How PLLA Spherulites Fragment or Disintegrate Under Deformation: Insights from AFM

What happens to the crystalline structures inside a semicrystalline polymer when the material is stretched? A study by Xabier Larrañaga and colleagues, published in Macromolecules, reveals two contrasting deformation pathways in poly(L-lactic acid) (PLLA): fragmentation, in which crystalline order is locally retained, and mechanical disintegration, in which the structure becomes substantially less ordered. Atomic force microscopy (AFM), combined with Raman spectroscopy and polarized-light microscopy, helps explain these different responses.

Physical aging and the deformation of PLLA

PLLA is a polymer whose mechanical response depends strongly on its molecular structure and processing history. In semicrystalline PLLA, ordered structures called spherulites coexist with less ordered, amorphous regions. How these components interact under mechanical stress influences whether the material fractures or undergoes plastic deformation.

An important factor is physical aging. As the amorphous polymer relaxes toward a lower-energy state over time, molecular mobility can become more restricted, making plastic deformation more difficult under the investigated conditions.

Larrañaga and colleagues examined two related aspects of PLLA deformation: the influence of controlled physical aging on the brittle-to-ductile transition of glassy PLLA, and the deformation behaviour of spherulites in semicrystalline PLLA. These were distinct experimental investigations.

In the glassy PLLA experiments, the least-aged samples exhibited a brittle-to-ductile transition at −30 °C, whereas more strongly aged samples required higher temperatures to deform in a ductile manner under the reported testing conditions.

These findings provide context for understanding the importance of the amorphous chain network in polymer deformation, while the separate semicrystalline experiments reveal how spherulites respond when the surrounding material undergoes plastic deformation.

Two deformation pathways: fragmentation and disintegration

When semicrystalline PLLA films were stretched, the researchers observed two distinct responses of the spherulites.

In fragmentation, a spherulite separates into smaller regions, while characteristic crystalline structure remains locally preserved within the fragments.

In mechanical disintegration, the response is different. The former spherulite remains recognizable, but its crystalline structure undergoes substantial disordering during deformation.

The authors interpret these contrasting pathways in terms of the mechanical interaction between crystalline regions and the surrounding amorphous chain network. They propose that connectivity through bridging polymer chains plays a role in whether crystalline domains separate into fragments or undergo more extensive structural rearrangement.

To examine these changes at a local scale, the researchers combined AFM surface imaging with mechanical measurements.

Tapping-mode AFM provided information about surface topography, while AFM force–displacement measurements and nanoindentation-derived Young’s modulus maps revealed variations in local stiffness.

Young’s modulus describes a material’s resistance to elastic deformation. Mapping this property across a deformed spherulite helps distinguish mechanically different regions that may not be apparent from surface morphology alone.

In fragmented spherulites, the AFM measurements revealed alternating regions of higher and lower stiffness. Locally stiff domains reached approximately 2.4 GPa. Together with the complementary Raman evidence, these measurements support the interpretation that crystalline regions remained within the fragments.

The mechanically disintegrated spherulites showed a different response.

Although the former spherulite could still be identified in the AFM topography, its measured stiffness was much closer to that of the surrounding amorphous material. The reported AFM-derived Young’s modulus was 1.01 ± 0.35 GPa for the disintegrated spherulite, compared with 0.92 ± 0.14 GPa for adjacent amorphous regions.

This reduced mechanical contrast is consistent with substantial structural reorganization. It does not mean that the spherulite had physically disappeared or that the two regions were mechanically identical.

Two stacked AFM maps of a disintegrated PLLA spherulite: grayscale surface topography above and orange Young’s modulus map below, with measurement scales.
AFM topography (upper map) and nanoindentation-derived Young’s modulus (lower map) of a mechanically disintegrated PLLA spherulite. Figure 5b excerpt reproduced from Larrañaga et al., Macromolecules (2026), DOI: 10.1021/acs.macromol.6c00777, CC BY 4.0. Panel extracted from the original figure; scientific content unchanged.

AFM probes used in the study

For AFM imaging and force–displacement measurements, the authors used noncoated silicon NanoWorld Pointprobe® NCH AFM probes.

The study combined tapping-mode topography with local force measurements to examine the relationship between surface morphology and nanomechanical response in deformed PLLA.

Raman spectroscopy provides complementary structural evidence

While AFM revealed changes in morphology and local stiffness, these measurements alone could not establish whether crystalline order had been retained or lost.

The researchers therefore used polarized Raman spectroscopy to investigate changes in molecular structure.

For fragmented spherulites, Raman spectra retained characteristic signatures of crystalline PLLA within the separated regions. This supported the interpretation that fragmentation had occurred without complete loss of local crystalline organization.

In mechanically disintegrated spherulites, several characteristic crystalline Raman features disappeared or became substantially weaker. The spectra instead showed features more consistent with a deformed, less ordered molecular structure. This indicates substantial disordering rather than necessarily proving the complete disappearance of all crystalline material.

Together, the AFM and Raman results distinguish mechanical fragmentation from the more extensive structural disordering associated with spherulite disintegration. Polarized-light microscopy provided additional context for the deformation morphology.

Understanding ductility in semicrystalline polymers

The study highlights why the mechanical behaviour of PLLA cannot be understood from crystallinity alone.

The experiments on glassy PLLA demonstrate the influence of physical aging on the brittle-to-ductile transition under the investigated conditions. The separate semicrystalline experiments show how interactions between crystalline regions and the amorphous chain network are associated with contrasting deformation responses.

By combining local AFM measurements with complementary structural characterization, Larrañaga and colleagues connect changes in morphology and stiffness with their interpretation of the underlying deformation mechanisms.

The findings contribute to a more detailed understanding of how molecular organization, physical aging and interactions between crystalline and amorphous regions influence the mechanical response of PLLA.

Full Citation

Xabier Larrañaga, Brian J. Rodriguez, Jose R. Sarasua and Ester Zuza
Against the Segmental Relaxation of the Chain Network: Activating the Ductile Mechanism of Both Glassy and Semicrystalline PLLA
Macromolecules 2026
https://doi.org/10.1021/acs.macromol.6c00777

Creative Commons Attribution 4.0 International (CC BY 4.0)
https://creativecommons.org/licenses/by/4.0/

Watching Designed Proteins Assemble with High-Speed AFM

How the surface beneath a protein can influence the structures it forms

What determines whether nanoscale building blocks assemble into an ordered structure or remain disordered?

In a study published in Nature Communications, Sakshi Yadav Schmid and colleagues investigated this question using designed protein nanorods at liquid–solid interfaces. Combining high-speed atomic force microscopy (HS-AFM), machine-learning analysis and computational modelling, they explored how the surface beneath the proteins influences their movement and organization.

The researchers observed a striking contrast: the same protein nanorods formed different arrangements on two types of mica, even though both surfaces have the same potassium-ion sublattice to which the proteins were designed to bind. Their findings reveal why understanding nanoscale assembly requires looking beyond the interactions deliberately built into the protein design.

Following protein assembly in real time

Self-assembly occurs when individual building blocks organize into larger structures through interactions with one another and their surroundings. To understand how these structures form, it helps to observe the process as it unfolds—not just examine the final arrangement.

High-speed AFM allowed the researchers to observe the protein nanorods in liquid as the assembly process unfolded. Using NanoWorld ARROW-UHFAuD and USC-F1.2-k0.15 cantilevers in their high-speed AFM experiments, they could follow how the nanorods moved and how their arrangements changed over time, rather than relying solely on images of the final structures.

The experiments compared two substrates: muscovite mica (m-mica) and fluorophlogopite mica (f-mica). Under the high-salt conditions investigated, the nanorods behaved very differently on the two surfaces.

On muscovite mica, the nanorods were highly mobile. Small groups of aligned rods appeared, grew and rearranged until a more stable, layered structure emerged. This arrangement is known as smectic order: the elongated building blocks point predominantly in the same direction and are organized into layers.

Time-lapse high-speed AFM images and corresponding nanorod identification images showing protein assembly on muscovite mica. Graphs below show the number of rods, nematic alignment and development of smectic order over time.
Figure 3. Development of smectic order on muscovite mica. Time-lapse high-speed AFM images (a) show designed protein nanorods assembling on muscovite mica. The corresponding machine-learning-based identification of the rods (b) supports quantitative measurements of the number of rods (c), nematic order (d) and smectic order (e). Together, the measurements reveal how alignment and layered organization develop over time.
Source: Yadav Schmid et al., Nature Communications 17, 2446 (2026), https://doi.org/10.1038/s41467-026-69170-0. © Battelle Memorial Institute and The Authors 2026. Licensed under CC BY 4.0. Reproduced without modification.

On fluorophlogopite mica, the rods showed translational and rotational movement but were less mobile overall. Their orientations followed three directions associated with the substrate’s potassium-ion sublattice. As more rods accumulated, they formed a high-density disordered assembly, rather than the layered structure seen on muscovite mica.

Time-lapse high-speed AFM images and corresponding nanorod identification images showing protein assembly on fluorophlogopite mica. Graphs below show rod counts and comparatively low nematic and smectic order parameters over time.
Figure 4. High-density disordered assembly on fluorophlogopite mica. Time-lapse high-speed AFM images (a) and corresponding machine-learning-based identification of the rods (b) document protein nanorods on fluorophlogopite mica. Measurements of the number of rods (c), nematic order (d) and smectic order (e) show that the assembly remains comparatively disordered.
Source: Yadav Schmid et al., Nature Communications 17, 2446 (2026), https://doi.org/10.1038/s41467-026-69170-0. © Battelle Memorial Institute and The Authors 2026. Licensed under CC BY 4.0. Reproduced without modification.

The difference was particularly intriguing because the potassium-ion patterns that guide protein binding are identical on the two substrates. Something beyond that shared pattern was influencing the outcome.

Measuring how order develops

Watching nanorods move provides a view of self-assembly in action. But how can researchers measure the transition from individual movements to collective organization?

The team used machine-learning methods to identify nanorods in the high-speed AFM images and determine their positions and orientations. From these measurements, they could track surface coverage and calculate how the degree of order changed over time.

Two quantities were especially useful. Nematic order describes how strongly elongated objects align in a common direction. Smectic order additionally captures their organization into layers.

On muscovite mica, the nanorods aligned relatively quickly, while their layered organization developed more gradually as surface coverage increased. On fluorophlogopite mica, the rods accumulated without developing comparable long-range alignment or smectic order.

This quantitative analysis showed not only that the final structures differed, but also how the two assembly processes followed different paths.

Why the surface matters

What could explain such different behavior on substrates with identical potassium-ion sublattices?

An important clue lies in the water next to the solid surface. The atomic structures of muscovite and fluorophlogopite mica differ in ways that influence the arrangement of nearby water molecules. These interfacial hydration structures can, in turn, affect the interactions experienced by the protein nanorods.

To investigate this explanation, the researchers used Monte Carlo simulations that considered the mobility of rod-shaped building blocks and the influence of preferred orientations imposed by the substrate. When the model introduced a directional preference representing the broken symmetry associated with muscovite mica, smectic ordering could emerge. Without that preference, the model reproduced behavior consistent with disordered assembly.

The distinction between the methods is important: high-speed AFM revealed how the nanorods moved and assembled, machine-learning analysis quantified the developing order, and the simulations helped explain how surface-related effects could produce the observed differences.

The findings highlight a factor that is easy to overlook when designing protein assemblies: the surrounding liquid and the structure of the underlying surface can influence the outcome, even when the intended protein–surface binding pattern is the same.

By revealing these effects as assembly happens, high-speed AFM provides an experimental window into processes that may be difficult to understand from finished structures alone. Combined with quantitative analysis and modelling, such observations can help researchers develop a more complete understanding of how designed proteins organize at interfaces.

Full citation

Yadav Schmid, S.; Helfrecht, B.; Stegmann, A.; Legg, B. A.; Pyles, H.; Chen, J.; Edison, J. R.; Ziatdinov, M.; Preisler, Z.; Dollar, O.; Whitelam, S.; Kalinin, S.; Baker, D.; Mundy, C. J.; Zhang, S.; De Yoreo, J. J.
Impact of solvent forces and broken symmetry on the assembly of designed proteins at a liquid-solid interface.
Nature Communications 2026, 17, 2446.
https://doi.org/10.1038/s41467-026-69170-0

Creative Commons Attribution 4.0 International (CC BY 4.0)

https://creativecommons.org/licenses/by/4.0/