{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T09:43:48Z","timestamp":1775987028818,"version":"3.50.1"},"posted":{"date-parts":[[2025,4,2]]},"group-title":"Bioengineering","reference-count":73,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,4,2]],"date-time":"2025-04-02T00:00:00Z","timestamp":1743552000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,31]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Adaptive optics (AO) enables cellular-resolution retinal imaging, yet mirror-based AOSLO systems are constrained by off-axis aberrations that restrict high-quality imaging to narrow fields of view, limiting in vivo studies of dynamic, large-scale retinal processes. Clinical translation of regenerative cell therapy to neurologic disease is hampered by attrition of donor neurons following transplantation. We hypothesized that early innate neuroinflammatory responses to retinal ganglion cell (RGC) transplantation underlie early death of donor cells and that next-generation imaging technologies would provide evidence for microglial attack of grafted neurons. We present a compact refractive lens-based AOSLO system that achieves two-color fluorescence imaging across up to a 16\u00b0 field of view in mouse retina. Dual-wavelength excitation (488 nm and 552 nm) enables visualization of two fluorescence labels, while AO correction improves axial resolution and depth fidelity, allowing robust separation of structures through anatomical layers in retina. Using this platform, we performed 3D time-lapse imaging of microglia and longitudinal imaging in an optic nerve crush model, revealing layer-dependent differences in microglial motility, early activation signatures, and large-scale redistribution longitudinally. The system enabled widefield visualization of injury-associated vascular changes and spatial coupling between microglia and vasculature. Finally, depth-resolved two-color imaging captured immune responses to intravitreally transplanted RGCs, including host-cell recruitment, rapid neurite retraction following local immune-cell contact, and microglial phagocytosis of donor RGCs. Together, these results demonstrate that refractive AOSLO enables in vivo observations of microvascular organization, neuroimmune dynamics, injury responses, and transplanted-cell behavior with spatiotemporal resolution. Our data also suggests that modulation of microglial reactivity may improve outcomes of RGC transplantation.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>Wide-field, depth-resolved imaging is essential for understanding how different cell types interact across a large retinal area, yet existing AOSLO systems retain limited imaging fields due to a conventional optical design using reflective spherical mirrors. Our refractive large-FOV AOSLO overcomes this limitation, enabling simultaneous two-color, diffraction-limited 3D imaging across an up to 16\u00b0 field in vivo. This platform reveals previously inaccessible biological phenomena\u2014including layer-specific microglial dynamics after optic nerve injury, microvascular remodeling, and rapid microglial rejection of transplanted RGCs\u2014providing critical insight into neuroimmune behavior and retinal repair mechanisms at single-cell and subcellular resolution.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.31.645335","type":"posted-content","created":{"date-parts":[[2025,4,2]],"date-time":"2025-04-02T05:35:22Z","timestamp":1743572122000},"source":"Crossref","is-referenced-by-count":2,"title":["Compact refractive dual-channel AOSLO for wide-field imaging in mice reveals microglial interactions with transplanted neurons"],"prefix":"10.64898","author":[{"given":"Zhongqiang","family":"Li","sequence":"first","affiliation":[{"name":"Wilmer Eye Institute, Johns Hopkins Medicine, Baltimore, Maryland, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stella","family":"Mary","sequence":"additional","affiliation":[{"name":"Wilmer Eye Institute, Johns Hopkins Medicine, Baltimore, Maryland, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5372-5457","authenticated-orcid":false,"given":"Thomas V","family":"Johnson","sequence":"additional","affiliation":[{"name":"Wilmer Eye Institute, Johns Hopkins Medicine, Baltimore, Maryland, United States"},{"name":"Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States"},{"name":"Solomon H Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ji","family":"Yi","sequence":"additional","affiliation":[{"name":"Wilmer Eye Institute, Johns Hopkins Medicine, Baltimore, Maryland, United States"},{"name":"Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041202005147000_2025.03.31.645335v2.1","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1186\/s40942-016-0037-8","article-title":"Human retinal microvascular imaging using adaptive optics scanning light ophthalmoscopy","volume":"2","year":"2016","journal-title":"Int J Retina Vitreous"},{"key":"2026041202005147000_2025.03.31.645335v2.2","doi-asserted-by":"publisher","DOI":"10.3390\/cells10061426"},{"key":"2026041202005147000_2025.03.31.645335v2.3","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.62.6.26"},{"key":"2026041202005147000_2025.03.31.645335v2.4","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1186\/s40478-018-0571-8","article-title":"Optic nerve as a source of activated retinal microglia post-injury","volume":"6","year":"2018","journal-title":"Acta Neuropathol Commun"},{"key":"2026041202005147000_2025.03.31.645335v2.5","doi-asserted-by":"publisher","DOI":"10.1126\/science.1194637"},{"key":"2026041202005147000_2025.03.31.645335v2.6","doi-asserted-by":"crossref","unstructured":"M. Mujat , R. D. Ferguson , D. X. Hammer , A. H. Patel , N. Iftimia (2024) High-Resolution Retinal Imaging: Technology Overview and Applications. in Photonics (MDPI), p 522.","DOI":"10.3390\/photonics11060522"},{"key":"2026041202005147000_2025.03.31.645335v2.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.survophthal.2021.05.012"},{"key":"2026041202005147000_2025.03.31.645335v2.8","doi-asserted-by":"publisher","DOI":"10.1097\/OPX.0b013e3181ff9a8b"},{"key":"2026041202005147000_2025.03.31.645335v2.9","doi-asserted-by":"crossref","unstructured":"L. X. Liu et al., Application of Adaptive Optics in Ophthalmology. Photonics 9 (2022).","DOI":"10.3390\/photonics9050288"},{"key":"2026041202005147000_2025.03.31.645335v2.10","doi-asserted-by":"publisher","DOI":"10.1364\/BOE.2.000717"},{"key":"2026041202005147000_2025.03.31.645335v2.11","doi-asserted-by":"publisher","DOI":"10.1364\/BOE.3.000715"},{"key":"2026041202005147000_2025.03.31.645335v2.12","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.14-14542"},{"key":"2026041202005147000_2025.03.31.645335v2.13","doi-asserted-by":"publisher","DOI":"10.1097\/OPX.0b013e3181d39479"},{"key":"2026041202005147000_2025.03.31.645335v2.14","doi-asserted-by":"publisher","DOI":"10.1364\/OE.10.000405"},{"key":"2026041202005147000_2025.03.31.645335v2.15","doi-asserted-by":"publisher","DOI":"10.1364\/BOE.9.004275"},{"key":"2026041202005147000_2025.03.31.645335v2.16","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1364\/AO.504283","article-title":"Alignment, calibration, and validation of an adaptive optics scanning laser ophthalmoscope for high-resolution human foveal imaging","volume":"63","year":"2024","journal-title":"Appl Opt"},{"key":"2026041202005147000_2025.03.31.645335v2.17","doi-asserted-by":"crossref","first-page":"A265","DOI":"10.1364\/JOSAA.27.00A265","article-title":"Adaptive optics scanning laser ophthalmoscope with integrated wide-field retinal imaging and tracking","volume":"27","year":"2010","journal-title":"JOSA A"},{"key":"2026041202005147000_2025.03.31.645335v2.18","doi-asserted-by":"crossref","unstructured":"Y. Yang et al., Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy. eLife 12 (2023).","DOI":"10.7554\/eLife.84853"},{"key":"2026041202005147000_2025.03.31.645335v2.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41377-020-0317-9"},{"key":"2026041202005147000_2025.03.31.645335v2.20","doi-asserted-by":"publisher","DOI":"10.1364\/OE.20.017297"},{"key":"2026041202005147000_2025.03.31.645335v2.21","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1364\/OPTICA.5.001027","article-title":"Handheld Adaptive Optics Scanning Laser Ophthalmoscope","volume":"5","year":"2018","journal-title":"Optica"},{"key":"2026041202005147000_2025.03.31.645335v2.22","doi-asserted-by":"publisher","DOI":"10.1364\/OL.43.005162"},{"key":"2026041202005147000_2025.03.31.645335v2.23","doi-asserted-by":"publisher","DOI":"10.1364\/JOSAA.14.002884"},{"key":"2026041202005147000_2025.03.31.645335v2.24","doi-asserted-by":"crossref","first-page":"5995","DOI":"10.1364\/BOE.403509","article-title":"Kilohertz retinal FF-SS-OCT and flood imaging with hardware-based adaptive optics","volume":"11","year":"2020","journal-title":"Biomedical Optics Express"},{"key":"2026041202005147000_2025.03.31.645335v2.25","doi-asserted-by":"crossref","first-page":"5274","DOI":"10.1364\/BOE.399034","article-title":"High-speed adaptive optics line-scan OCT for cellular-resolution optoretinography","volume":"11","year":"2020","journal-title":"Biomedical optics express"},{"key":"2026041202005147000_2025.03.31.645335v2.26","doi-asserted-by":"crossref","first-page":"2191","DOI":"10.1364\/BOE.6.002191","article-title":"Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina","volume":"6","year":"2015","journal-title":"Biomedical optics express"},{"key":"2026041202005147000_2025.03.31.645335v2.27","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1364\/BOE.5.000439","article-title":"Adaptive optics SLO\/OCT for 3D imaging of human photoreceptors in vivo","volume":"5","year":"2014","journal-title":"Biomedical optics express"},{"key":"2026041202005147000_2025.03.31.645335v2.28","first-page":"743","article-title":"Adaptive optics scanning laser ophthalmoscope imaging: technology update","volume":"10","year":"2016","journal-title":"Clin Ophthalmol"},{"key":"2026041202005147000_2025.03.31.645335v2.29","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2112561118"},{"key":"2026041202005147000_2025.03.31.645335v2.30","doi-asserted-by":"publisher","DOI":"10.1364\/BOE.426157"},{"key":"2026041202005147000_2025.03.31.645335v2.31","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.02.007"},{"key":"2026041202005147000_2025.03.31.645335v2.32","first-page":"2759","article-title":"Microglia in the primate retina: regional distribution and morphological diversity","volume":"222","year":"2017","journal-title":"Brain Structure and Function"},{"key":"2026041202005147000_2025.03.31.645335v2.33","doi-asserted-by":"publisher","DOI":"10.1111\/j.1474-9726.2010.00660.x"},{"key":"2026041202005147000_2025.03.31.645335v2.34","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1016\/j.omtm.2021.03.004","article-title":"Transplantation of miPSC\/mESC-derived retinal ganglion cells into healthy and glaucomatous retinas","volume":"21","year":"2021","journal-title":"Molecular Therapy-Methods & Clinical Development"},{"key":"2026041202005147000_2025.03.31.645335v2.35","doi-asserted-by":"crossref","unstructured":"E. Kriukov et al., Single-cell transcriptome of retinal myeloid cells in response to transplantation of human neurons reveals reversibility of microglial activation. bioRxiv (2025).","DOI":"10.1101\/2025.05.16.654622"},{"key":"2026041202005147000_2025.03.31.645335v2.36","unstructured":"E. A. Aguzzi et al., Internal limiting basement membrane inhibits functional engraftment of transplanted human retinal ganglion cells. Science Translational medicine (2026)."},{"key":"2026041202005147000_2025.03.31.645335v2.37","doi-asserted-by":"publisher","DOI":"10.1364\/BOE.473447"},{"key":"2026041202005147000_2025.03.31.645335v2.38","doi-asserted-by":"publisher","DOI":"10.1364\/OL.40.005830"},{"key":"2026041202005147000_2025.03.31.645335v2.39","doi-asserted-by":"crossref","unstructured":"H. Terasaki , S. Sonoda , M. Tomita , T. Sakamoto , Recent Advances and Clinical Application of Color Scanning Laser Ophthalmoscope. J Clin Med 10 (2021).","DOI":"10.3390\/jcm10040718"},{"key":"2026041202005147000_2025.03.31.645335v2.40","doi-asserted-by":"crossref","unstructured":"I. Grulkowski et al., Swept source optical coherence tomography and tunable lens technology for comprehensive imaging and biometry of the whole eye. Optica 5 (2018).","DOI":"10.1364\/OPTICA.5.000052"},{"key":"2026041202005147000_2025.03.31.645335v2.41","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.14-13895"},{"key":"2026041202005147000_2025.03.31.645335v2.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2021.108805"},{"key":"2026041202005147000_2025.03.31.645335v2.43","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2022.860070"},{"key":"2026041202005147000_2025.03.31.645335v2.44","doi-asserted-by":"publisher","DOI":"10.1186\/s12868-022-00703-1"},{"key":"2026041202005147000_2025.03.31.645335v2.45","doi-asserted-by":"publisher","DOI":"10.1002\/1098-1136(200103)33:3<256::AID-GLIA1024>3.0.CO;2-J"},{"key":"2026041202005147000_2025.03.31.645335v2.46","doi-asserted-by":"publisher","DOI":"10.1126\/science.1110647"},{"key":"2026041202005147000_2025.03.31.645335v2.47","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0584-17.2017"},{"key":"2026041202005147000_2025.03.31.645335v2.48","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.09-4537"},{"key":"2026041202005147000_2025.03.31.645335v2.49","doi-asserted-by":"publisher","DOI":"10.1038\/nn1805"},{"key":"2026041202005147000_2025.03.31.645335v2.50","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0015973"},{"key":"2026041202005147000_2025.03.31.645335v2.51","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1186\/s13287-024-04052-0","article-title":"Subretinal microglia support donor photoreceptor survival in rd1 mice","volume":"15","year":"2024","journal-title":"Stem Cell Research & Therapy"},{"key":"2026041202005147000_2025.03.31.645335v2.52","doi-asserted-by":"crossref","unstructured":"V. V. Malechka et al., Improvement of human donor retinal ganglion cell survival through modulation of microglia. bioRxiv, 2024.2007. 2006.602307 (2024).","DOI":"10.1101\/2024.07.06.602307"},{"key":"2026041202005147000_2025.03.31.645335v2.53","doi-asserted-by":"publisher","DOI":"10.1007\/BF01224758"},{"key":"2026041202005147000_2025.03.31.645335v2.54","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.15252\/emmm.201505298","article-title":"Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration","volume":"7","year":"2015","journal-title":"EMBO molecular medicine"},{"key":"2026041202005147000_2025.03.31.645335v2.55","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.16490"},{"key":"2026041202005147000_2025.03.31.645335v2.56","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1719601115"},{"key":"2026041202005147000_2025.03.31.645335v2.57","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1186\/s13287-025-04509-w","article-title":"Metabolic stress and early cell death in photoreceptor precursor cells following retinal transplantation","volume":"16","year":"2025","journal-title":"Stem Cell Research & Therapy"},{"key":"2026041202005147000_2025.03.31.645335v2.58","doi-asserted-by":"publisher","DOI":"10.1155\/2015\/673090"},{"key":"2026041202005147000_2025.03.31.645335v2.59","doi-asserted-by":"crossref","first-page":"e0285633","DOI":"10.1371\/journal.pone.0285633","article-title":"Dose-dependent modulation of microglia activation in rats after penetrating traumatic brain injury (pTBI) by transplanted human neural stem cells","volume":"18","year":"2023","journal-title":"PLoS One"},{"key":"2026041202005147000_2025.03.31.645335v2.60","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0087-1"},{"key":"2026041202005147000_2025.03.31.645335v2.61","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3163"},{"key":"2026041202005147000_2025.03.31.645335v2.62","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-immunol-032713-120240"},{"key":"2026041202005147000_2025.03.31.645335v2.63","first-page":"1","article-title":"Clinical application of stem cell therapy in Parkinson\u2019s disease","volume":"10","year":"2012","journal-title":"BMC medicine"},{"key":"2026041202005147000_2025.03.31.645335v2.64","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2007.10.036"},{"key":"2026041202005147000_2025.03.31.645335v2.65","doi-asserted-by":"publisher","DOI":"10.1016\/j.neuron.2012.03.026"},{"key":"2026041202005147000_2025.03.31.645335v2.66","doi-asserted-by":"crossref","first-page":"3576","DOI":"10.1364\/BOE.457408","article-title":"Non-interferometric volumetric imaging in living human retina by confocal oblique scanning laser ophthalmoscopy","volume":"13","year":"2022","journal-title":"Biomedical Optics Express"},{"key":"2026041202005147000_2025.03.31.645335v2.67","unstructured":"M. R. R. Gardner , H. Grady ; Milner, Thomas E. (2016) Computational Model (Zemax) of a Mouse Eye for Visible and Near-Infrared Wavelength Ranges. (The University of Texas at Austin, The University of Texas at Austin)."},{"key":"2026041202005147000_2025.03.31.645335v2.68","doi-asserted-by":"crossref","unstructured":"E. G. Cameron et al., Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration. Bio Protoc 10 (2020).","DOI":"10.21769\/BioProtoc.3559"},{"key":"2026041202005147000_2025.03.31.645335v2.69","first-page":"1","article-title":"Retinal dendritic cell recruitment, but not function, was inhibited in MyD88 and TRIF deficient mice","volume":"11","year":"2014","journal-title":"Journal of Neuroinflammation"},{"key":"2026041202005147000_2025.03.31.645335v2.70","doi-asserted-by":"publisher","DOI":"10.5966\/sctm.2012-0106"},{"key":"2026041202005147000_2025.03.31.645335v2.71","doi-asserted-by":"publisher","DOI":"10.1002\/sctm.17-0059"},{"key":"2026041202005147000_2025.03.31.645335v2.72","unstructured":"W. R. Institute (2024) H7 Human Embryonic Stem Cell Line. (WiCell Research Institute, Madison, WI, USA)."},{"key":"2026041202005147000_2025.03.31.645335v2.73","doi-asserted-by":"crossref","first-page":"101328","DOI":"10.1016\/j.xpro.2022.101328","article-title":"Analyses of transplanted human retinal ganglion cell morphology and localization in murine organotypic retinal explant culture","volume":"3","year":"2022","journal-title":"STAR protocols"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.03.31.645335","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T09:01:04Z","timestamp":1775984464000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.03.31.645335"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,2]]},"references-count":73,"URL":"https:\/\/doi.org\/10.1101\/2025.03.31.645335","relation":{},"subject":[],"published":{"date-parts":[[2025,4,2]]},"subtype":"preprint"}}