{"paper_id":"aa052ff5-6071-43ed-8f95-cc0fcebad467","body_text":"Nano-dynamic imaging: NanoSpacer as a low-cost optical method for real-time visualisation of nanoparticle disassembly and functionalisation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nano-dynamic imaging: NanoSpacer as a low-cost optical method for real-time visualisation of nanoparticle disassembly and functionalisation Ljiljana Fruk, Andrew Baker, Anna Erdinger, Adaobi Chike, Andrew Te Water Naude, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8389168/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Understanding the dynamic behavior of nanomaterials in solution remains challenging due to reliance on specialized instrumentation, costly infrastructure, and highly trained personnel. Here we introduce the NanoSpacer, a low-cost, label-free fluidic confinement platform that enables real-time monitoring of supramolecular assemblies, termed nano-dynamic imaging , using simple darkfield microscopy. We apply this approach to investigate the assembly, disassembly and surface chemistry of indocyanine green (ICG) J-aggregate systems. Using the NanoSpacer, we characterize conventional ICG J-aggregates and show that solvent- and surfactant-induced disassembly correlates with a decrease in chemical potential. We further report the first synthesis of hybrid ICG/ ICG-azide J-aggregate nanorods and reveal pronounced structural changes within supramolecular assembly. Real-time NanoSpacer imaging directly captures the in-situ disassembly of these hybrid nanorods, exposing dynamic pathways that are obscured in ensemble-averaged measurements. Moreover, surface click reactions on individual nanorods can be monitored in real time, uncovering substantial heterogeneity in single-particle reactivity. Collectively, these results establish the NanoSpacer as a versatile platform for probing nanoscale dynamics and surface functionalization. By lowering technical and financial barriers, this approach broadens access to dynamic studies of nanoscale systems, bringing us closer to the rational design of nanostructures. Physical sciences/Nanoscience and technology/Nanoscale materials/Structural properties Physical sciences/Materials science/Nanoscale materials/Nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The ability to visualize materials at the nanoscale and understand their unique properties has driven transformative advances in nanotechnology, biotechnology and colloidal self-assembly. Central to these developments are high-resolution imaging techniques, particularly electron microscopy (EM), which have enabled important insights into nanoscale structure and organization. However, despite their resolution, techniques such as transmission and scanning transmission electron microscopy (TEM and STEM) remain constrained by high cost, labor-intensive sample preparation, specialized expertise, and the need for extensive infrastructure. These limitation are particularly pronounced when investigating dynamic processes in liquid environments, 1,2 where highly controlled imaging conditions and sample preparation do not allow dynamic imaging. Although recent advances in liquid-phase electron microscopy have enabled partial access to real-time measurements in solution, the technical complexity and associated expense continue to hinder widespread adoption. 3 Alternative imaging methods, including in situ scanning electron microscopy (SEM) 4 and high-speed atomic force microscopy (AFM) 5 , enable direct observation of chemical reactions, single-molecule biophysical processes, and the disassembly of nanomaterials. 6 However, these approaches remain inaccessible to many laboratories due to their high cost and the specialized expertise required for implementation and data interpretation. Owing to their strong light-scattering properties, metallic nanoparticles have long been visualized using dark-field microscopy, with applications dating back to the early twentieth century 7 . However, in most cases, dark-field imaging has remained largely restricted to metallic systems, where surface plasmon resonance strongly enhances scattering efficiency. These plasmonic properties have been successfully exploited to monitor nanoscale transformations and probe surface chemistry, including click reactions. 8 More recently, techniques such as interferometric scattering microscopy (iSCAT), have expanded label-free optical imaging beyond plasmonic materials, enabling visualization of protein disassembly, 9 as well as imaging of organic nanoparticles, 10,11 and viruses. 12 These optical methods nevertheless typically require sophisticated, custom-built instrumentation and have not achieved widespread adoption. Moreover, in many optical imaging approaches, nanoparticles freely diffuse in three dimensions above the coverslip surface, substantially reducing the signal-to-noise ratio due to the limited time spent within the focal plane. In contrast, nanoconfined systems restrict particle motion along the optical axis, thereby increasing dwell time in focus and significantly enhancing the signal-to-noise ratio. 13 This combination of confinement and optical imaging enables reliable observation and sizing of nanomaterials at the single-particle level and has even enabled label-free detection of biomolecules. 14 Here we present NanoSpacer, a microfluidic confinement device fabricated on a standard glass microscope slide that, when combined with dark-field microscopy, transforms a conventional optical microscope into a platform capable of real-time nanoscale imaging. Fabricated using readily available optical components, NanoSpacer is substantially cheaper than established nanoparticle imaging or tracking (NTA) methods (Table S1 ), with total costs approximately 50-, 130- and 250-fold lower than those of AFM, TEM and cryo-TEM, respectively. Using the NanoSpacer, we directly visualize indocyanine green (ICG) J-aggregate nanoparticles and monitor their solvent-triggered disassembly in situ . First reported by Jelley and Scheibe in the 1930s, 15–18 J-aggregates exhibit remarkably sharp, red-shifted absorption bands arising from coherent excitonic coupling between chromophores. This strong interplay between supramolecular structure and optical response makes J-aggregates a powerful model system for investigating self-assembly processes. 19 However, as with many other organic nanomaterials, the weak light-scattering properties of J-aggregates limit their suitability for conventional optical techniques, while their low electron density restricts contrast in traditional electron-based methods. In contrast, the NanoSpacer platform provides single-particle-level visualization of these assemblies. Moreover, we uncover a strong correlation between variations in chemical potential and the disassembly dynamics of J-aggregates. These observations are supported by correlative bulk measurements, including surface tension analysis, UV-Vis spectroscopy, cryogenic transmission electron microscopy (cryoTEM), and NMR spectroscopy. Additionally, we use the NanoSpacer platform to characterize a previously unreported hybrid supramolecular system: J-aggregate nanorods co-assembled from ICG and an azide-functionalized ICG derivative. These nanorods are characterized both optically and chemically, confirming successful incorporation of the azide functionality, while the NanoSpacer enables direct exploration of their individual disassembly dynamics, revealing behaviors that are inaccessible through conventional bulk-based techniques. While azide modification of the ICG molecule results in a new supramolecular assembly, it also introduces a chemically addressable azide group for covalent surface functionalization via click chemistry. By tracking the reaction of azide-bearing nanorods with dibenzocyclooctyne (DBCO)-conjugated nanoparticles in real time, we demonstrate single-particle visualization of click chemistry occurring directly at the surface of ICG nanorods. Collectively, these studies establish the NanoSpacer as a versatile and accessible platform for single-particle monitoring of self-assembly and nanoscale chemical transformations. By lowering both technical and financial barriers to time-resolved nanomaterials research, the NanoSpacer opens new avenues for observing and understating dynamic processes at the nanoscale. Results NANOSPACER for characterization of organic J-aggregate nanoparticles NanoSpacers were fabricated by positioning and fixing transparent coverslips at a precise distance of 1.2 µm above a standard microscope slide (Fig. 1 a, Figure S1 ). This configuration provides sufficient space for particle observation while simultaneously restricting motion along the optical axis. The resulting steric confinement increases the nanoparticle dwell time in the focal plane, thereby substantially enhancing the signal-to- noise ratio (Fig. 1 a). Filled devices were imaged on a modified benchtop light microscope operating in dark field mode, which detects scattered light. All images and videos were acquired using a USB3.0 FLIR industrial camera (BFS-U3-200S6C-C, FLIR IIS Inc.). Together, these features enable nanoscale imaging and video acquisition using only a simple, microscope setup. Data were analyzed and processed using the open-source software Fiji, the Fiji plugin Trackmate (Fiji), and the TraJClassifier plugin (version v0.8.1). Hydrodynamic size was estimated from particle trajectories using the Stokes-Einstein relation by tracking nanoparticle motion in recorded videos (Figure S2). 13 The NanoSpacer technology demonstrates broad versatility and has been successfully used previously to image and size multiple nanoparticle systems, including polystyrene beads of different sizes (44, 100 and 200 nm) (Figure S2, Video 1) and iron oxide nanoparticles (SPIONs) encapsulated withing extracellular vesicles (135nm). 20 Here, for the first time we are able to characterize sub-50 nm, weakly scattering organic ICG J-aggregate nanoparticles made by self-assembly of indocyanine green dye (Fig. 1 b and c, Video 2). 21 Using the NanoSpacer, we determine an average particle size of 38 ± 16 nm (Fig. 1 c), whereas dynamic light scattering (DLS) yielded substantially larger hydrodynamic sizes of approximately 200 nm (Figure S3). Cryo-TEM revealed particle sizes of 32 ± 8 nm with predominantly disk like or oval morphologies, (Fig. 1 d, Figure S4) in close agreement with our NanoSpacer sizing measurement. 21 Importantly, NanoSpacer not only enables size determination, but also allows estimation of nanoparticle concentration owing to its precisely defined geometry. Using this approach, we measured a particle concentration of 2.6 x 10 13 particles mL − 1 , a value that is inaccessible to standard DLS or NTA instruments due to their limited resolution. From this measurement, we estimate that each conventional spherical ICG J-aggregate nanoparticle comprises approximately 16,500 ± 4500 ICG dimers per particle (Figure S5). Accurate quantification of the number of nanoparticles administered per dose is particularly important in nanomedicine, where there is a lack of suitable methods despite repeated calls for such metrics to become standard practice. 22 , 23 NanoSpacer enables rapid sizing and quantification of nanoparticle populations without extensive sample preparation, providing essential information for quality control and process validation during the development and manufacture of nanoparticle-based therapeutics. 24 Observation of disassembly of J-aggregate nanoparticles Although indocyanine green (ICG) J-aggregate nanoparticles are well known to be sensitive to ethanol and surfactants, conditions that induce their dissociation into monomeric ICG, 25 the mechanistic details of this disassembly remain poorly understood. Previous studies, including our own, have demonstrated that ICG J-aggregates are composed of ICG dimers rather than monomeric ICG, contrary to earlier assumptions. 21 , 26 We first performed bulk measurements on the ICG J-aggregates to characterize ensemble-level responses to ethanol, as well as other solvents and surfactants (Fig. 2 a.). By monitoring the UV-Vis absorbance of the J-aggregate nanoparticle at 895 nm and the corresponding ICG dimer absorption at 780 nm, we quantified the solvent concentrations that induced nanoparticle disassembly (Fig. 2 b.). J-aggregate disassembly was observed at an ethanol concentration of approximately 18% (v/v,) as well as in aqueous solutions of methanol, glycerol, and DMSO, and in the presence of surfactants such as Triton-X-100 and SDS (Fig. 2 c). This broad sensitivity to both solvents and surfactants suggests a partitioning mechanism governed by the solvent’s chemical potential that drives the nanoparticle’s disassembly. To test this hypothesis, we compared the J-aggregate ratio, a measure of nanoparticle disassembly, to the interfacial surface tension of each surfactant solution and solvent mixture. For the simple alcohols and surfactants, disassembly followed a shared trend with surface tension, occurring within a range of 36–47 mN/m (Fig. 2 d, Figure S6). The distinct behavior of DMSO and glycerol, both of which exhibit strong hydrogen-bonding, arises because surface tension not only reflects the sum of molecular interactions in solution but also adsorption at the interface. Thus, for these compounds, the change in chemical potential of water is not correlated to a change in surface tension in these systems. However, the fact that nanoparticle disassembly still occurs suggests that the chemical potential has decreased despite the surface tension not drastically changing. The NanoSpacer platform enables direct visualization of J-aggregate disassembly. The experimental workflow is outlined in Fig. 2 e. Briefly, J-aggregates were first loaded into the NanoSpacer device, after which ethanol was introduced around the confined sample while imaging was performed beneath the coverslip. Intact nanoparticles were clearly detectable prior to ethanol addition (Fig. 2 f., Video 2), but disappeared rapidly upon exposure (Fig. 2 g., Video 3). Notably, smaller scattering entities remained observable and the particle count rate increased substantially, consistent with the formation of many rapidly diffusing molecules or oligomeric species. Owing to their high diffusion rates, however, accurate sizing of these species was not possible with the current optical configuration and would require higher numerical aperture (NA) immersion objectives. Complete disassembly from supramolecular assemblies to molecular species was further confirmed by NMR. J-aggregate nanoparticles dispersed in D 2 O (a non-disassembling solvent) exhibited spectra characteristic of aggregated states, whereas dissolution in deuterated methanol produced spectra consistent with ICG dimers, indicating exposure of previously buried chemical groups and increased solvent exchange (Figure S7). 27 , 28 Synthesis of novel J-aggregate supramolecular assemblies Historically, self-assembled supramolecular systems, including J-aggregates of ICG, have largely been composed of a single molecular species. Although sulfonate groups in other cyanine dye J-aggregate systems have been found to play an important structural “interlocking” role, analogous modifications of ICG-based assemblies remain unexplored. 29 To address this gap, we prepared hybrid J-aggregate assembly by co-assembling ICG and azide-functionalized ICG derivative at a 1:1 molar ratio (Fig. 3 a). This approach introduces functional diversity into the supramolecular framework and provides a foundation for subsequent chemical modification. The resulting hybrid assemblies exhibited a distinct λ max of 932 nm, compared to λ max of 895 nm for conventional ICG J-aggregates (Fig. 3 b). 30 This pronounced red shift indicates the formation of a new supramolecular architecture with altered molecular stacking and excitonic coupling. Morphologically, the hybrid assemblies formed elongated nanorods approximately 6.7 ± 4 µm in length and 77 ± 25 nm in width (TEM images, Fig. 3 c, Figure S8). Notably, these ICG NanoRods appear to consist of a single wall, in contrast to the double-walled structures reported for other cyanine J-aggregate systems. 29 The presence of the azide group was confirmed by FTIR spectroscopy and further validated through click reactions with dibenzocyclooctyne (DBCO)-conjugated rhodamine and sulfo-Cy5 dyes (Figure S9). In contrast to conventional J-aggregates, which are composed almost exclusively of ICG dimers (Figure S10), 21 structural analysis by mass spectrometry revealed that the hybrid ICG/ICG-azide nanorods comprise of a mixture of ICG-azide/ICG-azide and hybrid ICG/ICG-azide dimers (Figure S11). HPLC further indicated the presence of residual monomeric ICG and ICG-azide species (Figure S12). Together, these observations suggest that although the self-assembly of the hybrid system follows a pathway simillar to that of conventional ICG J-aggregates, beginning with covalent dimerization followed by higher-order aggregation, monomeric ICG and ICG-azide species also contribute to the final supramolecular architecture of the hybrid system (Figure S11). 21 , 26 The NanoSpacer enabled rapid characterization of the hybrid nanorods, and the resulting images showed strong agreement with TEM observations (Fig. 3 e). Interestingly, RGB color imaging revealed that the nanorods exhibited a yellow orange scattering color, alongside the presence of some red spherical structures (Fig. 3 f, Video 4). These features likely arise from alternative assembly configurations or structural defects formed during the self-assembly process. Although these colors differ from the green appearance of the bulk solution under ambient lighting, they closely match the scattering colors observed in the bulk (Figure S13, and Video 5). Together, these observations confirm the successful formation of hybrid ICG–ICG-azide J-aggregate nanorods with distinct optical and morphological characteristics to normal ICG J-aggregates. Having established their structural identity, we next report their disassembly dynamics under controlled solvent conditions using the NanoSpacer platform. Real time observation of novel ICG J-aggregate nanorod disassembly We next investigated the disassembly behavior of the hybrid ICG NanoRods using the experimental workflow shown in Fig. 4 a. For these studies, a Microspacer configuration was employed, enabling rapid imaging of both bulk disassembly (Fig. 4 a.i) and the behaviour of individual nanoparticles (Fig. 4 a.ii). Briefly, this device consists of two NanoSpacers stacked vertically, with the upper coverslip having a larger diameter than the lower one, thereby forming a microcapillary around the central NanoSpacer region. This geometry enables controlled solvent exchange while preserving optical confinement. Due to their larger size and structural complexity, ICG NanoRods provide an opportunity to resolve fine details of disassembly with high spatial and temporal resolution. Their absorbance ratio at (932 nm/780 nm) was also used to monitor disassembly in bulk measurements. Notably, the NanoRods exhibited only a slight reduction in ethanol-induced disassembly compared to conventional ICG J-aggregates (Fig. 4 b), with a disassembly threshold at 25.0% (v/v) ethanol compared to 18.0% (v/v) of ethanol for conventional ICG J-aggregates (Fig. 2 c.). This similarity indicates a comparable disassembly response driven by the decrease in chemical potential of the solvent. As the Microspacer is compatible with standard optical components, imaging could be performed across multiple scales, enabling direct comparison of ethanol-induced disassembly at the microscale (outer capillary region) and nanoscale (central confinement region) (Fig. 4 c., Figure S14, Videos 6 and 7). At larger length scales, disassembly proceeded rapidly and closely resembled bulk behavior (Fig. 4 c): ethanol was introduced 4.5 s after image acquisition commenced, and complete dissolution of the nanorods occurred by 5.4 s (Fig. 4 d). In contrast, NanoSpacer imaging enabled direct in situ observation of single -particle disassembly dynamics at the water-ethanol interface. Individual nanorods were observed to disassemble at different rates, with an average disassembly time of 20.4 ± 4.8 s (Figure S16.). A broad distribution of initial particle sizes was observed, together with substantial variation in disassembly rates (-1.0 ± 0.8 um/s). Importantly, no significant correlation was found between initial particle size and disassembly rate (p = 0.1102, Figure S15), indicating that internal properties of the NanoRods, such as differences in internal structure, packing order, and defect density, are likely to play a more important role in determining their stability. Importantly, we observed that the nanorods disassemble through different pathways, with two dominant modes identified. In the first mode, transverse regions of reduced molecular density emerge across the nanorods, creating localized ‘weak points’ that lead to fragmentation into discrete segments (Fig. 4 e, S16, and Videos 8, 9 and 10). In the second mode, the nanorods undergo longitudinal separation along the long axis, resulting in so- called long-axis splits (Fig. 4 f, Video 10). Interestingly, both disassembly pathways occur on comparable timescales (Figure S15). Together, these observations demonstrate that individual nanorods exhibit distinct disassembly pathways and kinetics, highlighting the intrinsic heterogeneity of supramolecular stability. By directly correlating optical signatures with single-particle behavior, the NanoSpacer bridges the gap between ensemble-averaged spectroscopy and nanoscale dynamics, providing a powerful platform for resolving transient events and structural heterogeneity that would otherwise remain obscured. Surface functionalization of azide-nanorod j-aggregates Having established real-time tracking of supramolecular disassembly and confirmed the presence and reactivity of azide groups within the hybrid ICG NanoRods, we next investigated whether the NanoSpacer platform could be extended beyond structural dynamics to directly visualize and quantify covalent chemical surface modification at the single-particle level. Click chemistry, due to its rapid kinetics and biorthogonality, provided an ideal model reaction to probe nanoscale functionalization events, and the experimental overview is illustrated in Fig. 5 a. To enable monitoring of click reaction, we first functionalized 80 nm polydopamine nanoparticles (PDA NPs) with dibenzocyclooctyne (DBCO) using NHS coupling to surface amine groups (Figure S17). DBCO-PDA NPs were initially characterized independently in the NanoSpacer to establish baseline diffusion behavior and enable size determination (Video 11). Following this assessment, DBCO-PDA NPs were mixed with azide containing NanoRods and allowed to react for 15 min prior to introduction into the NanoSpacer (Fig. 5 c). Upon reaction, distinct structural and dynamic changes were observed. These included large composite assemblies in which nanorods were densely coated with PDA NPs, as well as hybrid aggregates consisting of short nanorods (red) decorated with PDA NPs (blue). In addition, individual spherical PDA NPs were observed either attached to NanoRod surfaces or freely diffusing in solution. Successful click functionalization was further confirmed by bulk FTIR measurements performed under identical reaction conditions, which showed disappearance of the azide vibrational band (~ 2100 cm − 1 ) from the NanoRods following reaction with the DBCO-PDA NPs (Figure S17c). Notably, we observed a marked and statistically significant reduction in the mobility of PDA NPs following reaction, consistent with covalent attachment to the NanoRods. The average mean speed decreased from 737.2 nm/s (n = 5396 events) prior to reaction to 82.3 nm/s (n = 10219 events, p < 0.0001) after reaction. In parallel, NP size analysis revealed a pronounced increase in median diameter from 89 nm before reaction (n = 10221 events) to 216 nm after reaction (3310 events, p < 0.0001), indicating the formation of hybrid nanorod-NP complexes (Figure S18). Beyond bulk trends, the NanoSpacer uniquely enables spatially resolved single-particle analysis of click chemistry in real time. We directly tracked the diffusion of individual PDA NPs in proximity to azide-containing NanoRods, correlating particle velocity with the local structural environment (Fig. 5 e., Figure S18, Video 12). Freely diffusing particles exhibited high velocities (orange), while NPs bound to nanorods displayed markedly reduced mobility (blue), as quantified in Fig. 5 f. Notably, multiple NPs attached to the same nanorod displayed highly similar motion profiles, indicative of shared mechanical constrains and consistent with site-specific attachment. In contrast, freely diffusing particles exhibited more heterogeneous and uncorrelated trajectories (Figure S19). To our knowledge, these studies represents the first direct, real-time visualization and quantification of click chemistry occurring at the surface of organic supramolecular nanorods with single-particle resolution. Together, these results establish the NanoSpacer as a transformative platform that links chemical reactivity, nanoscale structure, and dynamic behavior in situ , providing a critical step toward understanding and rational engineering functional supramolecular materials with spatial and temporal precision. Conclusion We introduce a novel optical approach to observe and characterize dynamic nanomaterials in solution. Nano-dynamic imaging enables real-time visualization of individual nanoparticles using readily available optical components, lowering the barrier to nanoscale analysis. The NanoSpacer platform is readily adaptable across diverse experimental formats, from smartphone microscopy 31 to established techniques such as optical tweezers, 32 where direct visualization of nanometer-scale dynamics under force could yield new mechanistic insight. This flexibility positions NanoSpacer as a scalable framework for broad dissemination and future technological integration. Using this approach, we achieve, for the first time, straightforward quantification and characterization of ICG J-aggregates, a class of materials that has remained challenging to study due to weak optical scattering. Bulk measurements revealed solvent- and surfactant-induced disassembly correlated with decrease in chemical potential, while NanoSpacer enabled direct confirmation and visualization of these processes at the single-particle level. Together, these results establish a clear link between ensemble thermodynamics and nanoscale dynamics. We further identify a previously unreported supramolecular architecture: hybrid J-aggregate nanorods formed from ICG and ICG-azide. Given ICG’s FDA approved status, these and related hybrid materials could be useful for biomedical applications. The pronounced structural transformation induced by the azide modification of the sulfonate group underscores extraordinary sensitivity of cyanine dye packing to subtle chemical modifications and supports the role of sulfonates as key structural motifs. 29 This finding significantly expands the known structural landscape of ICG J-aggregates and defines a versatile design space in which substituents of varying polarity, hydrophobicity, and steric bulk can be leveraged to deliberately tune supramolecular geometry, optical response, and functional behavior. Notably, these nanorods comprise heterogenous mixture of ICG and ICG-azide dimers alongside pristine monomers, revealing new opportunities for chemical modification and property control that are inaccessible in conventional J-aggregate systems. Beyond structural elucidation, NanoSpacer enables direct visualization of supramolecular disassembly of nanomaterials in real time, revealing heterogenous disassembly pathways at the single-particle level. This capability is broadly applicable to self-assembled systems and nanomaterials, with implications for drug aggregation and release, 27 nanocarrier design 24 , protein self-assembly 33 , light-induced disassembly 6 , nanoscale motor design 34 , and biological processes such as viral assembly or biomolecular condensate formation. 35 By resolving particle-to-particle variations in stability, NanoSpacer further establishes a framework for studying nanoparticle fracture mechanics and identifying structural defects that govern mechanical resilience, catalytic efficiency, or functional integrity, particularly relevant for protein and enzyme self-assemblies, where structure directly dictates activity. 33 Crucially, we demonstrate that azide functionalities are retained within the supramolecular assemblies and remain chemically reactive. Using NanoSpacer, we were able to monitor and quantify surface click chemistry events at the single-particle level by correlating chemical binding to changes in diffusion behavior. This represents a unique capability to directly observe nanoscale surface chemistry as it occurs. Future work will focus on the design of scattering-enhancing contrast agents to expand the chemical scope of this approach, enabling high-resolution studies of diverse surface reactions and dynamic nanointerfaces. In summary, NanoSpacer provides a simple yet powerful platform for real-time visualization of nanoscale processes, bridging the gap between high-resolution imaging and accessible benchtop analysis. By enabling direct observation of supramolecular disassembly, morphological evolution, and surface click reactions at single-particle level, this technology advances our understanding of structure–function relationships in self-assembled materials and establishes a foundation for rapid quality control, stability assessment, and in-situ reaction monitoring. Nano-dynamic imaging thus defines a new frontier for optical observation of chemical and structural dynamics at the nanoscale. Materials and Methods NanoSpacer Assembly NanoSpacer devices consist of a transparent coverslip precisely attached to a microscope slide – creating a nano space between the layers. 36 Nano-, and MicroSpacers (NANOSPACER AS) were fabricated by contact dip-pen deposition of UV-curable photoresist on a pre-cleaned microscope slide (e.g. EPREDIA, New Erie Scientific LLC, US). After glue deposition, a 13 mm glass coverslip (VWR) is placed on top and dropped onto the substrate from approx. 1 mm height using a vacuum picker system. 36 After the glass coverslip is fixed onto the bottom substrate by exposing the photoresist with UV light according to glue’s specifications and a NanoSpacer device is obtained. To produce a Microspacer device, the procedure is repeated by dip-pen deposition of another drop of glue central on the formerly placed coverslip. Then a 22 mm diameter coverslip is placed with a vacuum picker on top and dropped accordingly. Finally, the device is again exposed to UV-light to fixate the top coverslip and the device is ready to use for imaging and filtration applications. Microscopy All images and videos were acquired using darkfield-microscopy using an Olympus bench-top industrial microscope (Model BX53M, Olympus) equipped with a FLIR industrial camera (BFS-U3-200S6C-C, FLIR IIS Inc.) and standard white LED illumination. Movies were acquired using a 50x 0.5NA objective (LMPlanFL 50x BD, Olympus). For large field of views (FOVs) standard 10x 0.25NA (MPlan N BD, Olympus) or 5x (MPlan N, Olympus) air objectives were used. Image Processing for NanoSpacer assays and MSD software analysis tools Time-series data (Tiff image stack) for the dilution/tracking assays were processed using standard Fiji (version v1.54g) software functions before further analysis. Open-source software Trackmate (Fiji) allows to count, trace nanoparticles and even measure scattering intensity levels of single particles. 37 , 38 The TraJClassifier Fiji plugin (version v0.8.1) was then used for mean square displacement (MSD) analysis to derive diffusion coefficients from single particle tracks and estimate their hydrodynamic radii according to their Brownian motion using the Stokes–Einstein relation as known from established Nanoparticle tracking analysis (NTA). Nanoparticle J-aggregate Synthesis This method was adopted from Baker et al. 2024. 21 Aqueous ICG (Acros Organics, 10321541) solution (1.0 mM, 60 mL) was sonicated for 10 min and then heated to 65 o C under stirring (500 rpm). Formation of J-aggregates (λ max of 895 nm) was monitored by a UV-Vis spectrophotometer indicating the completion of reaction at 24 h, after which the reaction mixture was centrifuged and washed three times (17000 rpm/ 31000 g at 4 o C for 30 min in a Sorvall LYNX 4000 high speed centrifuge). The pellet was redispersed in deionized water, filtered through a 0.2 µm filter, and lyophilized to obtain dark green solid ICG J-aggregate nanoparticles. Lyophilization was carried out using a Telstar LyoQuest benchtop freeze dryer (0.008 mbar, − 70°C). Chemical Composition of ICG J-aggregate Nanoparticles To determine the chemical composition LC-MS and 1H NMR were conducted on the obtained ICG J-aggregates LC-MS was performed using a Waters’ Xevo G2-S bench top QTOF mass spectrometer (Wilmslow, cheshire, UK) using electrospray ionization (ESI) source in positive mode and was performed by the Department of Chemistry Mass Spectrometry Service, University of Cambridge (UK). Samples were first dissolved in methanol to ensure the Nanoparticle structure was disassembled. 1 HNMR measurements were carried out using 400 MHz QNP Cryoprobe Spectrometer (Bruker) by the NMR service of the Department of Chemistry, University of Cambridge. Samples were dissolved in deuterated methanol to make sure the ICG J-aggregate nanoparticle structure was dissolved. ICG: 1H NMR (400 MHz, MeOD): δ 8.23 (d, J = 8.6 Hz, 1H), 8.13–7.94 (m, 2H), 7.70–7.56 (m, 1H), 7.54–7.41 (m, 1H), 6.73 − 6.49 (m, 1H), 6.39 (d, J = 13.4 Hz, 1H), 4.25 (t, J = 6.5 Hz, 1H), 2.94 (t, J = 6.8 Hz, 1H), 2.25–1.74 (m, 6H). HRMS: calculated for C43H47N2O6S2- (M + H) +: Mass predicted:752.29; Found:752.2930 ICG J-aggregates: 1H NMR (400 MHz, MeOD) δ 8.37–8.25 (m, J = 15.7, 8.0 Hz, 4H), 8.10–7.92 (m, 6H), 7.67 (dd, J = 18.5, 8.4Hz, 4H), 7.56–7.43 (m, 4H), 6.53–6.41 (m, 2H), 5.91 (d, J = 13.9 Hz, 1H), 4.30–4.21 (m, 2H), 4.14–4.06 (m, J = 7.4 Hz, 2H),4.02–3.92 (m, 2H), 2.91–2.75 (m, J = 14.8, 8.2 Hz, 5H), 2.14 (s, 5H), 2.07 (d, J = 8.2 Hz, 6H), 2.00–1.79 (m, 9H). HRMS: calculated for C86H92N4O12S42-: Mass predicted:750.28; Found:750.2880 J-aggregate NanoRod Synthesis Aqueous ICG (Acros Organics, 10321541) solution (1.0 mM, 10 mL) as well as ICG-azide (Iris Biotech GmbH) solution (1.0 mM, 10mL) were both sonicated for 10 min. After which they were then mixed in equivalent molar ratios and heated to 65 o C under stirring (500 rpm). Formation of J-aggregates nanorods (λ max of 932 nm) was monitored by a UV-Vis spectrophotometer indicating the completion of reaction at 24 h, after which the reaction mixture was centrifuged and washed three times (18000 g at 4 o C for 30 min in a Sorvall LYNX 4000 high speed centrifuge). The pellet was redispersed in deionized water and lyophilized to obtain dark green Hybrid ICG and ICG Azide NanoRods. Lyophilization was carried out using a Telstar LyoQuest benchtop freeze dryer (0.008 mbar, − 70°C). Chemical Composition of ICG J-aggregate NanoRods To determine the chemical composition LC-MS was conducted on the obtained hybrid ICG and ICG Azide J-aggregate NanoRods. LC-MS was performed using a Waters’ Xevo G2-S bench top QTOF mass spectrometer (Wilmslow, cheshire, UK) using electrospray ionization (ESI) source in positive mode and was performed by the Department of Chemistry Mass Spectrometry Service, University of Cambridge (UK). Samples were first dissolved in methanol to ensure the Nanoparticle structure was disassembled. ICG Azide: Mass spectra of ICG Azide Mass predicted from C48H56N6O4S (M + H) + : 813.41; Found: 813.4153. ICG- ICG-Azide Dimers: Mass spectra of ICG ICG-Azide NanoRods after dissolving in methanol, ICG-ICG Azide dimer predicted mass from C91H101N8O10S3- (M + 2H) +: 1564.68; Found: 1564.6995. ICG Azide-ICG Azide Dimers: ICG Azide-ICG Azide dimer predicted mass from C96H110N12O8S2 (M + H): 1624.80; Found 1624.8126. Polydopamine Nanoparticle synthesis and Modification The protocol to prepare PDA nanoparticles was adapted from Hartono et al 2024 39 . Trizma base (45 mg) was dissolved in 5 mL deionized water and then added to a solution of 15 mL DMSO and 45 mL deionized water, followed by 30-min magnetic stirring. Subsequently, dopamine (46 mg) was added to the reaction. The mixture was left overnight under stirring (500 rpm) at room temperature. Purification of nanoparticles was achieved by centrifugation at 4,000 xg (4°C) for 15 min, followed by centrifugation of the obtained supernatant at 18,000 xg for 20 min. The resulting pellet was washed with deionized water three times in deionized water. Prepared polydopamine nanoparticles were added (1mg) to a reaction mixture of NHS-DBCO (Sigma Aldrich) and left to react for two hours at 37°C. Purification of nanoparticles was achieved by centrifugation of the at 18,000 xg for 20 min. The resulting pellet was washed with deionized water three times and resuspended with deionized water. The presence of the DBCO was evaluated by chemical conjugation to azide fluorescein as well as ICG azide dyes. Characterization of Nanoparticles DLS and zeta potential measurements were performed using a Zetasizer Nano Range instrument (Malvern Analytical). UV-Vis spectra were recorded with an Agilent Cary 300 UV-Vis spectrophotometer. Fluorescence spectra and analysis were carried out using a CLARIOstar PLUS (BMG LABTECH). Cryo-TEM of J-aggregate Nanoparticles Cryo-TEM micrographs were obtained using a Thermo Scientific (FEI) Talos F200X G2 microscope operated at 200 kV. Images were recorded on a Ceta 16M CMOS camera and processed with Velox software. Specimens for investigation were prepared through vitrification by plunge freezing of the aqueous suspensions on copper grids (300 mesh) with lacey carbon film (EM Resolution). Prior to use, the grids were glow discharged using a Quorum Technologies GloQube instrument at a current of 25 mA for 60 s. Suspensions of the samples (2.5 µL of a 1 mg/mL solution) were pipetted onto the grid, blotted using filter paper, and immediately frozen by plunging in liquid ethane utilizing a fully automated and environmentally controlled blotting device, Vitrobot Mark IV. The Vitrobot chamber was set to 4 ̊C and 95% humidity. Samples after vitrification were kept under liquid nitrogen until they were inserted into a Gatan Elsa cryo holder and analyzed in the TEM at − 178°C. TEM of Hybrid ICG and ICG-Azide NanoRods TEM was also performed on a Thermo Scientific (FEI) Talos F200X G2 TEM operating at 200 kV using a Ceta 16M CMOS camera. To make the carbon film hydrophilic, TEM grids (continuous carbon film on 300 mesh Cu from EM Resolutions) were glow discharged using a Quorum Technologies GloQube instrument at a current of 25 mA for 60 s. Samples were prepared on TEM grids by applying 2.5 µL of suspension to a grid and the excess blotted off after 40 s. Bulk Surface Tension Measurements of Solvents and Surfactants Surface tension measurements were carried out using pendant drop tensiometry, using an experimental setup based on that presented by Berry et al. (2015), as well as their OpenDrop software to extract interfacial tensions. 40 Knowledge of the liquid and medium (air) density, as well as the diameter of the needle used to extrude the droplet, are all that are needed to determine the surface tension. A syringe pump (World Precision Instruments) is used to dispense fluid at a rate of 10 µL/min from a 10 mL syringe, through an 18-gauge dispending needle (Weller) with measured outer diameter 1.27 mm, used to scale the image. Liquid droplets were recorded by a CMOS camera (imea) at 5 frames per second, and the penultimate or final frame recorded of each droplet (5 droplets were analyzed for each fluid) is used for analysis in OpenDrop software, where the Laplace-Young equation is used to determine the surface tension. Microscopy of Ethanol induced Disassembly of ICG Azide NanoRods ICG-azide nanorods nanoparticles are pipetted into the NanoSpacer area by adding approx. 4 µL of aqueous ICG-azide nanorods to the microspacer edge. By capillary force the sample is pulled underneath the two discs in the central area while the microspacer area allows to ethanol addition under a controllable environment and a larger field of view. Ethanol was then added to the MicroSpacer to surround the inner confined sample with solvent. By imaging in the central NanoSpacer area close to the edge, the direct visualization of the disassembly processes (as the ethanol diffuses towards the inside) is achieved. Same procedure of (i) pre-fill (1ul) followed by (ii) surrounding the NanoSpacer with ethanol, can be applied to conventional NanoSpacers or MicroSpacers. Microscopy of Click Chemistry of ICG Azide NanoRods and Polydopamine Nanoparticles ICG-Azide nanorods (1 mg/mL) and DBCO-PDA (1 mg/mL) nanoparticles were mixed in a 1:1 volume ratio and left to react for 15 min before pipetting the mixture into the NanoSpacer and imaged. The volume of the NanoSpacer is about 1µL of volume, but 5 µL was pipetted to evenly spread out the sample around the NanoSpacer to decrease the time to reach equilibrium state and reducing induced flow inside the capillary. RGB-TIFF stacks (Bayer8RGB, 20Hz) were split into respective channels (RGB) using Fiji stack functions. Only the B (blue) channel was used for detailed FOV Trackmate and TraJ analysis, speed analysis, diffusional sizing and classification of bound and unbound PDA particles. 41 Fourier Transform Infrared spectroscopy (FT-IR) ICG-Azide NanoRods (1 mg/mL) and DBCO-PDA (1 mg/mL) nanoparticles were mixed in a 1:1 volume ratio and left to react for 15 min. After this time the mixture was flash frozen, lyophilized using a Telstar LyoQuest benchtop freeze dryer (0.008 mbar, − 50°C) and prepared for analysis. Spectra were acquired on a Nicolet™ iS50 Fourier Transform Infrared spectrometer (FTIR) Spectromer (Thermo Scientific™). Spectra of pure samples without modifications were prepared in the same way, using solutions of ICG (1mg/mL), ICG J-aggregates (1 mg/mL), and ICG-Azide NanoRods (1 mg/mL). Machine Learning Image Analysis WEKA-segmentation was implemented using the Trainable-WEKA-segmentation plugin from Fiji. 42 In brief: a single high-resolution RGB image (2736x1824) was binned by a factor of two, to obtain a smaller image with less than 1024x1024 pixels for the WEKA segmentation plugin to work accordingly to software recommendations. The image classifier was trained on this reduced image size by manually marking at least 15 regions of ICG azide nanorods and background as one group, and on the other hand by marking 15 single blue particles/ aggregates/ particles on fibrils as a second group. After the classifier was trained, it was used to obtain the probability map for a TIFF stack (200 images, less than 1024x1024 pixels) which then was rescaled to the original size and used as binary mask for the original data. The probability map was converted to a binary mask by thresholding (Huang filter) and sequential convolution with 2px to reduce image artefacts and again binary converted. After using the so obtained mask on the original data (Image calculator function, AND, Fiji) the stack was converted into a 32-bit format and then Trackmate and TraJ used for obtaining the diffusion coefficient, speed and classification of tracked particles.(Min. track length 10 spots) 41 For the diffusional sizing only directed and normal diffusing particles were considered to circumvent stationary background (e.g. Confined, Sub-diffusion) to not bias the measurement with immobile background scatterers. Statistical Analysis Statistical analyses were performed as described in the figure legend for each experiment. Statistical significance was determined by the Student t test (two-tailed), Mann Whitney Test for non-parametric, as well as linear regression using Prism 10 software (GraphPad) as indicated. A p-value below .05 was considered significant and indicated with asterisk: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.00001. Declarations Data availability The data supporting the findings of this study are available within the Article and its Supplementary Information files. Acknowledgements A.G.B., and L.F. would like to acknowledge EPSRC funding (EP/W035049/1). A.P.T. H. would like to thank the Cambridge Trust, Winton Programme, and EPSRC NanoDTC program (EP/S022953/1). J.F.M. would like to thank the BBSRC DTP PhD Programme (BB/X010899/1). C.W. would like to thank Cambridge Trust/CSC PhD Studentship funding. A.T.W.N would like to thank the W.D. Armstrong PhD Studentship. O.V, F.C and L.H.M would like to thank the Norwegian Research Council (project number 355965) for funding, as well as the UiT the Arctic University of Norway for their UiT Talent Innovation grant 2025/26 and the Aurora Outstanding program. The TEM was funded through the EPSRC Underpinning Multi-user Equipment Call (EP/P030467/1). Further we would like to thank Fruk Lab members past and present for their support throughout this project. Additionally, the authors would like to thank Anna Scheeder for their critical reading of the manuscript. Further, the technical staff at Chemical Engineering and Biotechnology (CEB) for logistical and day to day lab support. Additionally, we would like to thank Andrew Mason, Duncan Howe, and Pete Gierth of the Yusuf Hamied Department of Chemistry, University of Cambridge, NMR facility, and Asha Boodhun, Roberto Canales, and Dijana Matak-Vinkovic of the Department of Chemistry Mass Spectrometry for their continued support. The figures were designed using BioRender.com, Adobe Illustrator and PowerPoint. Conflicts of Interest O.V is inventor of NanoSpacer and filed a technology-related patent and is founder of NANOSPACER AS. A.G.B. and L.F. are co-founders of Senesys Bio, a company improving the formulation of senolytics using the NanoJAGG platform and have filed patents on the use of J-aggregates in biological systems. 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Nat Methods 19:829–832 Hartono M et al (2024) Photoacoustic polydopamine-indocyanine green (PDA-ICG) nanoprobe for detection of senescent cells. Sci Rep 14:29506 Berry JD, Neeson MJ, Dagastine RR, Chan DYC, Tabor RF (2015) Measurement of surface and interfacial tension using pendant drop tensiometry. J Colloid Interface Sci 454:226–237 Wagner T, Kroll A, Haramagatti CR, Lipinski H-G, Wiemann M (2017) Classification and Segmentation of Nanoparticle Diffusion Trajectories in Cellular Micro Environments. PLoS ONE 12:e0170165 Arganda-Carreras I et al (2017) Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics 33:2424–2426 Additional Declarations Yes there is potential Competing Interest. O.V is inventor of NanoSpacer and filed a technology-related patent and is founder of NANOSPACER AS. A.G.B. and L.F. are co-founders of Senesys Bio, a company improving the formulation of senolytics using the NanoJAGG platform and have filed patents on the use of J-aggregates in biological systems. Supplementary Files BakerSuplementaryVideolinks.docx Supplementary videos BakerNanoSpacerSupplementaryInformation.pdf Supplementary Information Onlinefloatimage1.png Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8389168\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":563095315,\"identity\":\"0a5f3e74-54b3-4ce9-8825-47d4063f9309\",\"order_by\":0,\"name\":\"Ljiljana 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08:15:18\",\"extension\":\"xml\",\"order_by\":16,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":113548,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"NCOMMS251026510structuring.xml\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/5c268ae71142a55dd30b29fd.xml\"},{\"id\":100864177,\"identity\":\"445c1449-384a-40f5-8b69-ce0c06aff274\",\"added_by\":\"auto\",\"created_at\":\"2026-01-22 08:15:18\",\"extension\":\"html\",\"order_by\":17,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":124925,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"earlyproof.html\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/2a590c2b2516e1a444c5ea32.html\"},{\"id\":100949797,\"identity\":\"185028d9-ea13-44ec-a8d3-e088d492e5d0\",\"added_by\":\"auto\",\"created_at\":\"2026-01-23 07:05:44\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":54224,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of Indocyanine Green J-aggregate Nanoparticles using the NanoSpacer. \\u003c/strong\\u003ea) Schematic of NanoSpacer technique, all samples are imaged using a dark-field microscope, and the NanoSpacer provides a 1.2 mm nanoconfinement that enhances scattering and signal of nanoparticles. b) Synthesis scheme of indocyanine green J-aggregates ICG is heated at 65 °C for 24 hours and then ultracentrifuged. c) Image from NanoSpacer videos following analysis with Trackmate (Fiji) as well as TraJClassifier Fiji plugin (38 ± 16 nm). d) Cryo-TEM images on the indocyanine green J-aggregates (32±8 nm).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/32c60abffe94df4832e55f74.png\"},{\"id\":100949492,\"identity\":\"d7cf32ef-82c6-41b1-929e-ceab7204e5d7\",\"added_by\":\"auto\",\"created_at\":\"2026-01-23 07:03:15\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":95823,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupramolecular disassembly of indocyanine green J-aggregates.\\u003c/strong\\u003e a) Schematic of ethanol mediated disassembly. \\u0026nbsp;b) UV-VIS of both Indocyanine Green J-aggregate and the dimer of ICG, as well as the disassembly plot of the ratio of the assembled nanostructure (895 nm) was taken in reference to the monomer (780 nm) in increasing ethanol amounts. c)Plot of the J-aggregate ratio in the presence of different surfactants and solvents to quantify disassembly. d) J-aggregate ratio plotted against both measured and literature values of Interfacial Tension (mN/m). e) Cartoon of NanoSpacer experiment where 1. ICG J-aggregates are plated in the chamber in water, 2. then once equilibrated the water is replaced with ethanol, and 3. the results are imaged using the NanoSpacer f) Images of ICG J-aggregate nanoparticles and quantification of the size before ethanol induced disassembly. g) NanoSpacer images of ICG J-aggregate nanoparticles and quantification of the size after ethanol disassembly.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/ff84ac0c36e2f3f53b003c2e.png\"},{\"id\":100950179,\"identity\":\"5dba2c5a-7986-49b4-8f8d-18d2cc5c33d6\",\"added_by\":\"auto\",\"created_at\":\"2026-01-23 07:07:07\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":81856,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of novel ICG and ICG-azide hybrid J-aggregate NanoRod nanostructures. \\u003c/strong\\u003ea) Schematic of synthesis and reaction conditions to create the hybrid nanomaterial ICG and ICG-azide nanorods. b) UV-VIS absorbance of the starting compounds (ICG and ICG-azide 780 nm, the normal ICG J-aggregates (895 nm), and finally the ICG and ICG-azide hybrid nanorods (932 nm). c) TEM image of the ICG and ICG azide nanorods. d) NanoSpacer image in grey scale of NanoRods. e) NanoSpacer image of the ICG and ICG-azide Nanorods using RGB color readout. Panels i) and ii) show enlarged sections to highlight NanoRod morphology, and alternative structures.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/c10a550f70c9c8d06d343756.png\"},{\"id\":100864172,\"identity\":\"0df04002-364e-4998-88d0-dc53487b09c8\",\"added_by\":\"auto\",\"created_at\":\"2026-01-22 08:15:18\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":81842,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMonitoring of the disassembly of novel ICG and ICG-azide hybrid J-aggregate NanoRods. \\u003c/strong\\u003ea) Schematic of ethanol mediated disassembly, and NanoSpacer set-up. b) UV-VIS monitoring of the disassembly process, the ratio of the assembled nanostructure (932 nm) was taken in reference to the monomer (780 nm). c) NanoSpacer images of ethanol disassembly over time, and different pathways, i) horizontal breaks, ii) long axis splits. d) Disassembly process of the nanorods viewed from in bulk using MicroSpacer. e.) Horizontal break observed overtime for a single nanorod. f) Vertical split observed overtime for a single nanorod.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/cf716bf856a6e84d9837541c.png\"},{\"id\":100864186,\"identity\":\"8a2674d5-d3d6-4bc2-9047-c407a3d418d4\",\"added_by\":\"auto\",\"created_at\":\"2026-01-22 08:15:19\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":87116,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMonitoring the click-chemistry attachment of nanoparticles onto ICG and ICG-azide hybrid nanostructures\\u003c/strong\\u003e. a) Cartoon of experiment where PDA nanoparticles are mixed with ICG-ICG-azide nanorods and then placed into NanoSpacer. b) Initial images of both PDA and ICG Azide NanoRods NPs to serve as base value for analysis. c) Images of NanoRods, after mixing with PDA NPs. d) Change in measured speed of PDA NPs before and after reacting with ICG azide NanoRods. e) Kinetics of PDA particles freely diffusing and attached to the ICG Azide NanoRods. f) Plot of the speed (nm/s) versus the Track mean quality (Fiji parameter related to particle scattering intensity) of the different types of particles in the image.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/9a0619e6a0873ced6f071cce.png\"},{\"id\":101207644,\"identity\":\"1e5c01d0-3bc4-4108-8f97-f31a53083397\",\"added_by\":\"auto\",\"created_at\":\"2026-01-27 10:06:06\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1714901,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/bd8cdc4c-c4e9-4945-bb56-63424a2a96b1.pdf\"},{\"id\":100864166,\"identity\":\"d718ed9c-85ce-4068-83e5-a665e0eb37f7\",\"added_by\":\"auto\",\"created_at\":\"2026-01-22 08:15:18\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":15801,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary videos\",\"description\":\"\",\"filename\":\"BakerSuplementaryVideolinks.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/ac658114f5394a856c687a03.docx\"},{\"id\":100864176,\"identity\":\"39af033f-93dd-45ba-89cd-139983e1a89a\",\"added_by\":\"auto\",\"created_at\":\"2026-01-22 08:15:18\",\"extension\":\"pdf\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":6147724,\"visible\":true,\"origin\":\"\",\"legend\":\"Supplementary Information\",\"description\":\"\",\"filename\":\"BakerNanoSpacerSupplementaryInformation.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/7c519c32fee072140fcdb78e.pdf\"},{\"id\":100949935,\"identity\":\"68f282c0-7115-4db3-b29b-bda25d767054\",\"added_by\":\"auto\",\"created_at\":\"2026-01-23 07:06:23\",\"extension\":\"png\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":118979,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Onlinefloatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8389168/v1/d762a01bd01a020efaf7d6cf.png\"}],\"financialInterests\":\"\\u003cb\\u003eYes\\u003c/b\\u003e there is potential Competing Interest.\\nO.V is inventor of NanoSpacer and filed a technology-related patent and is founder of NANOSPACER AS. A.G.B. and L.F. are co-founders of Senesys Bio, a company improving the formulation of senolytics using the NanoJAGG platform and have filed patents on the use of J-aggregates in biological systems.\",\"formattedTitle\":\"Nano-dynamic imaging: NanoSpacer as a low-cost optical method for real-time visualisation of nanoparticle disassembly and functionalisation\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe ability to visualize materials at the nanoscale and understand their unique properties has driven transformative advances in nanotechnology, biotechnology and colloidal self-assembly. Central to these developments are high-resolution imaging techniques, particularly electron microscopy (EM), which have enabled important insights into nanoscale structure and organization. However, despite their resolution, techniques such as transmission and scanning transmission electron microscopy (TEM and STEM) remain constrained by high cost, labor-intensive sample preparation, specialized expertise, and the need for extensive infrastructure. These limitation are particularly pronounced when investigating dynamic processes in liquid environments,\\u003csup\\u003e1,2\\u003c/sup\\u003e where highly controlled imaging conditions and sample preparation do not allow dynamic imaging. Although recent advances in liquid-phase electron microscopy have enabled partial access to real-time measurements in solution, the technical complexity and associated expense continue to hinder widespread adoption.\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eAlternative imaging methods, including \\u003cem\\u003ein situ\\u003c/em\\u003e scanning electron microscopy (SEM)\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e and high-speed atomic force microscopy (AFM)\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e, enable direct observation of chemical reactions, single-molecule biophysical processes, and the disassembly of nanomaterials.\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e However, these approaches remain inaccessible to many laboratories due to their high cost and the specialized expertise required for implementation and data interpretation.\\u003c/p\\u003e \\u003cp\\u003eOwing to their strong light-scattering properties, metallic nanoparticles have long been visualized using dark-field microscopy, with applications dating back to the early twentieth century\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u003c/sup\\u003e. However, in most cases, dark-field imaging has remained largely restricted to metallic systems, where surface plasmon resonance strongly enhances scattering efficiency. These plasmonic properties have been successfully exploited to monitor nanoscale transformations and probe surface chemistry, including click reactions.\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e More recently, techniques such as interferometric scattering microscopy (iSCAT), have expanded label-free optical imaging beyond plasmonic materials, enabling visualization of protein disassembly,\\u003csup\\u003e9\\u003c/sup\\u003e as well as imaging of organic nanoparticles,\\u003csup\\u003e10,11\\u003c/sup\\u003e and viruses.\\u003csup\\u003e\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eThese optical methods nevertheless typically require sophisticated, custom-built instrumentation and have not achieved widespread adoption. Moreover, in many optical imaging approaches, nanoparticles freely diffuse in three dimensions above the coverslip surface, substantially reducing the signal-to-noise ratio due to the limited time spent within the focal plane. In contrast, nanoconfined systems restrict particle motion along the optical axis, thereby increasing dwell time in focus and significantly enhancing the signal-to-noise ratio.\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e This combination of confinement and optical imaging enables reliable observation and sizing of nanomaterials at the single-particle level and has even enabled label-free detection of biomolecules.\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eHere we present NanoSpacer, a microfluidic confinement device fabricated on a standard glass microscope slide that, when combined with dark-field microscopy, transforms a conventional optical microscope into a platform capable of real-time nanoscale imaging. Fabricated using readily available optical components, NanoSpacer is substantially cheaper than established nanoparticle imaging or tracking (NTA) methods (Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e), with total costs approximately 50-, 130- and 250-fold lower than those of AFM, TEM and cryo-TEM, respectively.\\u003c/p\\u003e \\u003cp\\u003eUsing the NanoSpacer, we directly visualize indocyanine green (ICG) J-aggregate nanoparticles and monitor their solvent-triggered disassembly \\u003cem\\u003ein situ\\u003c/em\\u003e. First reported by Jelley and Scheibe in the 1930s,\\u003csup\\u003e15\\u0026ndash;18\\u003c/sup\\u003e J-aggregates exhibit remarkably sharp, red-shifted absorption bands arising from coherent excitonic coupling between chromophores. This strong interplay between supramolecular structure and optical response makes J-aggregates a powerful model system for investigating self-assembly processes.\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e However, as with many other organic nanomaterials, the weak light-scattering properties of J-aggregates limit their suitability for conventional optical techniques, while their low electron density restricts contrast in traditional electron-based methods. In contrast, the NanoSpacer platform provides single-particle-level visualization of these assemblies. Moreover, we uncover a strong correlation between variations in chemical potential and the disassembly dynamics of J-aggregates. These observations are supported by correlative bulk measurements, including surface tension analysis, UV-Vis spectroscopy, cryogenic transmission electron microscopy (cryoTEM), and NMR spectroscopy.\\u003c/p\\u003e \\u003cp\\u003eAdditionally, we use the NanoSpacer platform to characterize a previously unreported hybrid supramolecular system: J-aggregate nanorods co-assembled from ICG and an azide-functionalized ICG derivative. These nanorods are characterized both optically and chemically, confirming successful incorporation of the azide functionality, while the NanoSpacer enables direct exploration of their individual disassembly dynamics, revealing behaviors that are inaccessible through conventional bulk-based techniques.\\u003c/p\\u003e \\u003cp\\u003eWhile azide modification of the ICG molecule results in a new supramolecular assembly, it also introduces a chemically addressable azide group for covalent surface functionalization \\u003cem\\u003evia\\u003c/em\\u003e click chemistry. By tracking the reaction of azide-bearing nanorods with dibenzocyclooctyne (DBCO)-conjugated nanoparticles in real time, we demonstrate single-particle visualization of click chemistry occurring directly at the surface of ICG nanorods.\\u003c/p\\u003e \\u003cp\\u003eCollectively, these studies establish the NanoSpacer as a versatile and accessible platform for single-particle monitoring of self-assembly and nanoscale chemical transformations. By lowering both technical and financial barriers to time-resolved nanomaterials research, the NanoSpacer opens new avenues for observing and understating dynamic processes at the nanoscale.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eNANOSPACER for characterization of organic J-aggregate nanoparticles\\u003c/h2\\u003e \\u003cp\\u003eNanoSpacers were fabricated by positioning and fixing transparent coverslips at a precise distance of 1.2 \\u0026micro;m above a standard microscope slide (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea, Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). This configuration provides sufficient space for particle observation while simultaneously restricting motion along the optical axis. The resulting steric confinement increases the nanoparticle dwell time in the focal plane, thereby substantially enhancing the signal-to- noise ratio (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea).\\u003c/p\\u003e \\u003cp\\u003eFilled devices were imaged on a modified benchtop light microscope operating in dark field mode, which detects scattered light. All images and videos were acquired using a USB3.0 FLIR industrial camera (BFS-U3-200S6C-C, FLIR IIS Inc.). Together, these features enable nanoscale imaging and video acquisition using only a simple, microscope setup. Data were analyzed and processed using the open-source software Fiji, the Fiji plugin Trackmate (Fiji), and the TraJClassifier plugin (version v0.8.1). Hydrodynamic size was estimated from particle trajectories using the Stokes-Einstein relation by tracking nanoparticle motion in recorded videos (Figure S2).\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe NanoSpacer technology demonstrates broad versatility and has been successfully used previously to image and size multiple nanoparticle systems, including polystyrene beads of different sizes (44, 100 and 200 nm) (Figure S2, Video 1) and iron oxide nanoparticles (SPIONs) encapsulated withing extracellular vesicles (135nm).\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eHere, for the first time we are able to characterize sub-50 nm, weakly scattering organic ICG J-aggregate nanoparticles made by self-assembly of indocyanine green dye (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb and c, Video 2).\\u003csup\\u003e21\\u003c/sup\\u003e Using the NanoSpacer, we determine an average particle size of 38\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;16 nm (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec), whereas dynamic light scattering (DLS) yielded substantially larger hydrodynamic sizes of approximately 200 nm (Figure S3). Cryo-TEM revealed particle sizes of 32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;8 nm with predominantly disk like or oval morphologies, (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed, Figure S4) in close agreement with our NanoSpacer sizing measurement.\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eImportantly, NanoSpacer not only enables size determination, but also allows estimation of nanoparticle concentration owing to its precisely defined geometry. Using this approach, we measured a particle concentration of 2.6 x 10\\u003csup\\u003e13\\u003c/sup\\u003e particles mL\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, a value that is inaccessible to standard DLS or NTA instruments due to their limited resolution. From this measurement, we estimate that each conventional spherical ICG J-aggregate nanoparticle comprises approximately 16,500\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4500 ICG dimers per particle (Figure S5).\\u003c/p\\u003e \\u003cp\\u003eAccurate quantification of the number of nanoparticles administered per dose is particularly important in nanomedicine, where there is a lack of suitable methods despite repeated calls for such metrics to become standard practice.\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e NanoSpacer enables rapid sizing and quantification of nanoparticle populations without extensive sample preparation, providing essential information for quality control and process validation during the development and manufacture of nanoparticle-based therapeutics.\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eObservation of disassembly of J-aggregate nanoparticles\\u003c/h3\\u003e\\n\\u003cp\\u003eAlthough indocyanine green (ICG) J-aggregate nanoparticles are well known to be sensitive to ethanol and surfactants, conditions that induce their dissociation into monomeric ICG,\\u003csup\\u003e25\\u003c/sup\\u003e the mechanistic details of this disassembly remain poorly understood. Previous studies, including our own, have demonstrated that ICG J-aggregates are composed of ICG dimers rather than monomeric ICG, contrary to earlier assumptions.\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eWe first performed bulk measurements on the ICG J-aggregates to characterize ensemble-level responses to ethanol, as well as other solvents and surfactants (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea.). By monitoring the UV-Vis absorbance of the J-aggregate nanoparticle at 895 nm and the corresponding ICG dimer absorption at 780 nm, we quantified the solvent concentrations that induced nanoparticle disassembly (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb.). J-aggregate disassembly was observed at an ethanol concentration of approximately 18% (v/v,) as well as in aqueous solutions of methanol, glycerol, and DMSO, and in the presence of surfactants such as Triton-X-100 and SDS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec). This broad sensitivity to both solvents and surfactants suggests a partitioning mechanism governed by the solvent\\u0026rsquo;s chemical potential that drives the nanoparticle\\u0026rsquo;s disassembly. To test this hypothesis, we compared the J-aggregate ratio, a measure of nanoparticle disassembly, to the interfacial surface tension of each surfactant solution and solvent mixture. For the simple alcohols and surfactants, disassembly followed a shared trend with surface tension, occurring within a range of 36\\u0026ndash;47 mN/m (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed, Figure S6).\\u003c/p\\u003e \\u003cp\\u003eThe distinct behavior of DMSO and glycerol, both of which exhibit strong hydrogen-bonding, arises because surface tension not only reflects the sum of molecular interactions in solution but also adsorption at the interface. Thus, for these compounds, the change in chemical potential of water is not correlated to a change in surface tension in these systems. However, the fact that nanoparticle disassembly still occurs suggests that the chemical potential has decreased despite the surface tension not drastically changing.\\u003c/p\\u003e \\u003cp\\u003eThe NanoSpacer platform enables direct visualization of J-aggregate disassembly. The experimental workflow is outlined in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee. Briefly, J-aggregates were first loaded into the NanoSpacer device, after which ethanol was introduced around the confined sample while imaging was performed beneath the coverslip. Intact nanoparticles were clearly detectable prior to ethanol addition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef., Video 2), but disappeared rapidly upon exposure (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg., Video 3). Notably, smaller scattering entities remained observable and the particle count rate increased substantially, consistent with the formation of many rapidly diffusing molecules or oligomeric species. Owing to their high diffusion rates, however, accurate sizing of these species was not possible with the current optical configuration and would require higher numerical aperture (NA) immersion objectives.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eComplete disassembly from supramolecular assemblies to molecular species was further confirmed by NMR. J-aggregate nanoparticles dispersed in D\\u003csub\\u003e2\\u003c/sub\\u003eO (a non-disassembling solvent) exhibited spectra characteristic of aggregated states, whereas dissolution in deuterated methanol produced spectra consistent with ICG dimers, indicating exposure of previously buried chemical groups and increased solvent exchange (Figure S7).\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e\\n\\u003ch3\\u003eSynthesis of novel J-aggregate supramolecular assemblies\\u003c/h3\\u003e\\n\\u003cp\\u003eHistorically, self-assembled supramolecular systems, including J-aggregates of ICG, have largely been composed of a single molecular species. Although sulfonate groups in other cyanine dye J-aggregate systems have been found to play an important structural \\u0026ldquo;interlocking\\u0026rdquo; role, analogous modifications of ICG-based assemblies remain unexplored.\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e To address this gap, we prepared hybrid J-aggregate assembly by co-assembling ICG and azide-functionalized ICG derivative at a 1:1 molar ratio (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea). This approach introduces functional diversity into the supramolecular framework and provides a foundation for subsequent chemical modification.\\u003c/p\\u003e \\u003cp\\u003eThe resulting hybrid assemblies exhibited a distinct λ \\u003csub\\u003emax\\u003c/sub\\u003e of 932 nm, compared to λ\\u003csub\\u003emax\\u003c/sub\\u003e of 895 nm for conventional ICG J-aggregates (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb).\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e This pronounced red shift indicates the formation of a new supramolecular architecture with altered molecular stacking and excitonic coupling. Morphologically, the hybrid assemblies formed elongated nanorods approximately 6.7\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4 \\u0026micro;m in length and 77\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;25 nm in width (TEM images, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec, Figure S8). Notably, these ICG NanoRods appear to consist of a single wall, in contrast to the double-walled structures reported for other cyanine J-aggregate systems.\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe presence of the azide group was confirmed by FTIR spectroscopy and further validated through click reactions with dibenzocyclooctyne (DBCO)-conjugated rhodamine and sulfo-Cy5 dyes (Figure S9). In contrast to conventional J-aggregates, which are composed almost exclusively of ICG dimers (Figure S10),\\u003csup\\u003e21\\u003c/sup\\u003e structural analysis by mass spectrometry revealed that the hybrid ICG/ICG-azide nanorods comprise of a mixture of ICG-azide/ICG-azide and hybrid ICG/ICG-azide dimers (Figure S11). HPLC further indicated the presence of residual monomeric ICG and ICG-azide species (Figure S12). Together, these observations suggest that although the self-assembly of the hybrid system follows a pathway simillar to that of conventional ICG J-aggregates, beginning with covalent dimerization followed by higher-order aggregation, monomeric ICG and ICG-azide species also contribute to the final supramolecular architecture of the hybrid system (Figure S11).\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe NanoSpacer enabled rapid characterization of the hybrid nanorods, and the resulting images showed strong agreement with TEM observations (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee). Interestingly, RGB color imaging revealed that the nanorods exhibited a yellow orange scattering color, alongside the presence of some red spherical structures (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ef, Video 4). These features likely arise from alternative assembly configurations or structural defects formed during the self-assembly process. Although these colors differ from the green appearance of the bulk solution under ambient lighting, they closely match the scattering colors observed in the bulk (Figure S13, and Video 5).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTogether, these observations confirm the successful formation of hybrid ICG\\u0026ndash;ICG-azide J-aggregate nanorods with distinct optical and morphological characteristics to normal ICG J-aggregates. Having established their structural identity, we next report their disassembly dynamics under controlled solvent conditions using the NanoSpacer platform.\\u003c/p\\u003e\\n\\u003ch3\\u003eReal time observation of novel ICG J-aggregate nanorod disassembly\\u003c/h3\\u003e\\n\\u003cp\\u003eWe next investigated the disassembly behavior of the hybrid ICG NanoRods using the experimental workflow shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea. For these studies, a Microspacer configuration was employed, enabling rapid imaging of both bulk disassembly (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea.i) and the behaviour of individual nanoparticles (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea.ii). Briefly, this device consists of two NanoSpacers stacked vertically, with the upper coverslip having a larger diameter than the lower one, thereby forming a microcapillary around the central NanoSpacer region. This geometry enables controlled solvent exchange while preserving optical confinement.\\u003c/p\\u003e \\u003cp\\u003eDue to their larger size and structural complexity, ICG NanoRods provide an opportunity to resolve fine details of disassembly with high spatial and temporal resolution. Their absorbance ratio at (932 nm/780 nm) was also used to monitor disassembly in bulk measurements. Notably, the NanoRods exhibited only a slight reduction in ethanol-induced disassembly compared to conventional ICG J-aggregates (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb), with a disassembly threshold at 25.0% (v/v) ethanol compared to 18.0% (v/v) of ethanol for conventional ICG J-aggregates (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec.). This similarity indicates a comparable disassembly response driven by the decrease in chemical potential of the solvent.\\u003c/p\\u003e \\u003cp\\u003eAs the Microspacer is compatible with standard optical components, imaging could be performed across multiple scales, enabling direct comparison of ethanol-induced disassembly at the microscale (outer capillary region) and nanoscale (central confinement region) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec., Figure S14, Videos 6 and 7). At larger length scales, disassembly proceeded rapidly and closely resembled bulk behavior (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec): ethanol was introduced 4.5 s after image acquisition commenced, and complete dissolution of the nanorods occurred by 5.4 s (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed).\\u003c/p\\u003e \\u003cp\\u003eIn contrast, NanoSpacer imaging enabled direct \\u003cem\\u003ein situ\\u003c/em\\u003e observation of single -particle disassembly dynamics at the water-ethanol interface. Individual nanorods were observed to disassemble at different rates, with an average disassembly time of 20.4\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.8 s (Figure S16.). A broad distribution of initial particle sizes was observed, together with substantial variation in disassembly rates (-1.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.8 um/s). Importantly, no significant correlation was found between initial particle size and disassembly rate (p\\u0026thinsp;=\\u0026thinsp;0.1102, Figure S15), indicating that internal properties of the NanoRods, such as differences in internal structure, packing order, and defect density, are likely to play a more important role in determining their stability.\\u003c/p\\u003e \\u003cp\\u003eImportantly, we observed that the nanorods disassemble through different pathways, with two dominant modes identified. In the first mode, transverse regions of reduced molecular density emerge across the nanorods, creating localized \\u0026lsquo;weak points\\u0026rsquo; that lead to fragmentation into discrete segments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee, S16, and Videos 8, 9 and 10). In the second mode, the nanorods undergo longitudinal separation along the long axis, resulting in so- called long-axis splits (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef, Video 10). Interestingly, both disassembly pathways occur on comparable timescales (Figure S15).\\u003c/p\\u003e \\u003cp\\u003eTogether, these observations demonstrate that individual nanorods exhibit distinct disassembly pathways and kinetics, highlighting the intrinsic heterogeneity of supramolecular stability. By directly correlating optical signatures with single-particle behavior, the NanoSpacer bridges the gap between ensemble-averaged spectroscopy and nanoscale dynamics, providing a powerful platform for resolving transient events and structural heterogeneity that would otherwise remain obscured.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eSurface functionalization of azide-nanorod j-aggregates\\u003c/h3\\u003e\\n\\u003cp\\u003eHaving established real-time tracking of supramolecular disassembly and confirmed the presence and reactivity of azide groups within the hybrid ICG NanoRods, we next investigated whether the NanoSpacer platform could be extended beyond structural dynamics to directly visualize and quantify covalent chemical surface modification at the single-particle level. Click chemistry, due to its rapid kinetics and biorthogonality, provided an ideal model reaction to probe nanoscale functionalization events, and the experimental overview is illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea.\\u003c/p\\u003e \\u003cp\\u003eTo enable monitoring of click reaction, we first functionalized 80 nm polydopamine nanoparticles (PDA NPs) with dibenzocyclooctyne (DBCO) using NHS coupling to surface amine groups (Figure S17). DBCO-PDA NPs were initially characterized independently in the NanoSpacer to establish baseline diffusion behavior and enable size determination (Video 11). Following this assessment, DBCO-PDA NPs were mixed with azide containing NanoRods and allowed to react for 15 min prior to introduction into the NanoSpacer (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec).\\u003c/p\\u003e \\u003cp\\u003eUpon reaction, distinct structural and dynamic changes were observed. These included large composite assemblies in which nanorods were densely coated with PDA NPs, as well as hybrid aggregates consisting of short nanorods (red) decorated with PDA NPs (blue). In addition, individual spherical PDA NPs were observed either attached to NanoRod surfaces or freely diffusing in solution. Successful click functionalization was further confirmed by bulk FTIR measurements performed under identical reaction conditions, which showed disappearance of the azide vibrational band (~\\u0026thinsp;2100 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e) from the NanoRods following reaction with the DBCO-PDA NPs (Figure S17c).\\u003c/p\\u003e \\u003cp\\u003eNotably, we observed a marked and statistically significant reduction in the mobility of PDA NPs following reaction, consistent with covalent attachment to the NanoRods. The average mean speed decreased from 737.2 nm/s (n\\u0026thinsp;=\\u0026thinsp;5396 events) prior to reaction to 82.3 nm/s (n\\u0026thinsp;=\\u0026thinsp;10219 events, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001) after reaction. In parallel, NP size analysis revealed a pronounced increase in median diameter from 89 nm before reaction (n\\u0026thinsp;=\\u0026thinsp;10221 events) to 216 nm after reaction (3310 events, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), indicating the formation of hybrid nanorod-NP complexes (Figure S18).\\u003c/p\\u003e \\u003cp\\u003eBeyond bulk trends, the NanoSpacer uniquely enables spatially resolved single-particle analysis of click chemistry in real time. We directly tracked the diffusion of individual PDA NPs in proximity to azide-containing NanoRods, correlating particle velocity with the local structural environment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ee., Figure S18, Video 12). Freely diffusing particles exhibited high velocities (orange), while NPs bound to nanorods displayed markedly reduced mobility (blue), as quantified in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ef. Notably, multiple NPs attached to the same nanorod displayed highly similar motion profiles, indicative of shared mechanical constrains and consistent with site-specific attachment. In contrast, freely diffusing particles exhibited more heterogeneous and uncorrelated trajectories (Figure S19).\\u003c/p\\u003e \\u003cp\\u003eTo our knowledge, these studies represents the first direct, real-time visualization and quantification of click chemistry occurring at the surface of organic supramolecular nanorods with single-particle resolution. Together, these results establish the NanoSpacer as a transformative platform that links chemical reactivity, nanoscale structure, and dynamic behavior \\u003cem\\u003ein situ\\u003c/em\\u003e, providing a critical step toward understanding and rational engineering functional supramolecular materials with spatial and temporal precision.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eWe introduce a novel optical approach to observe and characterize dynamic nanomaterials in solution. Nano-dynamic imaging enables real-time visualization of individual nanoparticles using readily available optical components, lowering the barrier to nanoscale analysis. The NanoSpacer platform is readily adaptable across diverse experimental formats, from smartphone microscopy\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e to established techniques such as optical tweezers,\\u003csup\\u003e32\\u003c/sup\\u003e where direct visualization of nanometer-scale dynamics under force could yield new mechanistic insight. This flexibility positions NanoSpacer as a scalable framework for broad dissemination and future technological integration.\\u003c/p\\u003e \\u003cp\\u003eUsing this approach, we achieve, for the first time, straightforward quantification and characterization of ICG J-aggregates, a class of materials that has remained challenging to study due to weak optical scattering. Bulk measurements revealed solvent- and surfactant-induced disassembly correlated with decrease in chemical potential, while NanoSpacer enabled direct confirmation and visualization of these processes at the single-particle level. Together, these results establish a clear link between ensemble thermodynamics and nanoscale dynamics.\\u003c/p\\u003e \\u003cp\\u003eWe further identify a previously unreported supramolecular architecture: hybrid J-aggregate nanorods formed from ICG and ICG-azide. Given ICG\\u0026rsquo;s FDA approved status, these and related hybrid materials could be useful for biomedical applications. The pronounced structural transformation induced by the azide modification of the sulfonate group underscores extraordinary sensitivity of cyanine dye packing to subtle chemical modifications and supports the role of sulfonates as key structural motifs.\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e This finding significantly expands the known structural landscape of ICG J-aggregates and defines a versatile design space in which substituents of varying polarity, hydrophobicity, and steric bulk can be leveraged to deliberately tune supramolecular geometry, optical response, and functional behavior. Notably, these nanorods comprise heterogenous mixture of ICG and ICG-azide dimers alongside pristine monomers, revealing new opportunities for chemical modification and property control that are inaccessible in conventional J-aggregate systems.\\u003c/p\\u003e \\u003cp\\u003eBeyond structural elucidation, NanoSpacer enables direct visualization of supramolecular disassembly of nanomaterials in real time, revealing heterogenous disassembly pathways at the single-particle level. This capability is broadly applicable to self-assembled systems and nanomaterials, with implications for drug aggregation and release,\\u003csup\\u003e27\\u003c/sup\\u003e nanocarrier design\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e, protein self-assembly\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, light-induced disassembly\\u003csup\\u003e\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e, nanoscale motor design\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e, and biological processes such as viral assembly or biomolecular condensate formation.\\u003csup\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/sup\\u003e By resolving particle-to-particle variations in stability, NanoSpacer further establishes a framework for studying nanoparticle fracture mechanics and identifying structural defects that govern mechanical resilience, catalytic efficiency, or functional integrity, particularly relevant for protein and enzyme self-assemblies, where structure directly dictates activity.\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003eCrucially, we demonstrate that azide functionalities are retained within the supramolecular assemblies and remain chemically reactive. Using NanoSpacer, we were able to monitor and quantify surface click chemistry events at the single-particle level by correlating chemical binding to changes in diffusion behavior. This represents a unique capability to directly observe nanoscale surface chemistry as it occurs. Future work will focus on the design of scattering-enhancing contrast agents to expand the chemical scope of this approach, enabling high-resolution studies of diverse surface reactions and dynamic nanointerfaces.\\u003c/p\\u003e \\u003cp\\u003eIn summary, NanoSpacer provides a simple yet powerful platform for real-time visualization of nanoscale processes, bridging the gap between high-resolution imaging and accessible benchtop analysis. By enabling direct observation of supramolecular disassembly, morphological evolution, and surface click reactions at single-particle level, this technology advances our understanding of structure\\u0026ndash;function relationships in self-assembled materials and establishes a foundation for rapid quality control, stability assessment, and \\u003cem\\u003ein-situ\\u003c/em\\u003e reaction monitoring. Nano-dynamic imaging thus defines a new frontier for optical observation of chemical and structural dynamics at the nanoscale.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eNanoSpacer Assembly\\u003c/h2\\u003e \\u003cp\\u003eNanoSpacer devices consist of a transparent coverslip precisely attached to a microscope slide \\u0026ndash; creating a nano space between the layers.\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e Nano-, and MicroSpacers (NANOSPACER AS) were fabricated by contact dip-pen deposition of UV-curable photoresist on a pre-cleaned microscope slide (e.g. EPREDIA, New Erie Scientific LLC, US). After glue deposition, a 13 mm glass coverslip (VWR) is placed on top and dropped onto the substrate from approx. 1 mm height using a vacuum picker system.\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e After the glass coverslip is fixed onto the bottom substrate by exposing the photoresist with UV light according to glue\\u0026rsquo;s specifications and a NanoSpacer device is obtained. To produce a Microspacer device, the procedure is repeated by dip-pen deposition of another drop of glue central on the formerly placed coverslip. Then a 22 mm diameter coverslip is placed with a vacuum picker on top and dropped accordingly. Finally, the device is again exposed to UV-light to fixate the top coverslip and the device is ready to use for imaging and filtration applications.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMicroscopy\\u003c/h2\\u003e \\u003cp\\u003eAll images and videos were acquired using darkfield-microscopy using an Olympus bench-top industrial microscope (Model BX53M, Olympus) equipped with a FLIR industrial camera (BFS-U3-200S6C-C, FLIR IIS Inc.) and standard white LED illumination. Movies were acquired using a 50x 0.5NA objective (LMPlanFL 50x BD, Olympus). For large field of views (FOVs) standard 10x 0.25NA (MPlan N BD, Olympus) or 5x (MPlan N, Olympus) air objectives were used.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImage Processing for NanoSpacer assays and MSD software analysis tools\\u003c/h2\\u003e \\u003cp\\u003eTime-series data (Tiff image stack) for the dilution/tracking assays were processed using standard Fiji (version v1.54g) software functions before further analysis. Open-source software Trackmate (Fiji) allows to count, trace nanoparticles and even measure scattering intensity levels of single particles.\\u003csup\\u003e\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e The TraJClassifier Fiji plugin (version v0.8.1) was then used for mean square displacement (MSD) analysis to derive diffusion coefficients from single particle tracks and estimate their hydrodynamic radii according to their Brownian motion using the Stokes\\u0026ndash;Einstein relation as known from established Nanoparticle tracking analysis (NTA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eNanoparticle J-aggregate Synthesis\\u003c/h2\\u003e \\u003cp\\u003eThis method was adopted from Baker et al. 2024.\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e Aqueous ICG (Acros Organics, 10321541) solution (1.0 mM, 60 mL) was sonicated for 10 min and then heated to 65\\u003csup\\u003eo\\u003c/sup\\u003eC under stirring (500 rpm). Formation of J-aggregates (λ\\u003csub\\u003emax\\u003c/sub\\u003e of 895 nm) was monitored by a UV-Vis spectrophotometer indicating the completion of reaction at 24 h, after which the reaction mixture was centrifuged and washed three times (17000 rpm/ 31000 g at 4\\u003csup\\u003eo\\u003c/sup\\u003eC for 30 min in a Sorvall LYNX 4000 high speed centrifuge). The pellet was redispersed in deionized water, filtered through a 0.2 \\u0026micro;m filter, and lyophilized to obtain dark green solid ICG J-aggregate nanoparticles. Lyophilization was carried out using a Telstar LyoQuest benchtop freeze dryer (0.008 mbar, \\u0026minus;\\u0026thinsp;70\\u0026deg;C).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eChemical Composition of ICG J-aggregate Nanoparticles\\u003c/h2\\u003e \\u003cp\\u003eTo determine the chemical composition LC-MS and 1H NMR were conducted on the obtained ICG J-aggregates LC-MS was performed using a Waters\\u0026rsquo; Xevo G2-S bench top QTOF mass spectrometer (Wilmslow, cheshire, UK) using electrospray ionization (ESI) source in positive mode and was performed by the Department of Chemistry Mass Spectrometry Service, University of Cambridge (UK). Samples were first dissolved in methanol to ensure the Nanoparticle structure was disassembled.\\u003c/p\\u003e \\u003cp\\u003e \\u003csup\\u003e1\\u003c/sup\\u003eHNMR measurements were carried out using 400 MHz QNP Cryoprobe Spectrometer (Bruker) by the NMR service of the Department of Chemistry, University of Cambridge. Samples were dissolved in deuterated methanol to make sure the ICG J-aggregate nanoparticle structure was dissolved.\\u003c/p\\u003e \\u003cp\\u003eICG: 1H NMR (400 MHz, MeOD): δ 8.23 (d, J\\u0026thinsp;=\\u0026thinsp;8.6 Hz, 1H), 8.13\\u0026ndash;7.94 (m, 2H), 7.70\\u0026ndash;7.56 (m, 1H), 7.54\\u0026ndash;7.41 (m, 1H), 6.73 \\u0026minus;\\u0026thinsp;6.49 (m, 1H), 6.39 (d, J\\u0026thinsp;=\\u0026thinsp;13.4 Hz, 1H), 4.25 (t, J\\u0026thinsp;=\\u0026thinsp;6.5 Hz, 1H), 2.94 (t, J\\u0026thinsp;=\\u0026thinsp;6.8 Hz, 1H), 2.25\\u0026ndash;1.74 (m, 6H). HRMS: calculated for C43H47N2O6S2- (M\\u0026thinsp;+\\u0026thinsp;H) +: Mass predicted:752.29; Found:752.2930\\u003c/p\\u003e \\u003cp\\u003eICG J-aggregates: 1H NMR (400 MHz, MeOD) δ 8.37\\u0026ndash;8.25 (m, J\\u0026thinsp;=\\u0026thinsp;15.7, 8.0 Hz, 4H), 8.10\\u0026ndash;7.92 (m, 6H), 7.67 (dd, J\\u0026thinsp;=\\u0026thinsp;18.5, 8.4Hz, 4H), 7.56\\u0026ndash;7.43 (m, 4H), 6.53\\u0026ndash;6.41 (m, 2H), 5.91 (d, J\\u0026thinsp;=\\u0026thinsp;13.9 Hz, 1H), 4.30\\u0026ndash;4.21 (m, 2H), 4.14\\u0026ndash;4.06 (m, J\\u0026thinsp;=\\u0026thinsp;7.4 Hz, 2H),4.02\\u0026ndash;3.92 (m, 2H), 2.91\\u0026ndash;2.75 (m, J\\u0026thinsp;=\\u0026thinsp;14.8, 8.2 Hz, 5H), 2.14 (s, 5H), 2.07 (d, J\\u0026thinsp;=\\u0026thinsp;8.2 Hz, 6H), 2.00\\u0026ndash;1.79 (m, 9H). HRMS: calculated for C86H92N4O12S42-: Mass predicted:750.28; Found:750.2880\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eJ-aggregate NanoRod Synthesis\\u003c/h2\\u003e \\u003cp\\u003eAqueous ICG (Acros Organics, 10321541) solution (1.0 mM, 10 mL) as well as ICG-azide (Iris Biotech GmbH) solution (1.0 mM, 10mL) were both sonicated for 10 min. After which they were then mixed in equivalent molar ratios and heated to 65\\u003csup\\u003eo\\u003c/sup\\u003eC under stirring (500 rpm). Formation of J-aggregates nanorods (λ\\u003csub\\u003emax\\u003c/sub\\u003e of 932 nm) was monitored by a UV-Vis spectrophotometer indicating the completion of reaction at 24 h, after which the reaction mixture was centrifuged and washed three times (18000 g at 4\\u003csup\\u003eo\\u003c/sup\\u003eC for 30 min in a Sorvall LYNX 4000 high speed centrifuge). The pellet was redispersed in deionized water and lyophilized to obtain dark green Hybrid ICG and ICG Azide NanoRods. Lyophilization was carried out using a Telstar LyoQuest benchtop freeze dryer (0.008 mbar, \\u0026minus;\\u0026thinsp;70\\u0026deg;C).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eChemical Composition of ICG J-aggregate NanoRods\\u003c/h2\\u003e \\u003cp\\u003eTo determine the chemical composition LC-MS was conducted on the obtained hybrid ICG and ICG Azide J-aggregate NanoRods. LC-MS was performed using a Waters\\u0026rsquo; Xevo G2-S bench top QTOF mass spectrometer (Wilmslow, cheshire, UK) using electrospray ionization (ESI) source in positive mode and was performed by the Department of Chemistry Mass Spectrometry Service, University of Cambridge (UK). Samples were first dissolved in methanol to ensure the Nanoparticle structure was disassembled.\\u003c/p\\u003e \\u003cp\\u003eICG Azide: Mass spectra of ICG Azide Mass predicted from C48H56N6O4S (M\\u0026thinsp;+\\u0026thinsp;H) + : 813.41; Found: 813.4153. ICG- ICG-Azide Dimers: Mass spectra of ICG ICG-Azide NanoRods after dissolving in methanol, ICG-ICG Azide dimer predicted mass from C91H101N8O10S3- (M\\u0026thinsp;+\\u0026thinsp;2H) +: 1564.68; Found: 1564.6995. ICG Azide-ICG Azide Dimers: ICG Azide-ICG Azide dimer predicted mass from C96H110N12O8S2 (M\\u0026thinsp;+\\u0026thinsp;H): 1624.80; Found 1624.8126.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePolydopamine Nanoparticle synthesis and Modification\\u003c/h2\\u003e \\u003cp\\u003eThe protocol to prepare PDA nanoparticles was adapted from Hartono et al 2024\\u003csup\\u003e39\\u003c/sup\\u003e. Trizma base (45 mg) was dissolved in 5 mL deionized water and then added to a solution of 15 mL DMSO and 45 mL deionized water, followed by 30-min magnetic stirring. Subsequently, dopamine (46 mg) was added to the reaction. The mixture was left overnight under stirring (500 rpm) at room temperature. Purification of nanoparticles was achieved by centrifugation at 4,000 xg (4\\u0026deg;C) for 15 min, followed by centrifugation of the obtained supernatant at 18,000 xg for 20 min. The resulting pellet was washed with deionized water three times in deionized water.\\u003c/p\\u003e \\u003cp\\u003ePrepared polydopamine nanoparticles were added (1mg) to a reaction mixture of NHS-DBCO (Sigma Aldrich) and left to react for two hours at 37\\u0026deg;C. Purification of nanoparticles was achieved by centrifugation of the at 18,000 xg for 20 min. The resulting pellet was washed with deionized water three times and resuspended with deionized water. The presence of the DBCO was evaluated by chemical conjugation to azide fluorescein as well as ICG azide dyes.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCharacterization of Nanoparticles\\u003c/h2\\u003e \\u003cp\\u003eDLS and zeta potential measurements were performed using a Zetasizer Nano Range instrument (Malvern Analytical). UV-Vis spectra were recorded with an Agilent Cary 300 UV-Vis spectrophotometer. Fluorescence spectra and analysis were carried out using a CLARIOstar\\u003csup\\u003ePLUS\\u003c/sup\\u003e (BMG LABTECH).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCryo-TEM of J-aggregate Nanoparticles\\u003c/h2\\u003e \\u003cp\\u003eCryo-TEM micrographs were obtained using a Thermo Scientific (FEI) Talos F200X G2 microscope operated at 200 kV. Images were recorded on a Ceta 16M CMOS camera and processed with Velox software. Specimens for investigation were prepared through vitrification by plunge freezing of the aqueous suspensions on copper grids (300 mesh) with lacey carbon film (EM Resolution). Prior to use, the grids were glow discharged using a Quorum Technologies GloQube instrument at a current of 25 mA for 60 s. Suspensions of the samples (2.5 \\u0026micro;L of a 1 mg/mL solution) were pipetted onto the grid, blotted using filter paper, and immediately frozen by plunging in liquid ethane utilizing a fully automated and environmentally controlled blotting device, Vitrobot Mark IV. The Vitrobot chamber was set to 4 ̊C and 95% humidity. Samples after vitrification were kept under liquid nitrogen until they were inserted into a Gatan Elsa cryo holder and analyzed in the TEM at \\u0026minus;\\u0026thinsp;178\\u0026deg;C.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTEM of Hybrid ICG and ICG-Azide NanoRods\\u003c/h2\\u003e \\u003cp\\u003eTEM was also performed on a Thermo Scientific (FEI) Talos F200X G2 TEM operating at 200 kV using a Ceta 16M CMOS camera. To make the carbon film hydrophilic, TEM grids (continuous carbon film on 300 mesh Cu from EM Resolutions) were glow discharged using a Quorum Technologies GloQube instrument at a current of 25 mA for 60 s. Samples were prepared on TEM grids by applying 2.5 \\u0026micro;L of suspension to a grid and the excess blotted off after 40 s.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBulk Surface Tension Measurements of Solvents and Surfactants\\u003c/h2\\u003e \\u003cp\\u003eSurface tension measurements were carried out using pendant drop tensiometry, using an experimental setup based on that presented by Berry \\u003cem\\u003eet al.\\u003c/em\\u003e (2015), as well as their OpenDrop software to extract interfacial tensions.\\u003csup\\u003e\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e\\u003c/sup\\u003e Knowledge of the liquid and medium (air) density, as well as the diameter of the needle used to extrude the droplet, are all that are needed to determine the surface tension. A syringe pump (World Precision Instruments) is used to dispense fluid at a rate of 10 \\u0026micro;L/min from a 10 mL syringe, through an 18-gauge dispending needle (Weller) with measured outer diameter 1.27 mm, used to scale the image. Liquid droplets were recorded by a CMOS camera (imea) at 5 frames per second, and the penultimate or final frame recorded of each droplet (5 droplets were analyzed for each fluid) is used for analysis in OpenDrop software, where the Laplace-Young equation is used to determine the surface tension.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMicroscopy of Ethanol induced Disassembly of ICG Azide NanoRods\\u003c/h2\\u003e \\u003cp\\u003eICG-azide nanorods nanoparticles are pipetted into the NanoSpacer area by adding approx. 4 \\u0026micro;L of aqueous ICG-azide nanorods to the microspacer edge. By capillary force the sample is pulled underneath the two discs in the central area while the microspacer area allows to ethanol addition under a controllable environment and a larger field of view. Ethanol was then added to the MicroSpacer to surround the inner confined sample with solvent. By imaging in the central NanoSpacer area close to the edge, the direct visualization of the disassembly processes (as the ethanol diffuses towards the inside) is achieved. Same procedure of (i) pre-fill (1ul) followed by (ii) surrounding the NanoSpacer with ethanol, can be applied to conventional NanoSpacers or MicroSpacers.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eMicroscopy of Click Chemistry of ICG Azide NanoRods and Polydopamine Nanoparticles\\u003c/h2\\u003e \\u003cp\\u003eICG-Azide nanorods (1 mg/mL) and DBCO-PDA (1 mg/mL) nanoparticles were mixed in a 1:1 volume ratio and left to react for 15 min before pipetting the mixture into the NanoSpacer and imaged. The volume of the NanoSpacer is about 1\\u0026micro;L of volume, but 5 \\u0026micro;L was pipetted to evenly spread out the sample around the NanoSpacer to decrease the time to reach equilibrium state and reducing induced flow inside the capillary. RGB-TIFF stacks (Bayer8RGB, 20Hz) were split into respective channels (RGB) using Fiji stack functions. Only the B (blue) channel was used for detailed FOV Trackmate and TraJ analysis, speed analysis, diffusional sizing and classification of bound and unbound PDA particles.\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFourier Transform Infrared spectroscopy (FT-IR)\\u003c/h2\\u003e \\u003cp\\u003eICG-Azide NanoRods (1 mg/mL) and DBCO-PDA (1 mg/mL) nanoparticles were mixed in a 1:1 volume ratio and left to react for 15 min. After this time the mixture was flash frozen, lyophilized using a Telstar LyoQuest benchtop freeze dryer (0.008 mbar, \\u0026minus;\\u0026thinsp;50\\u0026deg;C) and prepared for analysis. Spectra were acquired on a Nicolet\\u0026trade; iS50 Fourier Transform Infrared spectrometer (FTIR) Spectromer (Thermo Scientific\\u0026trade;). Spectra of pure samples without modifications were prepared in the same way, using solutions of ICG (1mg/mL), ICG J-aggregates (1 mg/mL), and ICG-Azide NanoRods (1 mg/mL).\\u003c/p\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eMachine Learning Image Analysis\\u003c/h2\\u003e \\u003cp\\u003eWEKA-segmentation was implemented using the Trainable-WEKA-segmentation plugin from Fiji.\\u003csup\\u003e\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e In brief: a single high-resolution RGB image (2736x1824) was binned by a factor of two, to obtain a smaller image with less than 1024x1024 pixels for the WEKA segmentation plugin to work accordingly to software recommendations. The image classifier was trained on this reduced image size by manually marking at least 15 regions of ICG azide nanorods and background as one group, and on the other hand by marking 15 single blue particles/ aggregates/ particles on fibrils as a second group. After the classifier was trained, it was used to obtain the probability map for a TIFF stack (200 images, less than 1024x1024 pixels) which then was rescaled to the original size and used as binary mask for the original data. The probability map was converted to a binary mask by thresholding (Huang filter) and sequential convolution with 2px to reduce image artefacts and again binary converted. After using the so obtained mask on the original data (Image calculator function, AND, Fiji) the stack was converted into a 32-bit format and then Trackmate and TraJ used for obtaining the diffusion coefficient, speed and classification of tracked particles.(Min. track length 10 spots)\\u003csup\\u003e\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u003c/sup\\u003e For the diffusional sizing only directed and normal diffusing particles were considered to circumvent stationary background (e.g. Confined, Sub-diffusion) to not bias the measurement with immobile background scatterers.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eStatistical analyses were performed as described in the figure legend for each experiment.\\u003c/p\\u003e \\u003cp\\u003eStatistical significance was determined by the Student t test (two-tailed), Mann Whitney Test for non-parametric, as well as linear regression using Prism 10 software (GraphPad) as indicated. A p-value below .05 was considered significant and indicated with asterisk: *p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, **p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01, ***p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, and ****p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.00001.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData \\u0026nbsp;availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data supporting the findings of this study are available within the Article and its Supplementary Information files.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA.G.B., and L.F. \\u0026nbsp;would like to acknowledge EPSRC funding (EP/W035049/1). A.P.T. H. would like to thank the Cambridge Trust, Winton Programme, and EPSRC NanoDTC program (EP/S022953/1). J.F.M. would like to thank the BBSRC DTP PhD Programme (BB/X010899/1). C.W. would like to thank Cambridge Trust/CSC PhD Studentship funding. A.T.W.N would like to thank the W.D. Armstrong PhD Studentship. O.V, F.C and L.H.M would like to thank the Norwegian Research Council (project number 355965) for funding, as well as the UiT the Arctic University of Norway for their UiT Talent Innovation grant 2025/26 and the Aurora Outstanding program. The TEM was funded through the EPSRC Underpinning Multi-user Equipment Call (EP/P030467/1).\\u003c/p\\u003e\\n\\u003cp\\u003eFurther we would like to thank Fruk Lab members past and present for their support throughout this project. Additionally, the authors would like to thank Anna Scheeder for their critical reading of the manuscript. Further, the technical staff at Chemical Engineering and Biotechnology (CEB) for logistical and day to day lab support. Additionally, we would like to thank Andrew Mason, Duncan Howe, and Pete Gierth of the Yusuf Hamied Department of Chemistry, University of Cambridge, NMR facility, and Asha Boodhun, Roberto Canales, and Dijana Matak-Vinkovic of the Department of Chemistry Mass Spectrometry for their continued support. The figures were designed using BioRender.com, Adobe Illustrator and PowerPoint.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eO.V is inventor of NanoSpacer and filed a technology-related patent and is founder of NANOSPACER AS. A.G.B. and L.F. are co-founders of Senesys Bio, a company improving the formulation of senolytics using the NanoJAGG platform and have filed patents on the use of J-aggregates in biological systems.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eModena MM, R\\u0026uuml;hle B, Burg TP, Wuttke S (2019) Nanoparticle Characterization: What to Measure? Adv Mater 31:1901556\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChakraborty S, Misra SK, Lead JR, Lynch I (2025) Capturing rapid nanomaterial transformations with cross-platform operando characterization. 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Nature 138:1009\\u0026ndash;1010\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJELLEY EE, Molecular (1937) Nematic and Crystal States of I: I-Diethyl\\u0026ndash;Cyanine Chloride. Nature 139:631\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eScheibe G, Kandler L, Ecker H (1937) Polymerisation und polymere Adsorption als Ursache neuartiger Absorptionsbanden von organischen Farbstoffen. Naturwissenschaften 25:75\\u0026ndash;75\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBricks JL, Slominskii YL, Panas ID, Demchenko AP (2018) Fluorescent J-aggregates of cyanine dyes: Basic research and applications review. Methods Appl Fluoresc 6:012001\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eFeginn Berle B et al (2025) Multimodal Imaging of Brain Metastasis-Derived Extracellular Vesicles Using Superparamagnetic Iron Oxide Nanoparticle Labeling. 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PLoS ONE 12:e0170165\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eArganda-Carreras I et al (2017) Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics 33:2424\\u0026ndash;2426\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8389168/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8389168/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eUnderstanding the dynamic behavior of nanomaterials in solution remains challenging due to reliance on specialized instrumentation, costly infrastructure, and highly trained personnel. Here we introduce the NanoSpacer, a low-cost, label-free fluidic confinement platform that enables real-time monitoring of supramolecular assemblies, termed \\u003cem\\u003enano-dynamic imaging\\u003c/em\\u003e, using simple darkfield microscopy. We apply this approach to investigate the assembly, disassembly and surface chemistry of indocyanine green (ICG) J-aggregate systems. Using the NanoSpacer, we characterize conventional ICG J-aggregates and show that solvent- and surfactant-induced disassembly correlates with a decrease in chemical potential. We further report the first synthesis of hybrid ICG/ ICG-azide J-aggregate nanorods and reveal pronounced structural changes within supramolecular assembly. Real-time NanoSpacer imaging directly captures the \\u003cem\\u003ein-situ\\u003c/em\\u003e disassembly of these hybrid nanorods, exposing dynamic pathways that are obscured in ensemble-averaged measurements. Moreover, surface click reactions on individual nanorods can be monitored in real time, uncovering substantial heterogeneity in single-particle reactivity. Collectively, these results establish the NanoSpacer as a versatile platform for probing nanoscale dynamics and surface functionalization. By lowering technical and financial barriers, this approach broadens access to dynamic studies of nanoscale systems, bringing us closer to the rational design of nanostructures.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\",\"manuscriptTitle\":\"Nano-dynamic imaging: NanoSpacer as a low-cost optical method for real-time visualisation of nanoparticle disassembly and functionalisation\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-01-22 08:15:12\",\"doi\":\"10.21203/rs.3.rs-8389168/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-communications\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"NCOMMS\",\"sideBox\":\"Learn more about [Nature Communications](http://www.nature.com/ncomms/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://mts-ncomms.nature.com/\",\"title\":\"Nature Communications\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Nature Communications\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"b1303264-369b-4cb2-b106-51baf5fbcf31\",\"owner\":[],\"postedDate\":\"January 22nd, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[{\"id\":59967600,\"name\":\"Physical sciences/Nanoscience and technology/Nanoscale materials/Structural properties\"},{\"id\":59967601,\"name\":\"Physical sciences/Materials science/Nanoscale materials/Nanoparticles\"}],\"tags\":[],\"updatedAt\":\"2026-03-10T09:57:10+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-01-22 08:15:12\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8389168\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8389168\",\"identity\":\"rs-8389168\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}