo Load of Viral and Bacterial Nanoparticles by Orbitrap Mass Spectrometry","volume":"136","author":"Snijder","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Wang, J.-H., Gessler, D.J., Zhan, W., Gallagher, T.L., and Gao, G. (2024). Adeno-Associated Virus as a Delivery Vector for Gene Therapy of Human Diseases. Signal Transduct. Target. Ther., 9.","DOI":"10.1038\/s41392-024-01780-w"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Zhao, Q., Peng, H., Ma, Y., Yuan, H., and Jiang, H. (2025). In Vivo Applications and Toxicities of AAV-Based Gene Therapies in Rare Diseases. Orphanet J. Rare Dis., 20.","DOI":"10.1186\/s13023-025-03893-z"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3123","DOI":"10.1016\/j.ymthe.2023.10.015","article-title":"Lethal Immunotoxicity in High-Dose Systemic AAV Therapy","volume":"31","author":"Duan","year":"2023","journal-title":"Mol. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4694","DOI":"10.1016\/j.ymthe.2025.08.029","article-title":"The Future of Gene Therapy: Safer Vectors, Sharper Focus: High-Profile Failures Demand Deep Root Cause Analysis\u2014But the Transformative Potential of AAV Remains within Reach If the Field Is Willing to Learn and Evolve","volume":"33","author":"Chamberlain","year":"2025","journal-title":"Mol. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"723","DOI":"10.1016\/j.ymthe.2019.12.010","article-title":"AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer","volume":"28","author":"Kuranda","year":"2020","journal-title":"Mol. Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1016\/j.molmed.2020.09.010","article-title":"Journey to the Center of the Cell: Tracing the Path of AAV Transduction","volume":"27","author":"Dhungel","year":"2021","journal-title":"Trends Mol. Med."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Kelich, J.M., Ma, J., Dong, B., Wang, Q., Chin, M., Magura, C.M., Xiao, W., and Yang, W. (2015). Super-Resolution Imaging of Nuclear Import of Adeno-Associated Virus in Live Cells. Mol. Ther.-Methods Clin. Dev., 2.","DOI":"10.1038\/mtm.2015.47"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1038\/gt.2012.6","article-title":"Intracellular Transport of Recombinant Adeno-Associated Virus Vectors","volume":"19","author":"Nonnenmacher","year":"2012","journal-title":"Gene Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/j.ymthe.2019.11.005","article-title":"GPR108 Is a Highly Conserved AAV Entry Factor","volume":"28","author":"Dudek","year":"2020","journal-title":"Mol. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.coviro.2017.06.003","article-title":"Host Determinants of Adeno-Associated Viral Vector Entry","volume":"24","author":"Pillay","year":"2017","journal-title":"Curr. Opin. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1016\/j.chom.2011.10.014","article-title":"Adeno-Associated Virus 2 Infection Requires Endocytosis through the CLIC\/GEEC Patway","volume":"10","author":"Nonnenmacher","year":"2011","journal-title":"Cell Host Microbe"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Zhang, C., Zhou, X., Yao, T., Tian, Z., and Zhou, D. (2018). Precision Fluorescent Labeling of an Adeno-Associated Virus Vector to Monitor the Viral Infection Pathway. Biotechnol. J., 13.","DOI":"10.1002\/biot.201700374"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.ymthe.2005.12.002","article-title":"RAAV2 Traffics through Both the Late and the Recycling Endosomes in a Dose-Dependent Fashion","volume":"13","author":"Ding","year":"2006","journal-title":"Mol. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"7781","DOI":"10.1128\/JVI.00481-06","article-title":"A Common Mechanism for Cytoplasmic Dynein-Dependent Microtubule Binding Shared among Adeno-Associated Virus and Adenovirus Serotypes","volume":"80","author":"Kelkar","year":"2006","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"10270","DOI":"10.1128\/JVI.77.19.10270-10279.2003","article-title":"Exploitation of Microtubule Cytoskeleton and Dynein during Parvoviral Traffic toward the Nucleus","volume":"77","author":"Suikkanen","year":"2003","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.virol.2007.05.009","article-title":"Effect of Inhibition of Dynein Function and Microtubule-Altering Drugs on AAV2 Transduction","volume":"367","author":"Hirosue","year":"2007","journal-title":"Virology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3523","DOI":"10.1021\/nn102651p","article-title":"Enhanced Real-Time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates","volume":"5","author":"Joo","year":"2011","journal-title":"ACS Nano"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1673","DOI":"10.1128\/JVI.02520-14","article-title":"Syntaxin 5-Dependent Retrograde Transport to the Trans -Golgi Network Is Required for Adeno-Associated Virus Transduction","volume":"89","author":"Nonnenmacher","year":"2015","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Cabanes-creus, M., Liao, S.H.Y., Rojas, A.L., Kelich, J., Coyne, J., Knight, M., Nazareth, D., Roca-pinilla, R., Senapati, S., and Walsh, M.C. (2025). AAV2 Bypasses Direct Endosomal Escape by Using AAVR to Access the Trans-Golgi Network En Route to the Nucleus. bioRxiv.","DOI":"10.1101\/2025.11.22.689972"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1099\/0022-1317-83-5-973","article-title":"The VP1 Capsid Protein of Adeno-Associated Virus Type 2 Is Carrying a Phospholipase A2 Domain Required for Virus Infectivity","volume":"83","author":"Girod","year":"2002","journal-title":"J. Gen. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Kurian, J.J., Lakshmanan, R., Chmely, W.M., Hull, J.A., Yu, J.C., Bennett, A., McKenna, R., and Agbandje-McKenna, M. (2019). Adeno-Associated Virus VP1u Exhibits Protease Activity. Viruses, 11.","DOI":"10.3390\/v11050399"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5199","DOI":"10.1128\/JVI.02723-05","article-title":"Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly","volume":"80","author":"Grieger","year":"2006","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4132","DOI":"10.1128\/JVI.02660-13","article-title":"Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus","volume":"88","author":"Nicolson","year":"2014","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Porwal, M., Cohen, S., Snoussi, K., Popa-Wagner, R., Anderson, F., Dugot-Senant, N., Wodrich, H., Dinsart, C., Kleinschmidt, J.A., and Pant\u00e9, N. (2013). Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003671"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3209","DOI":"10.1099\/vir.0.82232-0","article-title":"Parvoviral Nuclear Import: Bypassing the Host Nuclear-Transport Machinery","volume":"87","author":"Cohen","year":"2006","journal-title":"J. Gen. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4863","DOI":"10.1128\/JVI.01999-10","article-title":"Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle","volume":"85","author":"Cohen","year":"2011","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1929","DOI":"10.1126\/science.1064103","article-title":"Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus","volume":"294","author":"Seisenberger","year":"2001","journal-title":"Science."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"11505","DOI":"10.1128\/JVI.76.22.11505-11517.2002","article-title":"Adenovirus-Facilitated Nuclear Translocation of Adeno-Associated Virus Type 2","volume":"76","author":"Xiao","year":"2002","journal-title":"J. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"11776","DOI":"10.1128\/JVI.79.18.11776-11787.2005","article-title":"Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking","volume":"79","author":"Lux","year":"2005","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"10462","DOI":"10.1128\/JVI.00935-12","article-title":"Cytoplasmic Trafficking, Endosomal Escape, and Perinuclear Accumulation of Adeno-Associated Virus Type 2 Particles Are Facilitated by Microtubule Network","volume":"86","author":"Xiao","year":"2012","journal-title":"J. Virol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"11040","DOI":"10.1128\/JVI.01056-06","article-title":"Adeno-Associated Virus Type 2 Capsids with Externalized VP1\/VP2 Trafficking Domains Are Generated Prior to Passage through the Cytoplasm and Are Maintained until Uncoating Occurs in the Nucleus","volume":"80","author":"Sonntag","year":"2006","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2777","DOI":"10.1128\/JVI.74.6.2777-2785.2000","article-title":"Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors","volume":"74","author":"Bartlett","year":"2000","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1038\/mt.2011.250","article-title":"Quantitative 3D Tracing of Gene-Delivery Viral Vectors in Human Cells and Animal Tissues","volume":"20","author":"Xiao","year":"2012","journal-title":"Mol. Ther."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2632","DOI":"10.1128\/JVI.02309-08","article-title":"Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus","volume":"83","author":"Johnson","year":"2009","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Sutter, S.O., Lkharrazi, A., Schraner, E.M., Michaelsen, K., Meier, A.F., Marx, J., Vogt, B., B\u00fcning, H., and Fraefel, C. (2022). Adeno-Associated Virus Type 2 (AAV2) Uncoating Is a Stepwise Process and Is Linked to Structural Reorganization of the Nucleolus. PLoS Pathog., 18.","DOI":"10.1371\/journal.ppat.1010187"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Fan, Y., Sanyal, S., and Bruzzone, R. (2018). Breaking Bad: How Viruses Subvert the Cell Cycle. Front. Cell. Infect. Microbiol., 8.","DOI":"10.3389\/fcimb.2018.00396"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"13634","DOI":"10.1073\/pnas.0504583102","article-title":"How Adeno-Associated Virus Rep78 Protein Arrests Cells Completely in S Phase","volume":"102","author":"Berthet","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"4351","DOI":"10.1093\/emboj\/19.16.4351","article-title":"Inhibition of S-Phase Progression by Adeno-Associated Virus Rep78 Protein Is Mediated by Hypophosphorylated PRb","volume":"19","author":"Saudan","year":"2000","journal-title":"EMBO J."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5777","DOI":"10.1128\/JVI.02651-06","article-title":"Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication","volume":"81","author":"Nash","year":"2007","journal-title":"J. Virol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"8915","DOI":"10.1073\/pnas.91.19.8915","article-title":"Adeno-Associated Virus Vectors Preferentially Transduce Cells in S Phase","volume":"91","author":"Russell","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"8282","DOI":"10.1128\/jvi.68.12.8282-8287.1994","article-title":"DNA-Damaging Agents Greatly Increase the Transduction of Nondividing Cells by AAV Vectors","volume":"68","author":"Alexander","year":"1994","journal-title":"J. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Leone, P., Shera, D., McPhee, S.W.J., Francis, J.S., Kolodny, E.H., Bilaniuk, L.T., Wang, D.-J., Assadi, M., Goldfarb, O., and Goldman, H.W. (2012). Long-Term Follow-Up After Gene Therapy for Canavan Disease. Sci. Transl. Med., 4.","DOI":"10.1126\/scitranslmed.3003454"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.neulet.2014.05.044","article-title":"Widespread Neuron-Specific Transgene Expression in Brain and Spinal Cord Following Synapsin Promoter-Driven AAV9 Neonatal Intracerebroventricular Injection","volume":"576","author":"McLean","year":"2014","journal-title":"Neurosci. Lett."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1089\/hgtb.2016.188","article-title":"Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities","volume":"28","year":"2017","journal-title":"Hum. Gene Ther. Methods"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1089\/hum.2023.018","article-title":"Complement Activation by Adeno-Associated Virus-Neutralizing Antibody Complexes","volume":"34","author":"West","year":"2023","journal-title":"Hum. Gene Ther."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Ertl, H.C.J. (2022). Immunogenicity and Toxicity of AAV Gene Therapy. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.975803"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1146\/annurev-virology-100422-012608","article-title":"Studying Retroviral Life Cycles Using Visible Viruses and Live Cell Imaging","volume":"11","author":"Guerrero","year":"2024","journal-title":"Annu. Rev. Virol."},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Golm, S.K., H\u00fcbner, W., and M\u00fcller, K.M. (2023). Fluorescence Microscopy in Adeno-Associated Virus Research. Viruses, 15.","DOI":"10.3390\/v15051174"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"698","DOI":"10.1038\/s41565-021-00875-7","article-title":"A Highly Homogeneous Polymer Composed of Tetrahedron-like Monomers for High-Isotropy Expansion Microscopy","volume":"16","author":"Gao","year":"2021","journal-title":"Nat. Nanotechnol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Shaib, A.H., Chouaib, A.A., Chowdhury, R., Mihaylov, D., Imani, V., Georgiev, S.V., Mougios, N., Reshetniak, S., Mimoso, T., and Chen, H. (2023). Visualizing Proteins by Expansion Microscopy. bioRxiv.","DOI":"10.1101\/2022.08.03.502284"},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Hu, X., Meister, R., Tode, J., Framme, C., and Fuchs, H. (2024). Long-Term in Vitro Monitoring of AAV-Transduction Efficiencies in Real-Time with Hoechst 33342. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0298173"},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Bustamante-Jaramillo, L.F., Yue, L., Fingal, J., Rydell, G., Johansson, M., Edreira, T., M\u00fcller, O.J., Hille, S., M\u00fcller, M., and Gallardo, F. (2025). Real-Time Genome Imaging of Host Interactions in Adeno-Associated Virus Genome Release. iScience, 28.","DOI":"10.1016\/j.isci.2025.112624"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1111\/j.1365-2818.2006.01706.x","article-title":"A Guided Tour into Subcellular Colocalization Analysis in Light Microscopy","volume":"224","author":"Bolte","year":"2006","journal-title":"J. Microsc."},{"key":"ref_59","unstructured":"Sofroniew, N., Burt, A., Witz, G., Abouakil, F., and Lambert, T. (2024, September 27). Napari Napari-Animation. Available online: https:\/\/github.com\/napari\/napari-animation."},{"key":"ref_60","doi-asserted-by":"crossref","unstructured":"Sun, Y., Cao, C., Peng, Y., Dai, X., Li, X., Li, J., Liang, T., Song, P., Ye, Y., and Yang, J. (2024). Application of Advanced Bioimaging Technologies in Viral Infections. Mater. Today Phys., 46.","DOI":"10.1016\/j.mtphys.2024.101488"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"938","DOI":"10.1083\/jcb.93.3.938","article-title":"Centrioles in the Cell Cycle. I. Epithelial Cells","volume":"98","author":"Vorobjev","year":"1982","journal-title":"J. Cell Biol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"17132","DOI":"10.1021\/acs.analchem.5c03104","article-title":"Insights into Adeno-Associated Virus Capsid Charge Heterogeneity","volume":"97","author":"Wu","year":"2025","journal-title":"Anal. Chem."},{"key":"ref_63","doi-asserted-by":"crossref","unstructured":"Heldt, C.L., Skinner, M.A., and Anand, G.S. (2024). Structural Changes Likely Cause Chemical Differences between Empty and Full AAV Capsids. Biomedicines, 12.","DOI":"10.3390\/biomedicines12092128"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"2745","DOI":"10.1089\/hum.1998.9.18-2745","article-title":"Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors","volume":"9","author":"Grimm","year":"1998","journal-title":"Hum. Gene Ther."},{"key":"ref_65","doi-asserted-by":"crossref","unstructured":"Van den Berghe, N., Costermans, E., Nuseibeh, S., Brouns, T., Van Hove, I., Moeyaert, B., and Henckaerts, E. (2024). Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction. J. Vis. Exp., 212.","DOI":"10.3791\/67252"},{"key":"ref_66","unstructured":"Brown, E.M., Toloudis, D., Sherman, J., Swain-Bowden, M., Lambert, T., and AICSImageIO Contributors (2025, December 01). AICSImageIO: Image Reading, Metadata Conversion, and Image Writing for Microscopy Images in Pure Python (Version 3.10). Available online: https:\/\/github.com\/AllenCellModeling\/aicsimageio."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1038\/s41592-020-01018-x","article-title":"Cellpose: A Generalist Algorithm for Cellular Segmentation","volume":"18","author":"Stringer","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"van der Walt, S., Sch\u00f6nberger, J.L., Nunez-Iglesias, J., Boulogne, F., Warner, J.D., Yager, N., Gouillart, E., and Yu, T. (2014). Scikit-Image: Image Processing in Python. PeerJ, 2.","DOI":"10.7717\/peerj.453"},{"key":"ref_69","unstructured":"Allan, D.B., Caswell, T., Keim, N.C., Van der Wel, C.M., and Verweij, R.W. (2025, December 01). Zenodo: Soft-Matter\/Trackpy:V0.6.4. Available online: https:\/\/zenodo.org\/records\/12708864."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/236\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T00:53:44Z","timestamp":1766710424000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/236"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,25]]},"references-count":69,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["ijms27010236"],"URL":"https:\/\/doi.org\/10.3390\/ijms27010236","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,25]]}}}