{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T22:46:42Z","timestamp":1790117202168,"version":"4.0.1"},"posted":{"date-parts":[[2025,10,8]]},"group-title":"Cell Biology","reference-count":90,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The nucleolus is essential for ribosome biogenesis and for regulating cellular responses to growth and stress, and its relationship to cellular metabolic activity and functional state highlights its potential as a biomarker of cellular health. However, challenges in contrast multiplexing and high-resolution isotropic three-dimensional (3D) imaging hinder the non-invasive, simultaneous assessment of nucleolar activity and subcellular metabolic maps across different tissue contexts, especially in complex 3D environments. To fully harness the nucleolus\u2019s potential as a biomarker and diagnostic target, we present a multimodal imaging platform that combines third harmonic generation (THG) imaging with metabolic autofluorescence of NAD(P)H and FAD to study structural and metabolic nucleolar dynamics. Enabled by a high-power multimode fiber source and an axial deblurring network, we achieved \u223c 400 nm isotropic resolution in deep 3D imaging and confirmed the high accuracy of our method for label-free nucleolus identification using co-registered immunostaining and electron microscopy. To establish the biological relevance of our approach, we demonstrate that nucleolar stress leads to an unexpected depletion of NADH across cellular compartments. Furthermore, in the human endometrium\u2014where nucleolar dynamics are central to the tissue\u2019s response to progesterone\u2014our label-free imaging strategy delineated endometrial structures in freshly excised tissues and revealed that progesterone treatment induces distinct changes in nucleolar translocation and metabolic adaptation in organoids derived from diseased patients compared to controls. This capacity to non-invasively visualize and quantify features of the nucleolus and its local metabolic microenvironment at single-cell resolution in human tissues\u2014and dynamically track these changes over time in patient-derived organoids\u2014provides a powerful tool for uncovering the roles of the nucleolus in development, disease progression, and therapeutic response. Together, these findings establish our platform as a significant advance for both fundamental research and organelle-based tissue diagnostics.<\/jats:p>","DOI":"10.1101\/2025.10.07.681013","type":"posted-content","created":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T19:55:14Z","timestamp":1759953314000},"source":"Crossref","is-referenced-by-count":1,"title":["Simultaneous Label-free Imaging of Nucleolar Dynamics and Subcellular Metabolic Shifts Across Tissue Contexts"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3923-3720","authenticated-orcid":false,"given":"Kunzan","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ellen L.","family":"Kan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5824-7557","authenticated-orcid":false,"given":"Honghao","family":"Cao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiashu","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giovanni de","family":"Nola","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1801-5548","authenticated-orcid":false,"given":"Linda G.","family":"Griffith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3006-2742","authenticated-orcid":false,"given":"Eliezer","family":"Calo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1243-1815","authenticated-orcid":false,"given":"Sixian","family":"You","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025101107250461000_2025.10.07.681013v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2184"},{"key":"2025101107250461000_2025.10.07.681013v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/nrc.2017.104"},{"key":"2025101107250461000_2025.10.07.681013v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-021-00386-2"},{"key":"2025101107250461000_2025.10.07.681013v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/nature03207"},{"key":"2025101107250461000_2025.10.07.681013v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-03255-3"},{"key":"2025101107250461000_2025.10.07.681013v1.6","doi-asserted-by":"publisher","DOI":"10.1093\/jb\/mvab090"},{"key":"2025101107250461000_2025.10.07.681013v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/ncb814"},{"key":"2025101107250461000_2025.10.07.681013v1.8","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E04-11-0992"},{"key":"2025101107250461000_2025.10.07.681013v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2010.09.024"},{"key":"2025101107250461000_2025.10.07.681013v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/cr.1992.10"},{"key":"2025101107250461000_2025.10.07.681013v1.11","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E07-02-0154"},{"key":"2025101107250461000_2025.10.07.681013v1.12","doi-asserted-by":"crossref","unstructured":"Preston, C. C. , Stoddard, A. C. & Faustino, R. S. A transient mystery: nucleolar channel systems. In Nuclear, Chromosomal, and Genomic Architecture in Biology and Medicine, 581\u2013593 (Springer, 2022).","DOI":"10.1007\/978-3-031-06573-6_20"},{"key":"2025101107250461000_2025.10.07.681013v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80733-3"},{"key":"2025101107250461000_2025.10.07.681013v1.14","doi-asserted-by":"publisher","DOI":"10.1126\/science.1099402"},{"key":"2025101107250461000_2025.10.07.681013v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(04)00413-1"},{"key":"2025101107250461000_2025.10.07.681013v1.16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.12.6887"},{"key":"2025101107250461000_2025.10.07.681013v1.17","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.M900338-MCP200"},{"key":"2025101107250461000_2025.10.07.681013v1.18","doi-asserted-by":"crossref","unstructured":"Moore, H. M. et al. Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to uv and ionizing radiation. Mol. Cell. Proteom. 10 (2011).","DOI":"10.1074\/mcp.M111.009241"},{"key":"2025101107250461000_2025.10.07.681013v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms11390"},{"key":"2025101107250461000_2025.10.07.681013v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32856-2"},{"key":"2025101107250461000_2025.10.07.681013v1.21","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.79903"},{"key":"2025101107250461000_2025.10.07.681013v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1172"},{"key":"2025101107250461000_2025.10.07.681013v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2011.09.005"},{"key":"2025101107250461000_2025.10.07.681013v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-05767-5"},{"key":"2025101107250461000_2025.10.07.681013v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/35014078"},{"key":"2025101107250461000_2025.10.07.681013v1.26","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.97.1.55"},{"key":"2025101107250461000_2025.10.07.681013v1.27","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1017150108"},{"key":"2025101107250461000_2025.10.07.681013v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s44319-024-00134-5"},{"key":"2025101107250461000_2025.10.07.681013v1.29","doi-asserted-by":"crossref","first-page":"7332","DOI":"10.1038\/s41467-024-51574-5","article-title":"Single-cell laser emitting cytometry for label-free nucleolus fingerprinting","volume":"15","year":"2024","journal-title":"Nat. Commun"},{"key":"2025101107250461000_2025.10.07.681013v1.30","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0028237"},{"key":"2025101107250461000_2025.10.07.681013v1.31","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.152272"},{"key":"2025101107250461000_2025.10.07.681013v1.32","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-67857-y"},{"key":"2025101107250461000_2025.10.07.681013v1.33","doi-asserted-by":"crossref","first-page":"e78079","DOI":"10.7554\/eLife.78079","article-title":"Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in rett syndrome","volume":"11","year":"2022","journal-title":"eLife"},{"key":"2025101107250461000_2025.10.07.681013v1.34","doi-asserted-by":"crossref","first-page":"eadp2438","DOI":"10.1126\/sciadv.adp2438","article-title":"Deep and dynamic metabolic and structural imaging in living tissues","volume":"10","year":"2024","journal-title":"Sci. Adv"},{"key":"2025101107250461000_2025.10.07.681013v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth1078"},{"key":"2025101107250461000_2025.10.07.681013v1.36","first-page":"682","article-title":"NAD(P)H and collagen as in vivo quantitative fluorescent biomarkers of epithelial precancerous changes","volume":"62","year":"2002","journal-title":"Cancer Res"},{"key":"2025101107250461000_2025.10.07.681013v1.37","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2017.7451"},{"key":"2025101107250461000_2025.10.07.681013v1.38","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aap9302"},{"key":"2025101107250461000_2025.10.07.681013v1.39","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0708425104"},{"key":"2025101107250461000_2025.10.07.681013v1.40","first-page":"77","article-title":"Classification of T-cell activation via autofluorescence lifetime imaging","volume":"5","year":"2021","journal-title":"Nat. Biomed. Eng"},{"key":"2025101107250461000_2025.10.07.681013v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-04470-8"},{"key":"2025101107250461000_2025.10.07.681013v1.42","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1038\/s41467-025-56078-4","article-title":"System-and sample-agnostic isotropic three-dimensional microscopy by weakly physics-informed, domain-shift-resistant axial deblurring","volume":"16","year":"2025","journal-title":"Nat. Commun"},{"key":"2025101107250461000_2025.10.07.681013v1.43","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.172368799"},{"key":"2025101107250461000_2025.10.07.681013v1.44","doi-asserted-by":"publisher","DOI":"10.1038\/nbt894"},{"key":"2025101107250461000_2025.10.07.681013v1.45","doi-asserted-by":"crossref","first-page":"2031","DOI":"10.1038\/s41467-024-46244-5","article-title":"Spectral-temporal-spatial customization via modulating multimodal nonlinear pulse propagation","volume":"15","year":"2024","journal-title":"Nat. Commun"},{"key":"2025101107250461000_2025.10.07.681013v1.46","first-page":"1","article-title":"Nucleolar and coiled-body phosphoprotein 1 (NOLC1) regulates the nucleolar retention of TRF2","volume":"3","year":"2017","journal-title":"Cell Death Discov"},{"key":"2025101107250461000_2025.10.07.681013v1.47","doi-asserted-by":"publisher","DOI":"10.1007\/s00432-023-04934-5"},{"key":"2025101107250461000_2025.10.07.681013v1.48","doi-asserted-by":"crossref","first-page":"RP88799","DOI":"10.7554\/eLife.88799","article-title":"Distinct states of nucleolar stress induced by anticancer drugs","volume":"12","year":"2023","journal-title":"eLife"},{"key":"2025101107250461000_2025.10.07.681013v1.49","doi-asserted-by":"publisher","DOI":"10.1002\/jemt.22121"},{"key":"2025101107250461000_2025.10.07.681013v1.50","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2012.05.038"},{"key":"2025101107250461000_2025.10.07.681013v1.51","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.01606"},{"key":"2025101107250461000_2025.10.07.681013v1.52","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2012.01.024"},{"key":"2025101107250461000_2025.10.07.681013v1.53","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2019.06.020"},{"key":"2025101107250461000_2025.10.07.681013v1.54","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2023.10.031"},{"key":"2025101107250461000_2025.10.07.681013v1.55","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2008.11.018"},{"key":"2025101107250461000_2025.10.07.681013v1.56","doi-asserted-by":"publisher","DOI":"10.1006\/excr.1996.0259"},{"key":"2025101107250461000_2025.10.07.681013v1.57","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1921649117"},{"key":"2025101107250461000_2025.10.07.681013v1.58","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-30755-5"},{"key":"2025101107250461000_2025.10.07.681013v1.59","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-20-3124"},{"key":"2025101107250461000_2025.10.07.681013v1.60","doi-asserted-by":"crossref","unstructured":"Yamaguchi, M. et al. Three-dimensional understanding of the morphological complexity of the human uterine endometrium. iScience 24 (2021).","DOI":"10.1016\/j.isci.2021.102258"},{"key":"2025101107250461000_2025.10.07.681013v1.61","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.27.2.293"},{"key":"2025101107250461000_2025.10.07.681013v1.62","first-page":"325","article-title":"Asynchronous glands in the endometrium of women with recurrent reproductive failure","volume":"46","year":"2014","journal-title":"Pathology"},{"key":"2025101107250461000_2025.10.07.681013v1.63","first-page":"393","article-title":"The distribution of immune cells and macrophages in the endometrium of women with recurrent reproductive failure. iii: Further observations and reference ranges","volume":"45","year":"2013","journal-title":"Pathology-Journal of the RCPA"},{"key":"2025101107250461000_2025.10.07.681013v1.64","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1093\/humupd\/dmab039","article-title":"Novel microarchitecture of human endometrial glands: Implications in endometrial regeneration and pathologies","volume":"28","year":"2022","journal-title":"Human Reproduction Update"},{"key":"2025101107250461000_2025.10.07.681013v1.65","doi-asserted-by":"crossref","first-page":"640319","DOI":"10.3389\/fcell.2021.640319","article-title":"The elusive endometrial epithelial stem\/progenitor cells","volume":"9","year":"2021","journal-title":"Front. Cell Dev. Biol"},{"key":"2025101107250461000_2025.10.07.681013v1.66","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1915389116"},{"key":"2025101107250461000_2025.10.07.681013v1.67","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1016\/j.medj.2023.07.004","article-title":"Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk","volume":"4","year":"2023","journal-title":"Med"},{"key":"2025101107250461000_2025.10.07.681013v1.68","doi-asserted-by":"publisher","DOI":"10.1093\/humupd\/8.4.333"},{"key":"2025101107250461000_2025.10.07.681013v1.69","doi-asserted-by":"publisher","DOI":"10.1210\/er.2014-1045"},{"key":"2025101107250461000_2025.10.07.681013v1.70","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2200252119"},{"key":"2025101107250461000_2025.10.07.681013v1.71","doi-asserted-by":"publisher","DOI":"10.1098\/rsfs.2019.0079"},{"key":"2025101107250461000_2025.10.07.681013v1.72","doi-asserted-by":"publisher","DOI":"10.1038\/ncb3516"},{"key":"2025101107250461000_2025.10.07.681013v1.73","doi-asserted-by":"crossref","first-page":"240330","DOI":"10.1098\/rsob.240330","article-title":"Nucleolar origins: Challenging perspectives on evolution and function","volume":"15","year":"2025","journal-title":"Open Biol"},{"key":"2025101107250461000_2025.10.07.681013v1.74","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0080237"},{"key":"2025101107250461000_2025.10.07.681013v1.75","doi-asserted-by":"publisher","DOI":"10.3390\/cells11193017"},{"key":"2025101107250461000_2025.10.07.681013v1.76","doi-asserted-by":"publisher","DOI":"10.1210\/er.2012-1043"},{"key":"2025101107250461000_2025.10.07.681013v1.77","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1038\/s41698-019-0104-3","article-title":"Real-time intraoperative diagnosis by deep neural network driven multiphoton virtual histology","volume":"3","year":"2019","journal-title":"npj Precis. Oncol"},{"key":"2025101107250461000_2025.10.07.681013v1.78","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1909243116"},{"key":"2025101107250461000_2025.10.07.681013v1.79","doi-asserted-by":"publisher","DOI":"10.1038\/srep03432"},{"key":"2025101107250461000_2025.10.07.681013v1.80","article-title":"Self-localized ultrafast pencil beam for volumetric multiphoton imaging","year":"2025","journal-title":"arXiv preprint"},{"key":"2025101107250461000_2025.10.07.681013v1.81","doi-asserted-by":"crossref","unstructured":"Chen, L. , Chu, X. , Zhang, X. & Sun, J. Simple baselines for image restoration. In European Conference on Computer Vision (ECCV), 17\u201333 (2022).","DOI":"10.1007\/978-3-031-20071-7_2"},{"key":"2025101107250461000_2025.10.07.681013v1.82","doi-asserted-by":"publisher","DOI":"10.1038\/s41377-023-01230-2"},{"key":"2025101107250461000_2025.10.07.681013v1.83","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-018-0216-7"},{"key":"2025101107250461000_2025.10.07.681013v1.84","doi-asserted-by":"crossref","unstructured":"Kirillov, A. et al. Segment anything. In Proceedings of the IEEE\/CVF international conference on computer vision, 4015\u20134026 (2023).","DOI":"10.1109\/ICCV51070.2023.00371"},{"key":"2025101107250461000_2025.10.07.681013v1.85","doi-asserted-by":"publisher","DOI":"10.1364\/oe.542640"},{"key":"2025101107250461000_2025.10.07.681013v1.86","doi-asserted-by":"crossref","unstructured":"Bogovic, J. A. , Hanslovsky, P. , Wong, A. & Saalfeld, S. Robust registration of calcium images by learned contrast synthesis. In IEEE International Symposium on Biomedical Imaging (ISBI), 1123\u20131126 (IEEE, 2016).","DOI":"10.1109\/ISBI.2016.7493463"},{"key":"2025101107250461000_2025.10.07.681013v1.87","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1038\/nphoton.2015.61","article-title":"Controllable spatiotemporal nonlinear effects in multimode fibres","volume":"9","year":"2015","journal-title":"Nat. Photon"},{"key":"2025101107250461000_2025.10.07.681013v1.88","doi-asserted-by":"crossref","first-page":"2985","DOI":"10.1364\/OL.30.002985","article-title":"Compensation for multimode fiber dispersion by adaptive optics","volume":"30","year":"2005","journal-title":"Opt. Lett"},{"key":"2025101107250461000_2025.10.07.681013v1.89","doi-asserted-by":"publisher","DOI":"10.1177\/1933719109332825"},{"key":"2025101107250461000_2025.10.07.681013v1.90","doi-asserted-by":"publisher","DOI":"10.1159\/000046994"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.10.07.681013","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:45:50Z","timestamp":1768470350000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.10.07.681013"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,8]]},"references-count":90,"URL":"https:\/\/doi.org\/10.1101\/2025.10.07.681013","relation":{},"subject":[],"published":{"date-parts":[[2025,10,8]]},"subtype":"preprint"}}