{"paper_id":"0c757a2e-415d-4508-b45f-073dbcd7946e","body_text":"Correlative Synchrotron X -ray Microscopy Reveals Dose - and Division-\nDependent Nanoparticle Redistribution in Macrophages  \n \nIsabella Scarpaa,b, Renata S. Rabeloa, Aline O. Pereiraa,c, Francine F. Fenandesa, Flávia E. \nGaldinoa, Maiara F. Terraa, Maria Harkiolakid, Florian Meneaua,c, Carla C. Poloa, f, André \nA. Thomazb, Ana J. Pérez Bernáe, Mateus B. Cardosoa,b* \n \na Brazilian Synchrotron Light Laboratory (LNLS), Brazilian Center for Research in Energy \nand Materials (CNPEM), Campinas, S P 13083-100, Brazil \nb “Gleb Wataghin” Institute of Physics, University of Campinas (UNICAMP), Campinas, SP \n13083-859, Brazil \nc Institute of Chemistry, University of Campinas (UNICAMP), Campinas, SP 13083-970, \nBrazil \nd University of Warwick, Coventry  CV4 7AL, United Kingdom \ne Alba Synchrotron Light Source, MISTRAL Beamline Experiments Divison, Cerdanyola \ndel Vallès, Barcelona, 08290, Spain \nf  Institute of Biology, University of Campinas  (UNICAMP), Campinas, SP  13083-862, \nBrazil \n \n* Corresponding author: cardosomb@lnls.br \n \n \nKeywords: silica nanoparticles;  synchrotron X -ray microscopy , cryo-SXT; X -ray \nptychography; intracellular trafficking; nano-bio interactions ; PXCT  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n2 \n \nABSTRACT \nUnderstanding the intracellular fate of nanoparticles is essential for designing safer \nand more effective nanomedicines, yet most studies rely on static observations and lack high-\nresolution, near -native volumetric information. Here, we establish a synchrotron -based \ncorrelative X-ray microscopy framework to investigate how fluorescent silica nanoparticles \n(SiNPs) redistribute within macrophages as a function of concentration and successive cell -\ndivision cycles. SiNPs were internalized by RAW 264.7 macrophag es at different \nconcentrations and analyzed using a synchrotron -based correlative X -ray microscopy \nworkflow integrating cryo genic soft X -ray tomography (cryo -SXT), cryogenic structured \nillumination microscopy (cryo -SIM), and coherent X -ray ptychography, with confocal \nfluorescence microscopy used to establish population -level uptake tendencies. Cryo-SXT \nreveals a concentration -dependent redistribution of nanoparticle -containing vesicles from \nperipheral endosomes toward the perinuclear region, while correlative cryo -SIM confirms \nstrict vesicular confinement, with no evidence of free nanoparticle d iffusion into the \nnucleoplasm. At higher  doses, nanoparticles approach the nuclear region via vesicles \nextending into nuclear-envelope invaginations, rather than by true nuclear entry. Successive \ncell divisions redistribute the intracellular nanoparticle load and promote stable perinuclear \nclustering, identifying a long -term sequestration route in macrophages. Coherent X -ray \nptychography further reveals nanoscale deformations of the nuclear envelope associated with \ndense perinuclear vesicles. Together, these results establish synchrotron-based correlative X-\nray microscopy as a mechanistic, multiscale platform for unveiling the dynamic intracellular \nfate of nanoparticles and providing mechanistic insight into their apparent nuclear \nlocalization.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n3 \n \nINTRODUCTION \nThe intracellular fate of engineered nanoparticles plays a central role in determining \ntheir safety, bioavailability, and therapeutic performance in nanomedicine 1–3. Their ability to \ncross biological barriers and interact with specific intracellular targets makes them powerful \ntools for therapeutic innovation 4,5. However, the biological performance of nanoparticles \ndepends not only on their composition and design but also on their intracellular fate  - the \ndynamic sequence of events that determines how they traffic, transform, and interact with \norganelles after uptake 6–10. Understanding this fate is critical for improving nanoparticle \nsafety, bioavailability, and targeting efficiency. Among the wide variety of nanomaterials,  \nsilica nanoparticles (SiNPs) stand out as versatile platforms due to their tunable size, porosity, \nand surface functionalization, which enable fine control over drug loading and molecular \ninteractions 2,11–13. Numerous studies have shown that the adsorption of biomolecules, mostly \nproteins, onto the nanoparticle surface results in the formation of the protein corona14,15 plays \na decisive role in defining the biological identity of nanomaterials, influencing their cellular \nuptake, distribution,  cytotoxicity 16 and overall biocompatibility 17,18. Despite these advances, \nthe complex and dynamic nature of nanoparticle –biological interactions  remains poorly \nunderstood, limiting our ability to design nanoparticles with predictable and reproducible \nbehavior across diverse biological environments 19. \nOne of the main reasons for this limitation is the difficulty of accurately determining \nthe precise intracellular localization and behavior of nanoparticles, as conventional imaging \nmethods offer either insufficient spatial resolution or lack the necessary biological context . \nFluorescence microscopy offers molecular specificity but relies on external labeling, thereby \nrestricting access to intrinsic ultrastructural information 20,21. Transmission electron \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n4 \n \nmicroscopy (TEM) provides atomic -level resolution but requires ultrathin-sectioned \nsamples, which prevents visualization of entire cells in their native architecture 22. These \nconstraints underscore the need for advanced nano -imaging approaches that combine \nvolumetric, label -free, and high-resolution capabilities under near-physiological conditions. \nIn this context, X -ray–based methods have gained attention for their abili ty to overcome \nmany of the limitations of conventional microscopy by exploiting the strong penetration and \nintrinsic contrast of biological materials 23. Although achieving high spatial resolution still \ndemands precise optical design and fabrication, the advent of synchrotron light sources has \nopened new possibilities for extending both the resolution and sensitivity of X -ray \nimaging24,25. \nThese technological advances are implemented through distinct physical contrast \nmechanisms that define how synchrotron -based X-ray imaging interrogates cells, ranging \nfrom absorption to coherent diffraction. Particularly, cryogenic soft X-ray tomography (cryo-\nSXT)26,27 and X -ray ptychography 28 show great promise for achieving high -resolution \nbiological X-ray imaging. For example, cryo-SXT has enabled label-free mapping of cellular \nultrastructure and nanoparticle localization in diverse systems, including gold \nnanoparticles 29,30, iron oxide nanoparticles (SPIONs)31, and silica -based nanomaterials 32, \nrevealing their confinement in endocytic and lysosomal compartments and their progressive \naccumulation near the nucleus. Correlative approaches combining cryo-SXT with cryogenic \nfluorescence microscopy, as recently demonstrated in the literature 32, have shown that \nmolecular identity can be directly linked to three -dimensional ultrastructural context . 33In \nparallel, X -ray ptychography has demonstrated its ability to provide high -resolution, \nquantitative electron -density maps of biological specimens and to locate nanomaterials \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n5 \n \nwithin cells with nanometer -scale sensitivity 33,34. Despite these advances, most biological \napplications of synchrotron X -ray imaging remain primarily descriptive, focusing on short \nincubation times, single concentrations, and static snapshots of nanoparticle localization. \nSystematic investigations that e xploit X -ray techniques to follow how nanoparticle \ndistribution evolves as a function of dose, time, and cell -division dynamics are still largely \nmissing, particularly in immune cells.  \nHere, we address this methodological and biological gap by establishing a \nsynchrotron X-ray–based imaging framework to investigate how nanoparticle distribution \nevolves as a function of concentration and successive cell  division cycles in macrophages. \nBy integrating cryo -SXT and X -ray ptychography  in 2D and combined with tomography  \nwithin a single experimental strategy, we move beyond static localization and use X -ray \nimaging as a dynamic tool to monitor nanoparticle accumulation, vesicular trafficking, and \nlong-term intracellular redistribution.  This approach reveals a concentration -dependent \npathway in which SiNPs larger than 100 nm can access the nuclear region at early time points \nand are subsequently redistributed through cell divisions. We then introduce a nuclear \nisolation protocol compatible with ptychographic imaging  that enables targeted high-\nresolution analysis of nanoparticle –nucleus interactions. Beyond structural visualization, \nsynchrotron X-ray techniques are established here as a central experimental platform for \naddressing time- and dose-dependent questions in nanomedicine, providing a methodological \nframework for studying nanoparticle dynamics within complex and highly phagocytic \nimmune cells.  \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n6 \n \nRESULTS AND DISCUSSION \nFluorescent silica nanoparticles (SiNPs) , doped with the dye A TTO 633 , were \nsuccessfully synthesized as spherical structures, with a mean diameter of 135 ± 16 nm  \ndetermined from the analysis of 250 individual particles by STEM imaging ( Figure S1, \nSupporting Information ). The obtained size range (<200 nm) is particularly relevant for \nbiomedical applications, as it favors efficient cellular uptake and intracellular trafficking 35. \nThe overall experimental workflow is illustrated in Figure 1a, where SiNPs were coated with \nbovine serum albumin (BSA) to form a protein corona , thereby improving biocompatibility \nby reducing cytotoxicity while enhancing the cellular uptake16,32. Cells were then incubated \nwith these BSA-coated SiNPs for controlled periods, washed, and analysed at different time \npoints, enabling us to monitor the intracellular distribution of nanoparticles across successive \ncell divisions.  The framework represents the experimental model adopted in this study, \nrelating nanoparticle concentration to post -exposure time, during which increasing cell \nnumber reflects ongoing proliferation (Figure 1b).  By systematically varying nanoparticle \nconcentration and post-exposure time, we correlated the extent of SiNPs internalization with \ncell proliferation dynamics, providing mechanistic insight into the redistribution of \nnanoparticle load during the mitotic cycle . \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n7 \n \n \nFigure 1. Experimental workflow and conceptual framework.  a) 1- SiNPs are treated with BSA for 8 \nminutes to form a protein corona . 2 - SiNPs are incubated in RAW 264.7 cells for 1 h, subsequently \nwashed with phosphate buffered saline  (PBS), and analysed at different time points . b) Schematic \nrepresentation of SiNP s distribution as a function of nanoparticle concentration and post -exposure \ntime. The increase in cell number arises from proliferation along the time axis.  \n \nUnderstanding how nanoparticles interact with immune cells is essential for \npredicting their biological behavior and long -term in vivo fate. Regardless of their intended \nbiomedical application, SiNPs inevitably encounter immune cells, which serve as the first \nline of recognition and clearance of foreign materials 36,37. To evaluate how nanoparticle load \ninfluences cellular responses, RAW 264.7 macrophages were exposed to three SiNP s \nconcentrations (0.003, 0.03, and 0.3 mg/mL). This range was selected to capture potential \ndose-dependent effects on internalization efficiency, vesicular organization, and overall \nintracellular distribution  38. Flow cytometry (Figure S5 Supporting Information) was used to \nquantify nanoparticle uptake across  the three tested concentrations. Cells were stained with \npropidium iodide (PI) while SiNP s (containing ATTO 633) w ere detected in the APC \nchannel, enabling discrimination between nanoparticle -positive (ATTO +PI) and \nnanoparticle-free (PI) populations. At the lowest concentration (0.003 mg/mL), only 79.6% \nof cells were nanoparticle-positive (Figure S5c), indicating limited extracellular availability. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n8 \n \nThis incomplete initial loading leads to progressive dilution of nanoparticles and a decrease \nin the fraction of nanoparticle -positive cells across successive divisions.  In contrast, at 0.03 \nand 0.3 mg/mL, nearly all cells contained SiNPs at the initial time point (Figure S5a, b). This \nnear-uniform loading ensures that most cells retain nanoparticles after division, maintaining \na high fraction of nanoparticle -positive cells over multiple cell cycles.  This strong \nconcentration dependence reflects the high pha gocytic activity of macrophages and their \ncapacity to modulate uptake in response to particle abundance  39,40.  \nA multimodal imaging strategy was used to resolve how intracellular nanoparticle \norganization evolves with concentration. Confocal fluorescence microscopy provided an \ninitial overview of nanoparticle distribution after 1 h of incubation (Figure 2a). The AT TO \n633 signal from the particles increased progressively from 0.003 to 0.3 mg/mL, reflecting the \nhigher nanoparticle availability in the extracellular medium and is consistent with the flow \ncytometry results. To solve the ultrastructural context of these fluorescent signals, cryo-SXT \nat B24 Beamline ( Diamond) was employed under near -native conditions (Figure 2b). \nTomographic slices revealed high -contrast dense objects, highlighted by orange arrows, \ncorresponding to SiNPs encapsulated within vesicles , indicating that across all \nconcentrations SiNPs were internalized via vesicular pathways 41,42. However, their spatial \norganization strongly depended on nanoparticle dose.  At low concentration, only a small \nvesicle containing SiNPs w as detected in the cytoplasm. At intermediate concentration, \nmultiple vesicles containing SiNPs were observed in the cytosol . Notably, at the highest \nconcentration (0.3 mg/mL), large vesicular structures containing SiNPs were distributed \nthroughout the cytosol  and within the nuclear region. This concentration -dependent \nprogression is summarized schematically in Figure 2c.  Correlative  cryogenic structured \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n9 \n \nillumination microscopy (cryo -SIM) and cryo -SXT imaging further validated these \nobservations (Figure 2d). Cryo -SIM identified SiNPs (red) within vesicular compartments \nthat colocalized with lysosomal markers (blue), confirming their endolysosomal \nconfinement 41. Overlay with cryo-SXT showed that the colocalization of lysosomal and SiNP \nsignals corresponds to the high -contrast structures observed in the tomograms.  Also, both \ncryo-SXT and cryo -SIM revealed vesicle -encapsulated SiNPs located within the nuclear \nregion. Cryo-SIM confirmed that these particles remained enclosed by vesicular membranes, \nexcluding free diffusion into the nucleoplasm, as also confirmed by the fluorescence confocal \n(Figure 2a), where the white arrows point to the overlap of the nuclear and nanoparticle \nsignals. While nuclear localization has previously been reported mainly for ultrasmall \nnanoparticles or systems engineered for nuclear targeting43–45, this observation demonstrates \nvesicle-encapsulated access of non-functionalized silica nanoparticles larger than 100 nm to \nthe nuclear region, without nucleoplasmic entry. The combined X-ray and fluorescence data \nsuggest that this phenomenon arises from vesicle -induced deformation and invagination of \nthe nuclear envelope, rather than active transport through nuclear pores, consistent with \nmechanical stress –induced nuclear envelope remodeling 46. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n10 \n \n \nFigure 2. Internalization and intracellular distribution of SiNPs in RAW 264.7 cells at different \nconcentrations. a) Fluorescence confocal micrographs of cells exposed to SiNPs at 0.003, 0.03, and \n0.3 mg/mL (green : actin; blue: nucleus; red: SiNP ). b ) Slices from cryo -SXT tomographic \nreconstructions. Orange arrows indicate internalized SiNPs.  c) Schematic representation illustrating \nthe evolution of SiNP  concentration inside macrophages as a function of exposure.  d) Correlative \nimaging combining cryo -SIM and cryo -SXT shows lysosomes (blue), mitochondria (green), and \nSiNPs (red), followed by grayscale tomographic slices  at 0.3 mg/mL and the merged image. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n11 \n \nBuilding upon these concentration -dependent results, we next examined how cell \nproliferation dynamics influence nanoparticle redistribution across successive division \ncycles. Determining the doubling time (DT) was crucial to ensure experimental \nreproducibility, as cell growth rates can vary significantly between laboratories and culture \nconditions. Monitoring DT also allowed normalization of exposure periods to the cells’ \nbiological rhythm, enabling comparison across equivalent proliferative stages.  Fluorescence \nconfocal and cryo-SXT imaging (Figure 3a –c) reveal a clear temporal evolution in SiNP s \nintracellular distribution at 0.3 mg/mL concentration over two division cycles. At the initial \ntime (Figure 3a), SiNPs appear dispersed throughout the cytoplasm  and within the nuclear \nregion, as indicated by white arrows, with minimal clustering. After the first doubling (Figure \n3b), fluorescence intensity increased and small perinuclear aggregates emerged, indicating \nprogressive clustering of SiNPs , and cryo-SXT also revealed vesicles containing SiNPs \nrelocating away from the nuclear region.  By the second doubling  (Figure 3c) , SiNPs are \npredominantly concentrated around the nucleus, forming distinct high -intensity clusters \nconsistent with late endosomal /lysosomal trafficking 9,41,42. Segmented cryo -SXT volumes \nprovide complementary ultrastructural confirmation of these patterns, highlighting both the \nredistribution of SiNP -containing vesicles during cell division and their preferential \nperinuclear accumulation.  Correlative cryo-SIM and cryo-SXT analyses (Figure 3d) further \nelucidate the subcellular context, revealing the spatial relationship between SiNPs (red) and \nlysosomes (blue). After two division cycles, SiNPs remain confined within vesicular \nmembranes in the perinuclear region 47 suggesting that the observed redistribution results \nfrom active vesicular transport and maturation rather than passive diffusion.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n12 \n \n \nFigure 3. Multimodal imaging of intracellular SiNP distribution across two cell -division cycles in \nmacrophages.(a–c) Fluorescence confocal micrographs (actin, green; nucleus, blue; SiNPs, red), \ncorresponding cryo–SXT tomographic slices, and segmented volumes corresponding  to the evolution \nof intracellular SiNP  localization over time: a) initial time, b) first doubling time, and c) second \ndoubling time. d) Correlative cryo –SIM and cryo –SXT imaging of lysosomes (blue), SiNPs (red), \nand mitochondria (green), followed by grayscale tomographic slices at the second d oubling time and \nthe merged correlation image.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n13 \n \nFurthermore, X-ray mosaic projections of whole cells  obtained at Mistral  Beamline \n(Alba Synchrotron) (Figure 4a–c) extend this analysis to near–whole-cell volumes, showing \nthat at the initial time SiNP s-containing vesicles are broadly distributed throughout the \ncytoplasm, whereas over successive division cycles they progressively concentrate toward \nthe perinuclear region. Three -dimensional rendered and segmented volumes obtained from \nstitched cryo-SXT tomograms (Figure 4d–f) confirm this redistribution at the ultrastructural \nlevel, revealing a gradual relocation of vesicle-confined SiNPs toward the nuclear periphery. \nThe schematic representations (Figure 4g –i) summarize this dynamic process, highlighting \nthe progressive perinuclear aggregation across division stages . Together, this whole -cell \nperspective rules out local sampling effects and confirms that the observed perinuclear \naccumulation reflects a global intracellular reorganization rather than a region -specific \nphenomenon. This localization likely reflects a combination of endocytic processing, vesicle \ntrafficking, and sequestration mechanisms that preserve cellular homeostasis during  \nproliferation 47. Together, these findings demonstrate that SiNP intracellular organization is \ndynamic and responsive to cell division, with progressive perinuclear accumulation \nreflecting coordinated endocytic activity and stable vesicular entrapment 38. Such behavior \nprovides structural insight into how proliferative cells adapt to nanoparticle exposure, \nrevealing vesicular confinement as a key mechanism governing long -term nanoparticle \nretention and intracellular fate.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n14 \n \n \nFigure 4. Time-dependent intracellular distribution of SiNPs at 0.3 mg /mL. ( a–c) X -ray mosaic \nprojections of whole cells at a) initial time, b) first doubling time, and c) second doubling time. Pink \nand blue squares indicate adjacent, partially overlapping regions used for tomographic reconstruction. \n(d–f) Three -dimensional rendered and segmented volumes obtained from stitched tomograms \ncorresponding to the regions highlighted in panels (a –c) at d) initial time, e) first doubling time, and \nf) second doubling time. Highlighted structures include the cell  nucleus (pink), vesicles (gold), and \nSiNPs (cyan); the cell boundary is indicated by a purple contour.(g –i) Schematic representations \nsummarizing the progressive perinuclear aggregation of SiNP s-containing vesicles at g) initial time, \nh) first doubling ti me, and i) second doubling time.  \n \n \nWe next employed X -ray ptychography, an advanced configuration of coherent \ndiffractive imaging (CDI), to investigate these interactions at nanoscale resolution , \nparticularly under higher SiNP concentrations. This technique provides quantitative, label -\nfree imaging based on phase shifts induced by variations in the specimen's electron density, \nenabling visualization of dense intracellular regions that are otherwise inaccessible to \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n15 \n \nconventional microscopy.  To enable this analysis, a nuclear isolation protocol 48,49 was \ndeveloped to lyse the plasma membrane while preserving the nuclear envelope selectively. It \nprovides an essential means to investigate the potential structural influence of nanoparticle \nexposure on the nucleus. The optimized treatment with 1% Triton X-100 for 3 min, followed \nby glutaraldehyde fixation and critical-point drying, provided optimal preservation of nuclear \nintegrity and compatibility with coherent X -ray diffraction imaging (Figure 5a). The \nexperimental optical layout of the ptychography setup at Cateretê Beamline (Sirius) is \nschematically illustrated in Figure 5b. Unlike conventional lens -based imaging, X -ray \nptychography reconstructs the complex electron density of a sample from overlapping \ndiffraction patterns acquired during a scanning sequence 28. Each illuminated position \ngenerates an interference pattern (“speckle”), which is computationally processed through \niterative phase-retrieval algorithms 50 such as the Ptychographic Iterative Engine (PIE) 51 or \nthe Difference Map (DM)52 to recover both amplitude and phase information. When extended \nto ptychographic X -ray computed tomography  (PXCT)53, this approach enables three -\ndimensional visualization of entire cells with spatial resolutions reaching tens of nanometers. \nDespite its high resolving power, the application of X-ray ptychography to biological systems \nremains challenging due to the intrinsically low scattering contrast of biological materials \nand their susceptibility to radiation damage. However, recent advance s in beam coherence \nand scanning stability  (enabled by fourth -generation synchrotron light sources such as \nSIRIUS)24,54,55  have begun to overcome these limitations, allowing high -resolution imaging \nof intact cells. In this context, our study extends the use of ptychography to probe \nnanoparticle–nucleus interactions in immune cells, providing a three-dimensional, nanoscale \nview of how intracellular SiNPs influence nuclear architecture.   \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n16 \n \n \n \nFigure 5. Experimental workflow and ptychographic imaging setup.  a) Stepwise preparation of SiNP-\ntreated cells for ptychography, including decellularization with 1% Triton for 3 min, fixation, \ndehydration, and critical point drying. b) Schematic representation of the ptychography optical layout, \nshowing the path of the coherent X -ray beam through the sample membrane toward the detector.  \n \nDespite the higher radiation dose required, this approach enables detailed visualization \nof nuclear morphology, subnuclear organization, and intracellular nanoparticle distribution \nat different exposure times (Figure 6). The time-dependent intracellular distribution of SiNPs \nrevealed by X -ray ptychography is summarized in schematic representations of the spatial \norganization shown in (Figure 6a, e, i, m ). Two-dimensional ptychographic reconstructions \nrevealed clear contrasts between the nucleus and cytoplas m (Figure 6b, f, j, n), with SiNPs \nappearing (Figure 6f, j, n) as high-density domains surrounding and occasionally deforming \nthe nuclear envelope. These findings are consistent with the previously observed vesicle -\nmediated internalization pathways, suggesting that mechanical interactions between enlarged \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n17 \n \nendosomal vesicles and the nucleus may drive local invaginations of the nuclear membrane  \n(Figure 6e, f, g, h) . Three-dimensional ptychographic tomograms (Figure 6h, l, p ) further \nconfirmed the progressive perinuclear accumulation of SiNPs over time 47. After 1  h of \nincubation (Figure 6e, f, g, h ), nanoparticles were observed near the nuclear periphery  and \nwithin the nuclear region (MovieS6, Supporting Information), while after 9 h (Figure 6i, j, k, \nl; MovieS7 ) and 18 h (Figures 6m, n, o, p ; MovieS8 ), dense clusters formed around the \nnucleus, in agreement with the confocal and cryo -SXT data. These results indicate that \nnanoparticle redistribution during successive cell -division cycles is accompanied by \nstructural remodeling of the perinuclear compart ment, reinforcing the notion of persistent \nvesicular entrapment rather than direct nuclear diffusion. Such PXCT analyses underscore \nthe potential of coherent X -ray imaging to elucidate nanoscale interactions at the bio -nano \ninterface with unprecedented clarity.  \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n18 \n \n \nFigure 6. Time-dependent intracellular distribution of SiNPs  revealed by X -ray ptychography. \nSchematic representation of the spatial distribution of silica nanoparticles (cyan) around the nucleus \n(purple) at each time point : a) Control cell; e) 0.3 mg /mL after 1 h; i) 0.3 mg /mL after 9 h (first \ndoubling time); m) 0.3 m g/mL after 18 h (second doubling time).  Two-dimensional ptychographic \nreconstructions of nucleus of RAW 264.7 cells: : b) Control cell; f) 0.3 mg/mL after 1 h; j) 0.3 mg/mL \nafter 9 h; n) 0.3 mg /mL after 18 h. Magnified views of the boxed regions in  two-dimensional \nptychographic reconstructions , highlighting nuclear morphology and nanoparticle localization : c) \nControl cell; g) 0.3 mg /mL after 1 h; k) 0.3 m g/mL after 9 h; o) 0.3 mg /mL after 18 h.  Three-\ndimensional renderings of the tomographic reconstructions : d) Control cell; h) 0.3 mg/mL after 1 h; \nl) 0.3 mg/mL after 9 h; p) 0.3 mg/mL after 18 h. \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n19 \n \nCONCLUSIONS \nIn this work, we establish synchrotron -based correlative X -ray microscopy as a \ncentral platform to unravel the dynamic intracellular fate of nanoparticles in immune cells. \nBy combining correlative cryo -soft X-ray tomography with X -ray ptychography, we show \nthat the intracellular distribution of silica nanoparticles is not static but undergoes a dose - \nand division-dependent redistribution in macrophages. Cryo-SXT reveals volumetric, label-\nfree maps of nanoparticle -containing vesicles, demonstrating concentration -dependent \naccess to the nuclear region and progressive perinuclear accumulation over successive \ndivisions. Correlative cryo-SIM confirms the endo lysosomal nature of th ese compartments \nand excludes free diffusion into the nucleoplasm, while large -volume reconstructions show \nthat this redistribution reflects a global intracellular reorganization. 2D ptychography and \nPXCT extends these observations to the nanoscale, revealing vesicle-induced deformation of \nthe nuclear envelope. Together, these results identify vesicular confinement, mitotic \nredistribution, and perinuclear accumulation as key mechanisms governing long -term \nnanoparticle retention in macrophages. More broadly, this work positions synchrotron-based \nX-ray imaging as a dynamic analytical platform for investigating time - and dose-dependent \nnano–bio interactions across complex cellular systems.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n20 \n \nMATERIALS AND METHODS \nSynthesis and purification of fluorescent SiNPs. The nanoparticles were synthesized using \na modified Stöber method 56. One milligram of A TTO633-NHS-ester (λex/em: 630/651 nm) \nwas dissolved in 800 µL of anhydrous dimethylformamide (DMF). Four hundred µL of this \nsolution were added to 2 mL of anhydrous ethanol, followed by 9.6 µL of (3-aminopropyl) \ntriethoxysilane (APTES). The reaction was maintained at room temperature with constant \nstirring for 20 hours, resulting in A TTO633-APTES. Then, 7 mL of ammonium hydroxide \nsolution was added to 120 mL of ethanol and kept under stirring at room temperature. After \n30 min, 2.4096 mL of A TTO6 33-APTES and 2.5 mL of tetraethoxysilane (TEOS) were \nadded. After 3 hours, an additional 2.5 mL aliquot of TEOS was added, and the reaction was \nmaintained under stirring for 24 hours. The nanoparticles were purified by centrifugation. \nThe final volume of the synthesis was divided into three Falcon tubes (approximately 40 mL \neach) and centrifuged at 10,000 rpm for 15 min at 20 °C. After centrifugation, the supernatant \nwas discarded, and 30 mL of ethanol was added to each Falcon tube. The nanoparticles were \nresuspended using a vortex and sonicated for 30 min. After another ce ntrifugation step, the \nsupernatant was discarded, and the nanoparticles were resuspended in 40 mL of ultrapure \nwater. They were then placed in an ultrasonic bath for 30 min. This washing procedure with \nwater was repeated four times. The final suspension was stored at 8°C, and the concentration \nwas determined by gravimetry.  \nConfocal fluorescence microscopy  of RA W 264.7. RAW 264.7 macrophages  cells were \nseeded at a density of 1.0 × 10 5 cells per well in 6 -well plates  containing sterile glass \ncoverslips. Prior to cell seeding, coverslips were sequentially cleaned under agitation in nitric \nacid (HNO₃, 15 min), rinsed thoroughly with distilled water, incubated in 1 N NaOH (15 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n21 \n \nmin), rinsed again with distilled water, and treated with 70% ethanol (30 min). Coverslips \nwere air-dried on filter paper, transferred to glass containers, and sterilized by autoclaving.  \nSiNPs were used at concentrations of 0.3, 0.03, and 0.003 mg /mL. Prior to cell exposure, \nnanoparticles were incubated for 8 min in DMEM containing 1% BSA. Cells after 24 h were \ntreated with the nanoparticle suspensions for 1 h at 37 °C.  After incubation, cells, including \nuntreated controls, were washed with PBS and fixed with 250 µL of 4% paraformaldehyde \nper coverslip for 10 min at room temperature. Fixation was followed by three washes with \ncold PBS (5 min each). This procedure was repeated for experimental time points of 9 h  \n(calculated DT for BR-cultured cells ) and 18 h following nanoparticle incubation at a \nconcentration of 0.3 mg /mL. For permeabilization and blocking, coverslips were incubated \nwith 250 µL of a solution containing Triton X-100 and BSA for 60 min at room temperature, \nfollowed by three additional washes with cold PBS (5 min each). The actin cytoskeleton and \ncell nuclei were subsequently labeled with Alexa Fluor 488–conjugated phalloidin and DAPI, \nrespectively. Fluorescence imaging was performed using an inverted Zeiss LSM confocal \nmicroscope equipped with an Airyscan detector, located at INFABiC (National Institute of \nScience and Technology in Applied Photonics to Cell Biology), University of Campinas \n(UNICAMP). Images were acquired using a 63× oil immersion objective (NA 1.4 ). \n \nCryo-SXT and Cryo-SIM of RA W 264.7 at Diamond Light Source. Gold and carbon grids \n(Quantifoil AU G200F1 finder) were cleaned with 70% ethanol, rinsed with PBS, and placed \nin 6-well plates containing 10% FBS in water for 24 h, with the carbon side facing upward. \nRAW 264.7 cells (250,000 cells/well) were seeded onto the carbon side of the grids for 24 h \n(37 °C, 5% CO₂). SiNPs at concentrations of 0.3, 0.03, and 0.003 mg/mL were preincubated \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n22 \n \nwith 1% BSA in DMEM for 8 min. Cells were then treated with the nanoparticles for 1 h and \nwashed with PBS. Control samples were prepared without nanoparticle treatment . \nAccordingly, after 1 h, 16 h (calculated DT for UK-cultured cells), and 32 h, cells were treated \nfor 30 min with LysoTracker Blue 100 nM (λex/em 373/422 nm) and MitoTracker Green 100 \nnM ( λex/em 490/516 nm), diluted in DMEM + 10% FBS . The cells were then vitrified \nimmediately in liquid ethane, without prior washing, and kept in liquid nitrogen until the \nanalyses were performed. All protocols pertaining to this sample preparation have been  \ndescribed elsewhere 57. Firstly, the grids were mapped using the Zeiss Axio Imager M2  \ncoupled to a Linkam cryostage. Subsequently, images were collected using the cryo-SIM, at \nbeamline B24 at the Diamond Synchrotron Light Source (Didcot, UK)  to determine the \nlocation of lysosomes and nanoparticles. The samples were kept on a cryo-stage (CMS196M, \nLinkam Scientific), and a 100x magnification objective, numerical aperture 0.9, and 2 mm \nworking distance were used (Nikon). Subsequently, analyses by cryo -SXT of these same \nregions were performed on an UltraXRM-S220C microscope (Carl Zeiss X-ray Microscopy \nInc.), also on beamline B24 at the Diamond Synchrotron Light Source (Didcot, UK). This \nanalysis was performed in the energy range known as the water window (284 eV - 543 eV) \nat 520  eV. A depth of focus of 1 µm was selected, achieving a resolution of 25 nm.  The \nreconstructed tomograms were segmented using Avizo software (Thermo Fisher Scientific). \nThis software was employed for surface rendering and three-dimensional visualization of the \nintracellular distribution of nanoparticles. Segmentation was performed manually based on \nthe reconstructed tomograms.  \nCryo-SXT of RA W 264.7 at Alba Synchrotron. Gold–carbon grids were cleaned by glow \ndischarge and exposed to UV radiation for 30 min. Subsequently, the grids were placed \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n23 \n \ncarbon-side up in 60 mm culture dishes containing FBS diluted in water and incubated for 1 \nh. RAW 264.7 cells (300,000 cells per well) were seeded onto the carbon side of the grids \nand cultured for 24 h at 37 °C under 5% CO₂.  SiNPs at a concentration of 0.3 mg/mL were \npre-incubated with 1% BSA in DMEM for 8 min. Cells were then treated with the \nnanoparticles for 1 h and washed with PBS. Control samples were prepared under the same \nconditions without nanoparticle treatment. The DT of RAW 264.7 cells cultured in Barcelona \nwas previously determined to be 15 h. Based on this value, samples were collected after 1 h, \n15 h, and 30 h, in addition to untreated controls. At each time point, samples were washed \nwith PBS to remove excess medium and ensure optimal X-ray transmission. For tomographic \nalignment, 1  µL of a 100 nm gold nanoparticle suspension (3.60  × 108 particles/mL, \nEMGC100, BBI Group, Cardiff, UK) was added onto the grids. The grids were rapidly \nvitrified by plunge freezing in liquid ethane using a Leica EMCPC system. Frozen grids were \nimaged and selected using a LINKAM CMS196 stage mounted on a Zeiss Axio Scope \nfluorescence microscope. The vitrified grids were subsequently stored in liquid nitrogen until \nimaging. Cryo-SXT was performed at the Mistral beamline of the ALBA Synchrotron using \nan UltraXRM-S220C microscope (Carl Zeiss X-ray Microscopy Inc.)58,59. Tomographic data \nwere collected at 520 eV , with exposure times of 1 –2 s per projection. Projection images \nacquired at different sample orientations were computationally combined to generate three -\ndimensional (3D) reconstructions of whole-cell subcellular ultrastructure 60. A tilt series was \nacquired for each cell area using an angular step of 1° over a ±70° range, employing a Fresnel \nzone plate (FZP) with a 40 nm outermost zone width and an effective pixel size of 13 nm.  \nEach transmission projection image of the tilt series was normalized using flat -field images, \naccounting for exposure time and storage ring current. Wiener deconvolution, considering \nthe experimental impulse response of the optical system, was applied to the normalized data \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n24 \n \nto enhance image quality 61. The Napierian logarithm was then used to reconstruct the linear \nabsorption coefficient (LAC). The resulting image stacks were loaded into IMOD software62, \nand individual projections were aligned to a common tilt axis using the 100 nm gold \nnanoparticles as fiducial markers. Aligned stacks were subsequently reconstructed using \nalgebraic reconstruction techniques (ART) 63. Tomographic reconstructions were segmented \nmanually using Avizo software (Thermo Fisher Scientific) based on the reconstructed \ntomograms. \n2D Ptychography and PXCT. RAW 264.7 cells were cultured in DMEM supplemented with \n10% FBS at 37 °C in a 5% CO₂ atmosphere. Cells were seeded at a density of 10,000 cells \nper well in 6-well plates onto 100 nm-thick silicon nitride membranes, previously sterilized. \nAfter 24hrs they were then incubated with SiNPs coated with 1% BSA at a concentration of \n0.3 mg/mL for 1 h. After this internalization period, cells were washed with PBS  and \nmaintained in DMEM . Untreated cells were used as controls. After 9 h and 18 h , the cells \nwere washed with PBS and treated with 1% Triton for 3 min to lyse the plasma membrane \nwhile preserving intact nuclei. Cells were subsequently fixed with 2.5% glutar aldehyde in \n0.1 M cacodylate buffer  for 24 h, washed with  0.1 M cacodylate buffer  and progressively \ndehydrated in a graded ethanol series  (15%, 30%, 50%, 80%, 90%, 100%) , followed by \ncritical point drying with CO₂. Samples were first screened under an optical microscope to \nidentify the most suitable regions for three-dimensional imaging, which was performed at the \nCateretê beamline of the LNLS (Brazilian Synchrotron Light  Laboratory). The area to be \nimaged was selected using the Arinax on-axis optical microscope. The 2D ptychographic and \nPXCT were carried out at the energy of 6 keV . The sample was illuminated by a beam defined \nby a set of central stop (CS), Fresnel Zone Plate (FZP) (50 um diameter and outermost zone \nwidth of 50 nm) and an order sorting aperture (OSA). The sample was placed 2.4 mm from \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n25 \n \nthe FZP focus, resulting in a beam size of 10 um, which scanned the sample with steps of \n1.75 µm. The acquisition of each scanning point was 25 ms. The s cattered X-rays were \ndetected the in vacuum PiMega 540d detector (PiTec- LNLS, 55 um pixel size) positioned 7 \nm downstream from the sample. Bidimensional projections and tomographic reconstructions \nwere performed using the PtychoShelves package 64. Each projection was reconstructed into \ntwo-dimensional (2D) images  using 500 iterations of the difference map (DM) algorithm. \nSubsequently, the set of 2D reconstruction were assembled to reconstruct the tomographic \nimage, using Filter Back Projection (FBP) algorithm. Tomographic reconstructions were \nsegmented using Avizo software (Thermo Fisher Scientific).  \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n26 \n \nACKNOWLEDGMENTS \nThe authors gratefully acknowledge the financial support provided by the Fundação de \nAmparo à Pesquisa do Estado de São Paulo (FAPESP – Processes 2021/12071 -6, \n2023/00103-6, 2024/00989-7). The authors also thank the Electron Microscopy Laboratory \nof LNNano for access to the electron microscopy facilities (Proposal SEM -20233386), \nLNBio for access to the flow cytometer (Proposal 20231954) . We thank the access to \nequipment and assistance provided by the National Institute of Science and Technology on \nPhotonics Applied to Cell Biology (INFABIC) at the State University of Campinas; \nINFABIC is co -funded by Fundação de Amparo a Pesquisa do Estado  de São Paulo \n(FAPESP) (2014/50938 -8) and Conselho Nacional de Desenvolvimento Cientifico e \nTecnológico (CNPq) (465699/2014 -6) for the ac cess to the confocal fluorescence \nmicroscopy, LNLS for access to ptychography at the Caterete beamline (20250854 and \n20250988), Diamond Light Source for access to cryo-SIM and cryo-SXT at the B24 beamline \n(BI34928), and Alba Synchrotron for access to cryo -SXT at the Mistral beamline \n(2024078502). The authors further acknowledge iNext (PID 24410 and VID 43520) for \nfinancial support during the experiments performed at Diamond Light Source. Special thanks \nare due to Vitor B. Pelgati for his assistance with confocal fluorescence microscopy, to Jessica \ndo N. Faria for support with sample preparation for cryo -SXT experiments at the ALBA \nSynchrotron and to Archana Jadhav and Kamal L. Nahas for experimental support at \nDiamond Light Source. The authors also appreciate the contributions of Tiago A. Kalile and \nPedro H. Z. Guidolim to the ptychography experiments.    \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 22, 2026. ; https://doi.org/10.64898/2026.02.21.707158doi: bioRxiv preprint \n\n27 \n \nREFERENCES \n(1) Kim, B. Y. S.; Rutka, J. 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