Real-time label-free imaging of living crystallization-driven self-assembly

Guo, Yujie; Xia, Tianlai; Walter, Vivien; Xie, Yujie; Rho, Julia Y.; Xiao, Laihui; O’Reilly, Rachel K.; Wallace, Mark I.

doi:10.1038/s41467-025-57776-9

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Published: 18 March 2025

Real-time label-free imaging of living crystallization-driven self-assembly

Nature Communications volume 16, Article number: 2672 (2025) Cite this article

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Abstract

Living crystallization-driven self-assembly (CDSA) of semicrystalline block copolymers is a powerful method for the bottom-up construction of uniform polymer microstructures with complex hierarchies. Improving our ability to engineer such complex particles demands a better understanding of how to precisely control the self-assembly process. Here, we apply interferometric scattering (iSCAT) microscopy to observe the real-time growth of individual poly(ε-caprolactone)-based fibers and platelets. This label-free method enables us to map the role of key reaction parameters on platelet growth rate, size, and morphology. Furthermore, iSCAT provides a contrast mechanism for studying multi-annulus platelets formed via the sequential addition of different unimers, offering insights into the spatial distribution of polymer compositions within a single platelet.

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Introduction

Monodisperse nanomaterials with precise morphologies are a vital component of many recent nanotechnologies^1,2. One promising synthetic route to prepare particles with defined non-spherical architectures is the crystallization-driven self-assembly (CDSA) of block copolymers^{3,4,5,6,7,8,9}. Living CDSA provides access to complex hierarchical nanostructures with impressive morphological and dimensional control; and this complexity enables wide-ranging applications in drug delivery^10,11,12,13, colloid stabilization^14,15, catalysis^16,17,18, optoelectronics^19,20, and information storage^21,22,23. The livingness of CDSA is achieved by initiating growth from uniform seed crystallites, which provide the nucleation that enables self-assembly to start from a common point (Fig. 1A)^{6,8,10,24,25,26}. Realizing the full potential of CDSA for nanostructure synthesis requires a precise understanding of how CDSA particles grow and how this self-assembly process can be controlled.

**Fig. 1: Schematic illustration of living CDSA monitored by iSCAT.**

Most previous reports monitoring the self-assembly of block copolymers derive kinetics from ensemble properties: For example, light scattering can be employed to report on the average size of CDSA micelles^27,28,29. However, these ensemble methods are insensitive to heterogeneity in the growth of individual nanoparticles and provide no direct information on particle morphology. Given the uniform complex hierarchical structures desired by CDSA, this ensemble averaging is a significant drawback in understanding and quantifying growth mechanics. Thus, a key hurdle to properly understanding CDSA lies in providing methods capable of revealing how assembly is controlled on a particle-by-particle basis.

Transmission electron microscopy (TEM) is arguably the most common method used to study the size evolution of individual polymer assemblies^{7,28,30,31,32}. However, conventional TEM provides only snapshots of the distribution of particle sizes during growth, lacking the capability to track the transformations of individual nanoparticles. Exciting recent advances in liquid cell TEM have opened a route to monitor the liquid phase reaction in situ with millisecond to second temporal resolution, albeit at the expense of significant instrumental complexity^33,34,35. Solution-phase atomic force microscopy (AFM) has also been applied to study the mechanism of interfacial seeded-growth of 1D micellar nanoparticles. However, tip-induced nanofiber fragmentation has been reported as a drawback by creating new active interfaces and affecting kinetics³⁶. Fluorescence microscopy offers a perhaps less invasive alternative, but at the expense of spatial resolution: For example, confocal laser scanning microscopy (CLSM) has been used to visualize 1D fiber growth at 100 ms temporal resolution with 120 nm lateral spatial resolution²⁸. Super-resolution fluorescence microscopy techniques can improve these limits with, for instance, 76 nm precision and 15 ms temporal resolution of the growth of 1D fiber³⁷. Although providing easy access to nanoparticle kinetics, fluorescence microscopy’s significant drawback is the requirement for extrinsic labels; given the relative size of a fluorophore to component monomers, it is perhaps unsurprising that impacts on the CDSA kinetics are reported³⁰. Label-free techniques such as phase contrast or differential interference contrast microscopy can circumvent this issue but are typically limited in detection sensitivity^38,39,40,41.

To address the need for an in situ non-invasive method of characterizing living CDSA kinetics with high spatio-temporal single-particle resolution, here we apply interferometric scattering (iSCAT) microscopy to track CDSA in individual polymer particles. iSCAT is an intrinsically label-free technique capable of single-molecule resolution^42,43,44. To date, iSCAT has mainly been used to study biological systems, including the mass measurement of individual proteins, the formation of lipid membranes, and biological diffusion using single-particle tracking of metal nanoparticles^45,46,47. iSCAT has also been effectively utilized to monitor protein aggregation and actin polymerization processes⁴⁵. Given the similarities shared between actin polymerization and 1D CDSA fiber formation, we reasoned that iSCAT might also be productively applied to the study of CDSA.

iSCAT relies on the interference between light scattered from an object of interest supported on a glass coverslip, and reference light reflected back from the same glass-sample interface (Fig. 1B). Compared to conventional interferometry, which is highly sensitive to environmental factors such as vibrations and temperature changes and often requires sophisticated vibration isolation, the common path configuration of iSCAT allows the reference and scattered light to travel along the same optical path. This allows a simple and robust approach to measure changes in light scattering from small objects in a less controlled environment. For a diffraction-limited object, iSCAT signals appear as an Airy disc of concentric dark and light rings caused by the interference between the reflected and scattered signals. iSCAT offers lateral and axial resolutions of around 200–300 nm and 10–100 nm, respectively, and can further exploit optical super-resolution techniques to improve spatial precision^43,48. In addition, iSCAT can operate at high temporal resolutions (up to 1 μs)⁴⁹ and monitor processes over long observation times without photobleaching.

CDSA kinetics are highly dependent on core chemistry²⁸, and to date, the majority of studies examining CDSA-mediated growth have focused on 1D fibers, with typically slow (hours to days) formation kinetics^30,36,50. However, limited attention has been given to the study of 2D platelet kinetics, which are characterized by relatively rapid self-assembly processes, distinct morphology, and ease of modification. Here, we chose poly(ε-caprolactone) (PCL)-based polymers as the core-forming block for the preparation of well-defined 1D fibers and 2D platelets. Compared with other commonly used core materials, the biocompatibility and biodegradability of PCL make it an attractive starting point for a range of materials for biomedical applications^11,51. In addition, the crystallinity of the core-forming PCL block plays an essential role in determining the morphology and kinetics of CDSA particles: High crystallinity (i.e., high degree of polymerization) contributes to fast crystallization rates, commonly accompanied by self-nucleation and agglomeration; whereas core-forming blocks with lower crystallinity typically exhibit slower crystallization rates, resulting in irregular assembly shapes and a prolonged assembly process^52,53. Here, PCL₄₅ was selected, as it provides a good balance between fast kinetics and tight morphological control. Using iSCAT, we mapped the kinetics and morphology of fiber and platelet growth from poly(ε-caprolactone)-b-poly(N,N-dimethylacrylamide) (PCL-b-PDMA) block copolymer and mixtures of PCL and PCL-b-PDMA, respectively.

Results

Real-time monitoring of living CDSA

To first summarize our overall method: Uniform seeds were prepared from polydisperse fibers of PCL₄₅-b-PDMA₃₄₈ formed in ethanol (Supplementary Fig. 3A), followed by sonication to produce short fibers with consistent size and length (mean length 24.6 ± 4.9 nm, Supplementary Figs. 3B,C). Seeds were then attached to the coverslip via spin-coating, with a silicone spacer placed on top to form a reaction chamber. This sample was then mounted on a custom-built microscope, ready for imaging (Supplementary Fig. 4)⁵⁴. Unimer solutions were then introduced into the reaction chamber, followed by immediate iSCAT imaging to monitor the CDSA process (Fig. 1). An in-depth description of the experimental protocol and our instrumentation is provided in the “Methods” section and the Supplementary Information.

1D fibers

We initially applied iSCAT microscopy to monitor seeded-growth of 1D fibers: 50 μL of 2.51 nM (0.1 μg mL⁻¹) PCL₄₅-b-PDMA₃₄₈ seed solution was spin-coated onto the coverslip. PCL₇₃-b-PDMA₂₀₄ unimer was added into methanol to reach a final concentration of 0.06 μM (1.67 μg mL⁻¹) before being introduced into the reaction chamber and imaged using iSCAT.

Figure 2 A depicts a representative time series of iSCAT images collected during the formation of 1D fibers (637 nm, 4 μW μm⁻², 400 μs exposure time, 3 s⁻¹ per frame time-lapse). Upon unimer addition, uniform fiber elongation can be monitored as the reaction proceeds (the size of PCL₄₅-b-PDMA₃₄₈ seeds is below the detection limit of our setup, thus they are not observable; Supplementary Movie 1). Following image segmentation (see Supplementary Methods), the length evolution of individual fibers can be determined as shown in Fig. 2B. Applying a previously established kinetic model for 1D fiber growth³⁰, rate constants were extracted by fitting to the trajectories of length vs. time (Supplementary Methods section, Supplementary Fig. 5, 5.1 × 10⁻³ s⁻¹). Comparing these data to previous work on poly(ferrocenyldimethylsilane)-b-polydimethylsiloxane (PFS₆₃-b-PDMS₅₁₃) 1D fibers (10–30 mg mL⁻¹ unimer and 0.1 mg mL⁻¹ seed), we observe faster kinetics in our PCL-based system (5.1 × 10⁻³ s⁻¹ compared to 1.8 × 10⁻⁴ to 2.13 × 10⁻⁴ s⁻¹) despite lower unimer and seed concentration.

**Fig. 2: iSCAT monitoring of living CDSA.**

2D platelets

Following our initial imaging of 1D fiber formation, we next focused on 2D platelet growth, where the characteristic morphology and fast controllable assembly of platelets might be used to assess the capabilities of iSCAT for CDSA measurement. Seeds were deposited on glass coverslips at a surface density of ~0.16 μm⁻². Following the addition of 150 μL of 0.35 μM (3.33 μg mL⁻¹) PCL₄₅:PCL₄₅-b-PDMA₃₄₈ unimer mixtures, hexagonal platelets appear on the surface and uniformly grow in size as the reaction proceeds (Fig. 2C, Supplementary Movie 2; 637 nm, 4 μW μm⁻², 400 μs exposure time, 1.5 s⁻¹ per frame time-lapse). We selected a PCL₄₅:PCL₄₅-b-PDMA₃₄₈ unimer mixture with the concentration of 0.35 μM (3.33 μg mL⁻¹) to ensure slow growth of well isolated platelets. Keeping the seed surface density fixed, increasing the unimer concentration results in faster kinetics. Further details are provided in the “Methods” section below. Control experiments without the presence of seeds show no characteristic fast growth (Supplementary Fig. 6). We also explored the effect of changing the core chemistry: Platelets prepared with a poly(η-octalactone) (POL)-based core-forming block exhibited less uniform morphology and size compared to the PCL-based system (Supplementary Fig. 7). Furthermore, we observed changes in platelet morphology with solvent (Supplementary Fig. 8), however, iSCAT was able to resolve CDSA assemblies under a wide range of solution conditions (Supplementary Fig. 9).

Image sequences were then segmented (Supplementary Methods) to extract parameters describing platelet morphology (area, long and short axis length, aspect ratio, and perimeter). Analysis of the time evolution of platelet area yields the kinetics of individual platelet growth (Fig. 2D). 210 platelets were recorded from 3 experimental repeats over a 25 min time-lapse. The individual growth trajectories of 40 representative platelets are shown in Fig. 2D, with their mean area evolution and standard deviation displayed in Supplementary Fig. 10. All platelets show similar growth kinetics and as shown in Supplementary Fig. 11A, at the final time point (1500 s), the platelet areas followed a Gaussian distribution with a mean of 1.12 μm² and a standard deviation of 0.077 μm². This relatively low standard deviation indicates that the majority of platelet areas converged toward a stable mean value over time. In addition, the area distributions of platelets collected at the end of in situ recording (Supplementary Fig. 11A) were independent of the position of the platelet in the sample (Supplementary Fig. 11B). These data confirm the ability of living CDSA to produce uniform assemblies with controlled size and demonstrate that the in situ iSCAT imaging captures growth kinetics representative of the entire population.

During the early stages of CDSA, the platelet size is below the diffraction limit, and thus platelet area cannot be used to examine growth kinetics. However, the evolution of particle contrast can be used to report on this early stage (Fig. 2E, F, Supplementary Movie 3). As platelets are detected, individual spots begin to appear on the surface with their absolute contrast values increasing over time (becomes more negative, i.e., darker). Eventually, these diffraction-limited spots grow sufficiently that the characteristic shape becomes discernible (Fig. 2C). Similar to our measurements at later stages (Fig. 2D), during this early stage of growth, all platelets appear to increase in size at the same rate.

iSCAT imaging can also be used to monitor platelet growth at higher time resolution (Fig. 2G, H). To demonstrate this capability, we examined the progression of the position of a platelet edge, with contrast profiles extracted from platelet cross-sections as depicted in Fig. 2G. Data were collected with a temporal resolution of 333 μs (Supplementary Fig. 12, Supplementary Movie 4). A plot of the contrast profile evolution at 33.3 ms intervals with 100-frame averaging is shown in Fig. 2H.

Comparison of characterization methods

With iSCAT established as a method to image CDSA assemblies, we sought to assess the reliability and accuracy of iSCAT for CDSA characterization in comparison to conventional methods for examining platelet morphology and size.

We compared platelet parameters extracted from iSCAT imaging with those obtained from identical samples using AFM, TEM, and CLSM. Assembly of fluorescently-labeled platelets was initiated by adding 10 μL of 1 mM (10 mg mL⁻¹) aminochloromaleimide (ACM)⁵⁵ -coupled homopolymer and block copolymer unimer mixtures (ACM-PCL₄₅:ACM-PCL₄₅-b-PDMA₃₄₈, 1:1, w:w) into 1 mL 0.25 μM (10 μg mL⁻¹) PCL₄₅-b-PDMA₃₄₈ seed solution in ethanol, followed by mixing via shaking. At predetermined time points, identical aliquots were taken, quenched by water addition, and analyzed using each technique. Further details for AFM, TEM, and CLSM measurement are provided in the “Methods” section.

Figure 3 shows that platelet morphology and area information extracted from AFM, TEM, CLSM, and iSCAT are comparable (Supplementary Fig. 13). Under these reaction conditions, platelet formation is rapid, with the assembly completed within 2 min of our first time point; essentially faster than the time resolution of AFM, TEM, and CLSM measurements.

**Fig. 3: Comparison of 2D platelet characterization methods.**

Platelet growth kinetics

To characterize the kinetics of living CDSA, we adapted a kinetic model recently developed by Gao and co-workers⁵⁶, where edge-directed 2D platelet kinetics is described by

$$\frac{d{N}_{{{{{\rm{t}}}}}}}{dt}=-k{[4\pi B{n}_{{{{{\rm{a}}}}}}^{2}({N}_{0}-{N}_{{{{{\rm{t}}}}}})]}^{\frac{1}{2}}{N}_{{{{{\rm{t}}}}}},$$

(1)

where N_t is the number of unimers remaining in the reaction at time t; k is the overall growth rate constant; n_a is the number of unimers in the exposed areas per unit perimeter; N₀ is the initial number of unimers; and B is the area contributed per unimer, calculated from the ratio of final platelet area to initial number of unimers (Supplementary Equation (6)). The area per unimer (B) is considered as a constant, linking the experimentally determined platelet area to the consumption of unimers⁵⁶. Consequently, the evolution of platelet area (A_t) with time (t) can be expressed simply in terms of an effective overall rate constant, ${k}^{{\prime} }$ (see Supplementary Methods):

$${A}_{{{{{\rm{t}}}}}}=\frac{B{N}_{0}}{{N}_{{{{{\rm{seed}}}}}}}\left(1-\frac{2}{\cosh ({k}^{{\prime} }t)+1}\right).$$

(2)

We first validated our method by ensuring that the time-dependence of platelet morphology sampled from a CDSA reaction at predetermined intervals and then analyzed by both iSCAT and AFM was consistent (Supplementary Fig. 14).

To quantify the kinetics of platelet growth, we then examined the dependence of platelet size and morphology on unimer concentration, seed concentration, and solvent conditions (Fig. 4). Platelets were prepared in bulk under various reaction conditions, with sample aliquots removed at predefined time points to fully capture the reaction kinetics. These samples were then characterized by iSCAT (2 μW μm⁻², 637 nm, 900 μs exposure time; see further details in the “Methods” section). A minimum of 100 platelets were analyzed at each time point for each condition.

**Fig. 4: Dependence of platelet kinetics and morphology on reaction conditions.**

As expected, a higher initial unimer concentration leads to faster platelet growth, producing platelets with larger final surface area (Fig. 4A). A linear dependence of the overall rate constant on unimer concentration was observed with an overall reaction order of 0.41 (Supplementary Fig. 15A). In addition, the coefficient of determination (R²) reported for all concentrations indicates that the model provides a good fit. This deviation from simple first-order kinetics has been reported previously and attributed to conformational effects on the block copolymer during assembly^30,32. We also examined the dependence of platelet formation on seed concentration (0.25–1.13 nM), keeping unimer concentration and solvent conditions fixed (Fig. 4B). As expected, a higher seed concentration produces platelets with smaller final sizes.

The impact of tetrahydrofuran (THF), a good solvent for the crystallizable core-forming block (PCL), was investigated. Figure 4C illustrates the overall inhibitory effect of THF addition: Increasing the volume fraction of good solvent hinders the platelet growth rate, as crystallization is slowed due to enhanced polymer solubility³⁰. Generally, we observe that the final area of platelets generated from fixed unimer and seed concentrations remains consistent, regardless of solvent compositions (0–3% of THF). However, notably, this does not hold true for the highest THF concentration we explored (Fig. 4C, purple, 5%). Similar results have been observed previously, attributing the increased final size to the solubilization of the seeds themselves at high THF content. This solubilization leads to a decrease in crystalline nuclei, thereby providing an additional source of unimers for remaining nuclei¹⁵.

Alongside measurements of growth kinetics based on platelet area, the impact of unimer, seed, and solvent variation on the platelet morphology was also examined. Figures 4D–F show the time evolution of the platelet aspect ratio (L₁: L₂). Figures 4G–I show the underlying changes in long (L₁) and short (L₂) axis lengths with experimental conditions. Both unimer and seed concentrations had little impact on the platelet shape during assembly. Conversely, alterations in the THF content in the system (from 0 to 5%) significantly impacted platelet shape. While the platelet aspect ratio (L₁: L₂) remained relatively constant during platelet formation for a particular THF concentration (Fig. 4F), an increased THF content led to a more pronounced preference for unimer addition along the longer axis (L₁), resulting in the formation of elongated platelets as illustrated in Fig. 4I and Supplementary Fig. 16. However, the mechanism leading to this asymmetry remains unknown and warrants further study.

Overall, for all conditions tested (unimer concentration, seed concentration, and solubility), our simple kinetic model (Eq. (2)) for edge-directed growth provides a good description of our experimental observations. It can also be noted that the core chemistry significantly affects the kinetics. Compared with 1D fiber formation using a poly(ferrocenyldimethylsilane)-based core-forming blocks³⁰ or 2D nanosheet formation from poly(cyclopentenylene vinylene)-based core-forming blocks³², our PCL-based system exhibits faster kinetics in both 1D and 2D assembly.

Sequential compositional control via edge-directed platelet growth

iSCAT is highly sensitive to changes in object thickness and refractive index. This can be exploited to provide additional information on nanoscopic assembly/disassembly processes. For instance, changes in scattering properties have previously been utilized to study 2D lipid bilayer formation and phase transitions^46,57. Following CDSA, the platelet perimeter remains active for the templated edge-directed growth of additional platelet area²⁶. Therefore, multi-annulus platelets can be prepared by sequential unimer addition. The growth occurs radially from the edges of the platelets, with each successive annulus extending the preceding one. By varying the unimer composition, the composition/thickness of each annulus can be adjusted, which allows us to utilize the contrast mechanism of iSCAT to investigate the kinetics and properties of individual annuli during platelet assembly.

4-annulus platelets were prepared by sequentially adding mixtures of PCL₄₅:PCL₄₅-b-PDMA₃₄₈ (1:1, w:w) and pure PCL₄₅ (Fig. 5A, Supplementary Movie 5, further details in “Methods” section). Figure 5B shows that platelet annuli formed from PCL₄₅:PCL₄₅-b-PDMA₃₄₈ mixtures exhibit a higher (more negative) iSCAT contrast (i.e., darker) compared to annuli prepared from pure PCL₄₅. This difference is likely due to a combination of both refractive index and thickness variations resulting from differences in polymer composition. As iSCAT is highly sensitive to variations in refractive index, with a detection limit as low as 0.005^43,58, the refractive index difference between PCL (n = 1.476) and PDMA (n = 1.430–1.456)^59,60 can contribute to the observed contrast variation. Additionally, the increased thickness of platelet annuli formed from PCL₄₅:PCL₄₅-b-PDMA₃₄₈, compared to pure PCL₄₅, as shown in the AFM measurements of two-annulus platelets prepared following the same protocol (Figures 5D, E and Supplementary Fig. 17), can also lead to a path length difference between the scattered and reference signals. However, despite AFM providing direct evidence that thickness variations contribute significantly to the observed iSCAT contrast, it remains challenging to clearly decouple the impact of refractive index and thickness, as both factors influence the optical path length at the nanoscale.

**Fig. 5: Multi-annulus platelet formation kinetics.**

Figure 5 C illustrates the size evolution for the platelet shown in Fig. 5B. Growth kinetics are dependent on platelet composition, with the first and third annuli exhibiting different kinetics as compared to the second and fourth annuli. To explore this observation further, we prepared 3-annulus platelets where the unimer composition was fixed, but the concentration of PCL₄₅:PCL₄₅-b-PDMA₃₄₈ was varied for each annulus (Fig. 5F, Supplementary Movie 6, further details in “Methods” section). As shown in Fig. 5F, with no expected obvious difference in annulus thickness, the boundaries between each annulus are less distinct, albeit still identifiable (to clarify the boundaries, each annulus was color-coded as shown in Supplementary Fig. 18A). The outermost boundary within each annulus appears discernibly darker (with a larger negative contrast) compared to the center. We speculate that this effect is potentially caused by a nonuniform distribution of PCL₄₅ and PCL₄₅-b-PDMA₃₄₈ within each annulus due to differences in their crystallization rates^61,62. However, although providing some contrast variations, this effect was insufficient to render significant changes in growth kinetics. Figure 5G illustrates the progression of averaged platelet area (calculated from averaging three 3-annulus platelets displayed in Supplementary Fig. 18B), with each successive unimer addition synchronized to the corresponding time of addition. The rate constants extracted for each annulus were essentially unchanged from those in Fig. 4A. As expected, a higher unimer concentration resulted in a faster assembly rate. As a result, besides the dimensional and morphological information, iSCAT can also probe into the nanoscopic surface information of individual platelets based on their contrast variations.

Discussion

Taken together, our results demonstrate the potential of using iSCAT microscopy as a label-free tool for the in situ reporting the morphological and dimensional information of CDSA assemblies, ranging from 1D fiber formation to 3D thickness information of multi-annulus structures. With its excellent sensitivity and sub-millisecond temporal resolution, iSCAT successfully provides detailed insights into the CDSA process, including early-stage growth and high-resolution imaging of the growth dynamics.

This method has enabled us to quantify the dependence of living CDSA kinetics on unimer concentration, seed concentration, and selective solubility. Living CDSA shares obvious similarities with living covalent polymerization, where unimer and exposed active sites at the platelet edges are analogous to the monomer and initiator in the reaction. Here, 2D platelet formation of PCL₄₅:PCL₄₅-b-PDMA₃₄₈ was well described by a kinetic model based on such assembly at the living edge of the forming platelet, with a sub-unitary overall order of reaction and dependence of growth kinetics on the nature of the block copolymer^32,56. This deviation from simple first-order kinetics has been previously attributed to conformational dispersity of the block copolymer^28,30. This is particularly intriguing when considering the variation in contrast within growth annuli in our examination of the growth of multi-annulus platelets (Fig. 5). Secondly, observed variations in contrast across individual annuli are of note (Fig. 5F). One interpretation could be a change in composition, perhaps caused by a variation in the relative rates of deposition of PCL₄₅ and PCL₄₅-b-PDMA₃₄₈. These observations warrant further investigation, mapping how different building units might assemble differently as 2D platelet shape evolves. Lastly, in a multi-annulus platelet, the contrast variations between each annulus can be attributed to changes in both refractive index and thickness resulting from variations in composition. The interplay between these two factors is challenging to fully decouple without additional information. iSCAT’s ability to provide rapid, label-free insights into structures and composition suggests significant potential for detecting relative changes in polymeric particle properties. We anticipate that further development of methods for decoupling thickness and refractive index effects will enhance the quantitative capabilities of iSCAT, leading to broader applications.

Beyond its capabilities for in situ monitoring, iSCAT also holds much promise as a tool for ex situ characterization of particle morphology due to its fast data collection, low cost, simple sample preparation, and high throughput. Additionally, this technique can be implemented using simple commercially available optical components compatible with fluorescence microscopy^48,63. However, iSCAT is not without limitations: Microscopy limits the field of view in any single image (20 × 20 μm in our setup), requiring parallelization or surface scanning to monitor the growth of larger numbers of particles simultaneously. iSCAT also requires a reference light-field and thus is limited to studies at or near a surface. We note that here we saw no difference in kinetics between platelets grown in solution and those growing in situ at the coverslip surface. However, the presence of this surface is inevitably different at some level from growth conditions in the bulk solution. Nevertheless, confocal variants of iSCAT hold the potential for 3D imaging much deeper into the sample solution^44,48. Here we have focused on 2D CDSA, but the interferometric nature of iSCAT also embeds axial information; enabling, for example, the measurement of nanoparticle axial position and size^{47,48,64,65,66}. As illustrated in Supplementary Fig. 19, we applied recent methods that exploit defocused image stacks to generate quantitative phase information⁶⁷. However, to establish an accurate relationship between the sample thickness and the corresponding phase information, calibration is required, using samples with controlled refractive indices and varied thicknesses. We envisage iSCAT has great potential to be applied to profiling more complex CDSA processes, with three-dimensional shape at high spatio-temporal resolution^68,69,70. However, it is important to emphasize that in terms of the scope of our current study, which mainly focuses on the two-dimensional CDSA and growth, limited information on the 3D height variation in our 2D system can be extracted. In addition, there remains considerable scope to further enhance our detection sensitivity; for example, by applying recent reports of machine learning⁷¹ and optimization of reflected vs. scattered signals⁷² to improve iSCAT mass sensitivity.

Overall, iSCAT is a potent technique that offers a different perspective on improving CDSA-based nanoparticle engineering. Here, we have demonstrated the applicability of iSCAT to study PCL-based 1D fibers and 2D platelets. Real-time label-free single platelet imaging provides a rich source of information hitherto inaccessible by most commonly used techniques. This combination of speed, resolution, and simplicity further places iSCAT as a valuable tool for the design and characterization of complex polymer particles generated by CDSA.

Methods

Materials

Unless otherwise stated, all chemicals and solvents were used as received without further purification. Sodium ethanethiolate (90%) and borane tetrahydrofuran complex solution (1.0 M in THF) were purchased from Alfa Aesar. Carbon disulfide (≥99%), solid iodine (≥99%) and 4,4’-azobis(4-cyanovaleric acid) (ACVA, 98%) were purchased from Merck.

The synthesis of dual head chain transfer agent (CTA) of 2-cyano-5-hydroxypentan-2-yl ethyl carbonotrithioate (CHPET) follows previous reports¹¹. CTA and diphenylphosphate (DPP, 99%, Merck) were dried over P₂O₅ in a desiccator under static vacuum for 1 week before use. ε-caprolactone (99%, ACROS Organics) was vacuum distilled twice over CaH₂ before being introduced in the glove box and used. 2,2’-azobis(2-methylpropionitrile) (AIBN, 98%, Merck) was recrystallized twice from methanol and stored at 4 ^∘C in the dark. 1,4-dioxane (anhydrous, 99.8%) and N, N-dimethylacrylamide (DMA, 99%, contains 500 ppm monomethyl ether hydroquinone as inhibitor) were purchased from Merck. DMA was passed through a basic alumina plug to remove the inhibitor before use. Dry solvents were used directly from a drying and degassing inert solvent tower system. Aminochloromaleimide (ACM) fluorescent dye was synthesized following the method provided below⁵⁵.

Polymer synthesis and characterization

Synthesis of PCL₄₅

In a nitrogen-filled glove box (oxygen and water content lower than 0.1 ppm), solutions of diphenylphosphate (DPP, 17.0 mg, 1 eq) in dry toluene (2.5 mL) and dual-head CTA (17.0 mg, 1 eq) in dry toluene (1 mL) were added to ε-caprolactone dry toluene solution (543.3 mg, 70 eq in 1.5 mL). After stirring for 6 h at room temperature, the solution was removed from the glove box, precipitated three times into cold diethyl ether dropwise, and collected by centrifugation. ¹H NMR (400 MHz, CDCl₃): δ (ppm): 4.10 (t, 2H, CH₂OCO), 4.04 (t, 90H, CH₂CH₂O), 3.62 (m, 2H, ${{{{\rm{C}}}}}({{{{\rm{CN}}}}}){{{{\rm{C}}}}}{{{{{\boldsymbol{H}}}}}}_{2}{{{{{\rm{CH}}}}}}_{2}$), 3.32 (q, 2H, SCH₂CH₃), 2.28 (90H, OCOCH₂CH₂), 1.86 (s, 3H, ${{{{\rm{C}}}}}({{{{\rm{CN}}}}})({{{{\rm{C}}}}}{{{{{\boldsymbol{H}}}}}}_{3}){{{{{\rm{CH}}}}}}_{2}$), 1.72–1.54 (180H, OCOCH₂CH₂CH₂CH₂), 1.45-1.28 (90H, OCOCH₂CH₂CH₂CH₂). SEC (Chloroform, PMMA standard): M_n = 12.9 kg mol⁻¹, Đ_M = 1.05.

To synthesize ACM, 2,3-dichloromaleic anhydride (1 eq) and 4-aminobutanoic acid (1 eq) were dissolved in 10 mL acetic acid (AcOH) in a round-bottom flask. The reaction mixture was heated at 150 ^∘C for 6 h under constant stirring. Upon completion, the mixture was concentrated under reduced pressure to remove the AcOH. The crude residue was dissolved in 5 mL dichloromethane (DCM) and purified via flash column chromatography using 5% AcOH in DCM as the eluent. The product was isolated as an orange solid. Then the above product was dissolved in 20 mL THF in a reaction flask. Sodium carbonate (2.5 eq) and butylamine (1.05–1.1 eq) were added, and the reaction mixture was stirred at room temperature. The reaction progress was monitored by thin layer chromatography, confirming the consumption of the above product within 30 min to 4 h. The solvent was evaporated under reduced pressure, and the residue was dissolved in 150 mL DCM. The organic layer was washed with water (2 × 150 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the product was purified via column chromatography on silica gel using petroleum ether/ethyl acetate as the eluent. The product was obtained as a yellow-orange solid.

To synthesize ACM-labeled PCL₄₅, ACM was coupled to the PCL₄₅ polymer backbone by esterification. In a typical coupling reaction, PCL₄₅ (100.0 mg, 1 eq), ACM (16.1 mg, 3 eq), 4-dimethylaminopyridine (DMAP) (2.3 mg, 1 eq) and N,N’-dicyclohexylcarbodiimide (DCC) (38.3 mg, 10 eq) were mixed together in an ampule with 2 mL CHCl₃. The solution was left stirring at room temperature for 2 days. The solution was then filtered with the filtrate precipitated in diethyl ether three times and the resultant polymer was dried under vacuum.

Synthesis of PCL₄₅-b-PDMA₃₄₈

PCL₄₅ (100.0 mg, 1 eq), DMA (736.3 mg, 400 eq), and AIBN (0.3 mg, 0.1 eq) were dissolved in 1 mL 1,4-dioxane and placed in an ampule. The solution was then freeze-pump-thawed three times and heated for 2 h at 70 ^∘C. The reaction was quenched by immersion of the ampule in liquid nitrogen and the polymer was precipitated in ice-cold diethyl ether three times before being dried under vacuum and analyzed. SEC (chloroform, PMMA standard): M_n = 46.3 kg mol⁻¹, Đ_M = 1.24.

To synthesize ACM-labeled PCL₄₅-b-PDMA₃₄₈, ACM was coupled to the PCL₄₅-b-PDMA₃₄₈ polymer backbone by esterification. In a typical coupling reaction, PCL₄₅-b-PDMA₃₄₈ (200 mg, 1 eq), ACM (4.3 mg, 3 eq), DMAP (0.6 mg, 1 eq), and DCC (10.3 mg, 10 eq) were mixed in an ampule with 2 mL CHCl₃. The solution was left stirring at room temperature for 2 days. The solution was then filtered, and the filtrate precipitated in diethyl ether three times and the resultant polymer was dried under vacuum.

NMR characterization

All ¹H NMR spectra were recorded on a Bruker 400 MHz (DPX-400) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to internal standard tetramethylsilane at δ = 0. Samples were prepared in deuterated chloroform (CDCl₃) and referenced to residual non-deuterated signal of solvent (CDCl₃ at 7.26 ppm ¹H-NMR). The resonance multiplicities are described as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet).

SEC characterization

SEC analysis was performed on an Agilent 1260 Infinity II system combined with refractive index (RI) and ultraviolet (UV) detectors (λ = 309 and 360 nm), equipped with a PLGel 3 μm (50 × 7.5 mm) guard column and two PLGel 5 μm (300 × 7.5 mm) mixed-D columns, mobile phase (eluent) is CHCl₃ with 0.5% triethylamine (TEA). Molecular weight and molecular weight distributions were calibrated against poly(methyl methacrylate) (PMMA) standards and analyzed using Agilent SEC software.

CDSA preparation

Preparation of PCL₄₅-b-PDMA₃₄₈ seed solution

20 mg PCL₄₅-b-PDMA₃₄₈ was added into 4 mL ethanol. The mixtures were heated at 70 ^∘C without stirring on a heating block for 3 h before cooling to room temperature and then aging for 5 days to yield micron-length polydisperse fibers (Supplementary Fig. 3A). The crystalline fibers were then sonicated using a Bandelin Sonopuls sonication probe in a dry-ice/acetone bath for 20 min. The polymer solution was exposed to 60 cycles of 20 s bath sonication with an interval of 100 s to yield a short crystalline seed stock solution (Supplementary Fig. 3B). For determination of the average length of the seed, a minimum number of 100 seeds were analyzed with TEM (Supplementary Fig. 3C).

Bulk preparation of PCL₄₅:PCL₄₅-b-PDMA₃₄₈ platelets

10 mg mL⁻¹ unimer stock solution (PCL₄₅:PCL₄₅-b-PDMA₃₄₈ or ACM-PCL₄₅:ACM-PCL₄₅-b-PDMA₃₄₈) was prepared by mixing PCL₄₅ (or ACM-PCL₄₅) with PCL₄₅-b-PDMA₃₄₈ (or ACM-PCL₄₅-b-PDMA₃₄₈) at a 1:1 weight ratio in a good solvent (THF or Chloroform). A working seed solution was prepared by diluting the stock seed solution in ethanol to form a targeted concentration, then aged for at least 1 h. Then a small volume of unimer stock solution was added to a dispersion of seed solution in a screw cap vial followed by vigorously shaking by hand for 5 s, the final unimer concentration was varied by adjusting the volume of stock solutions added to each reaction. At predetermined time points, 140 μL sample mixture was withdrawn and added to 140 μL of deionized water to quench the reaction, followed by subsequent analysis.

In situ 1D fiber growth

50 μL of 2.51 nM (0.1 μg mL⁻¹) PCL₄₅-b-PDMA₃₄₈ seed solution was spin-coated onto the cleaned coverslip twice at 3200 rpm for 50 s, immediately followed by 4000 rpm for 30 s. A cleaned silicone spacer was then placed onto the seed-coated coverslip to form a reaction chamber. This reaction chamber has then been mounted above the objective with the objective being adjusted to focus on the surface (Supplementary Fig. 4). PCL₇₃-b-PDMA₂₀₄ unimer stock solutions were diluted in methanol to achieve the targeted concentration (freshly prepared every time, 0.06 μM, 1.67 μg mL⁻¹) and added into the reaction chamber, followed by iSCAT imaging immediately. A laser power density of 4 μW μm⁻² at 637 nm, a camera exposure time of 400 μs, and an overall time-lapsed frame rate of 3 s⁻¹ were selected.

In situ 2D platelet growth

To obtain data shown in Fig. 2C, D, plasma-cleaned coverslips were placed in 50 mL 0.002 μg mL⁻¹ poly(ε-caprolactone)-b-poly(2-vinylpyridine) (PCL₄₅-b-P2VP₁₃₄) seed ethanol solution for 36 h to achieve 2D surface seed density of ~0.16 μm⁻². Before the reaction, the coverslip was gently rinsed with ethanol and dried under a stream of nitrogen. 150 μL of 0.35 μM (3.33 μg mL⁻¹) PCL₄₅:PCL₄₅-b-PDMA₃₄₈ unimer mixtures were then added onto the seed-modified surface followed by recording immediately (637 nm, 4 μW μm⁻², 400 μs exposure time, 1.5 s⁻¹ per frame time-lapse).

For other experiments, spin coating was used to prepare seeds-coated surface: 50 μL of 2.51 nM (0.1 μg mL⁻¹) PCL₄₅-b-PDMA₃₄₈ seed solution was spin-coated onto the cleaned coverslip twice at 3200 rpm for 50 s, immediately followed by 4000 rpm for 30 s. A reaction chamber was constructed following the same protocol introduced above, PCL₄₅:PCL₄₅-b-PDMA₃₄₈ unimer stock solutions were diluted in methanol to achieve the targeted concentration and added into the reaction chamber, followed by iSCAT imaging immediately.

For data shown in Fig. 2E, F, 150 μL of 0.35 μM (3.33 μg mL⁻¹) PCL₄₅:PCL₄₅-b-PDMA₃₄₈ unimer mixtures were added onto the surface. A laser power density of 4 μW μm⁻² at 637 nm, a camera exposure time of 400 μs, and an overall time-lapsed frame rate 10 s⁻¹ were selected. For data shown in Fig. 2G, H, 150 μL of 0.29 μM (2.78 μg mL⁻¹) PCL₄₅:PCL₄₅-b-PDMA₃₄₈ unimer mixtures were added onto the surface. A laser power density of 24 μW μm⁻² at 637 nm, a camera exposure time of 80 μs, and an overall time-lapsed frame rate of 3000 s⁻¹ were selected.

Multi-annulus platelets were prepared by alternately adding PCL₄₅:PCL₄₅-b-PDMA₃₄₈ mixtures and PCL₄₅ alone. PCL₄₅:PCL₄₅-b-PDMA₃₄₈ mixtures were first added onto a seed-coated surface to form the first annulus. After ~5 min, the reaction solution was removed from the chamber and the chamber was rinsed gently with methanol to remove unimer residues, followed by the addition of PCL₄₅ solution. This alternating sequence was repeated until the desired number of annuli was achieved. Recording started immediately after each unimer addition. A laser power density of 4 μW μm⁻² at 637 nm, a camera exposure time of 400 μs, and an overall time-lapsed frame rate of 1 s⁻¹ were selected. For the experiment in Fig. 5F, PCL₄₅:PCL₄₅-b-PDMA₃₄₈ mixtures with sequentially increasing unimer concentration were added at each annulus. A laser power density of 4 μW μm⁻² at 637 nm, a camera exposure time of 400 μs, and an overall time-lapsed frame rate 0.5 s⁻¹ were selected.

Comparison of analysis methods

TEM characterization

TEM imaging was performed on a JEOL 1400 Bio (1720/GB06) microscope at an acceleration voltage of 80 kV. CDSA samples were deposited onto copper grids without staining and dried naturally before analysis. For the determination of the average size of the platelets, a minimum number of 100 platelets were analyzed.

AFM characterization

AFM samples were prepared by drop-casting and drying 10 μL of assemblies in ethanol onto mica attached to a Sigmacote-treated silicon wafer. Imaging and analysis were performed on a JPK Nanowizard 4 in quantitative imaging (QI) mode. AFM tips (PPP-NCHAuD) were purchased from NANOSENSORSTM, with a resonance frequency window of 204–497 kHz and a force constant of 10–130 nN. For the determination of the average size of the platelets, a minimum number of 100 platelets were analyzed.

CLSM characterization

An inverted FV3000 (Olympus) confocal microscope with 20× and 60× oil immersion objectives was used for imaging. Scan rates of 1 μs pixel⁻¹ at 512 by 512 pixels to 1024 by 1024 pixels were used. For platelet characterization, a minimum number of 100 platelets were segmented manually using Fiji image analysis software⁷³.

iSCAT characterization

A wide-field iSCAT was previously constructed (detailed in Supplementary Fig. 4)⁵⁴. Briefly, a 637 nm multimode diode laser (RLM-6000L, Kvant lasers, Slovakia) was fiber-coupled before homogenization (Albedo system, Errol, France), generating a top-hat profile. Following collimation, the beam was focused at the back focal plane of the objective (100× Plan-Apo 1.45 NA, Nikon, Japan). A polarizing beam splitter transmits plane-polarized light with a specific orientation to the quarter-wave plate, which converts it to circularly polarized light. When used together, these components function as an optical isolator, efficiently separating the signal of interest from the illuminating light. Light reflected back from the sample-glass interface interferes with the light scattered by the objects within the sample, was then separated from the incident light, and directed onto a high-speed CMOS camera (PCO.dimax CS1, ExcelitasPCO GmbH, Germany). Focus control is then provided by a piezo-stage (P-545-3R8S, Physik Instrumente, Germany). The camera, laser, and piezo-stage are controlled using a custom LabVIEW program (National Instruments, USA).

Glass coverslips (24 × 60 mm, #1.5 thickness, Epredia) were cleaned by sequential sonication in chloroform, acetone, and isopropanol for 15 min before drying with N₂. Circular silicone spacers (ϕ9 × 2.5 mm thickness, Merck) were washed with the same protocol, dried under vacuum, and then placed on top of the cleaned coverslip to form a chambered coverslip (Supplementary Fig. 4). The reaction chamber was sealed with an additional coverslip (22 × 22 mm, Merck) which was cleaned using the same procedure. To prepare plasma-cleaned coverslips, the solvent-cleaned coverslips were treated with oxygen plasma for 6 min (Diener Electronic, Femto, Germany).

PCL₄₅:PCL₄₅-b-PDMA₃₄₈ platelets synthesized in bulk were spin-coated onto the cleaned coverslip at 3200 rpm for 15 s until the desired surface density was achieved. Then a silicone spacer was placed onto the platelet-coated surface before the addition of methanol: water (30:70, v: v) solution and imaging. A laser power density of 2 μW μm⁻² at 637 nm and a camera exposure time of 900 μs were selected unless otherwise stated. A minimum number of 100 platelets were analyzed to determine the average size.

Data analysis

Image analysis of raw images collected from iSCAT microscopy experiments proceeded via the following steps: (1) Dark counts subtraction was conducted by subtracting each frame by a frame recorded under the same conditions without laser illumination; (2) Temporal fluctuations in laser intensity were removed via the division of each frame by the frame modal pixel value; (3) Background correction was then performed via subtraction of a background corresponding to the median-average of 10 frames corresponding to the image area prior to platelet growth; (4) A binary mask was created from a low-pass Gaussian filtered (σ = 2 px) replica of the image and used to isolate individual platelets; (5) Platelet parameters, including area, long and short axis length, aspect ratio, and perimeter were then collected using the built-in Analyze Particles function in the Fiji image analysis software⁷³.

Data availability

The raw data generated in this study have been deposited at kcl.figshare.com doi:10.18742/28309298. Data is also available from the authors on request.

Code availability

The main data analysis was written in Python 3.6 and used standard Python packages. The code for each analysis can be provided together with the raw datasets from the corresponding author.

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Acknowledgements

Y.G. thanks the King’s-China Scholarship Council PhD Scholarship program for funding. M.I.W. is funded by the Wellcome Trust (224327/Z/21/Z). This work was supported by the BBSRC (BB/R001790/1). T.X., J.Y.R., and R.O.R. thank the University of Birmingham for financial support.

Author information

These authors contributed equally: Yujie Guo, Tianlai Xia.

Authors and Affiliations

Department of Chemistry, King’s College London, London, UK
Yujie Guo & Mark I. Wallace
School of Chemistry, University of Birmingham, Birmingham, UK
Tianlai Xia, Yujie Xie, Julia Y. Rho, Laihui Xiao & Rachel K. O’Reilly
Department of Engineering, King’s College London, London, UK
Vivien Walter

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Yujie Guo
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Tianlai Xia
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Vivien Walter
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Yujie Xie
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Contributions

M.I.W. and R.O.R. conceived this work. Y.G. and T.X. contributed to this project equally, they performed the experimental studies and data analysis. V.W., Y.X., J.Y.R., and L.X. participated in the project discussion and commented on the manuscript.

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Correspondence to Rachel K. O’Reilly or Mark I. Wallace.

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Guo, Y., Xia, T., Walter, V. et al. Real-time label-free imaging of living crystallization-driven self-assembly. Nat Commun 16, 2672 (2025). https://doi.org/10.1038/s41467-025-57776-9

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Received: 29 January 2024
Accepted: 04 March 2025
Published: 18 March 2025
DOI: https://doi.org/10.1038/s41467-025-57776-9

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