Cryogenic transmission electron microscopy reveals assembly and nanostructure of PEDOT:PSS

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: minor_revision

Abstract
Soft and conducting organic materials are ideal candidates for stretchable bioelectronics and wearable devices. Despite recent advances, our understanding of conducting polymer nanostructures and how they arise remains incomplete, given the limited high-resolution studies and molecular-level descriptions of these systems. Here, we employ cryogenic transmission electron microscopy (cryo-EM) to investigate the evolution of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) morphology in solution and the resulting solid state structure in the presence of ionic and molecular additives. Our results reveal the formation of heterostructural elongated fibers consisting of PEDOT:PSS micelles in solution. Cryo-EM further reveals that additives increase the number of fibrils, in addition to inducing the formation of crystalline domains. We observe that fibril and crystalline phases in solutions act as a template for the growth of these nanostructures in the solid state. Furthermore, exploiting cryo-EM reveals the role of solid-liquid interactions in PEDOT:PSS through the imaging of PEDOT:PSS nanostructures after the hydration of thin films. Hydration leads to the swelling of heterostructural fibers while reducing the crystalline domain size. Such behavior explains the mechanical robustness of PEDOT:PSS thin films processed with various additives as well as the high electrical conductivity of PEDOT:PSS in applications such as organic electorchemical transistors.

Introduction
Mixed ionic-electronic conductors have attracted tremendous attention due to their unique properties and applicability in a wider range of devices, such as batteries1, fuel cells2, and organic electrochemical transistors3,4. Organic mixed-ionic electronic conductors, particularly conjugated polymeric systems, have opened new prospects in applications in biosensing5 and neuromorphic computing6. Poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) is one of the most widely used polymeric mixed ionic-electronic conductors due to its commercial availability, high electrical/ionic conductivity7,8, and mechanical compliance7,9. This prototypical mixed conductor relies on a mixture of conjugated and ionic polymers, although how interactions between the two components, processing, and additives govern assembly, microstructure and macroscopic properties remain an open question.

PEDOT:PSS in its pristine form possesses a low electrical conductivity, near 1 S/cm3,7,10,11. The addition of high boiling point solvents11, “secondary dopants” such as ionic liquids and salts12,13, and post-deposition acid treatment14 can substantially enhance electrical conductivity by three orders of magnitude over their pristine counterparts. In addition to the significant boost to conductivity, ionic and molecular additives have also been linked to an increase in the stretchability and flexibility of PEDOT:PSS, a key property for fabricating wearable electronics7,12,13.

A large body of work has focused on studying structure-function relationships in PEDOT:PSS under different processing conditions7,12,13,15–19. Despite these efforts, a holistic picture describing the PEDOT:PSS morphology remains elusive, in part due to morphological complexities and the lack of real space imaging techniques conveying the solution and solid-state properties. For instance, we commonly accept that PEDOT:PSS microstructure consists of core-shell micelles with PEDOT-rich cores and PSS-rich coronas. Despite this common assumption, the reported core-shell size extracted from neutron, X-ray, and light scattering techniques ranges from a few nanometers15 to hundreds of nanometers20,21. The role of additives further complicates the morphological picture3. X-ray and neutron scattering measurements point to the formation of rod-like structures21 in PEDOT:PSS with added salts, as well as an increase in the degree of crystallinity7 of PEDOT:PSS through the breakdown of core-shell and phase segregation of PEDOT and PSS. Nevertheless, important aspects of the PEDOT:PSS morphology remain unresolved, such as the size and distribution of crystalline domains and the correlation between rod-like aggregates and crystalline phases. Despite the partial success of the core-shell breakdown model, this description fails to explain multiple PEDOT:PSS properties, such as the simultaneous increase in electrical conductivity and increase in mechanical compliance, in particular of modern formulations of PEDOT:PSS7,22. This behavior is in contrast with other conjugated polymer systems, where a trade-off exists between charge transport (and crystallinity) and mechanical compliance23,24. We propose that the lack of a comprehensive morphological model predicting the physical properties of PEDOT:PSS mainly stems from reliance on scattering and spectroscopy techniques to characterize the complex global and local morphology of PEDOT:PSS.

Here, we reveal how the microstructure impacts the mechanical and electrical properties of PEDOT:PSS using a combination of cryogenic electron microscopy (cryo-EM), electron energy loss spectroscopy (EELS), and X-ray scattering. Exploiting high-resolution cryo-EM reveals the nanostructure evolution of PEDOT:PSS dispersions upon the addition of ionic and nonionic additives. We used bis(trifluoromethane)sulfonimide lithium (Li-TFSI) and 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIM-TFSI) salts due to their strong cation exchange capability with PEDOT:PSS22. Polyethylene glycol diacrylate (PEGDA) was selected as a nonionic additive because it can enable highly-conductive, stretchable PEDOT:PSS-based bioelectronics9,10. Our results reveal that ionic and PEGDA additives lead to the formation of elongated fibers and chain packing in solutions. Further analysis of cryo-EM results reveals that elongated fibers form through the coalescence and self-assembly of PEDOT:PSS micelles that often exhibit an outer layer of extended and stacked chains. The presence of these heterostructural fibers has direct implications on the mechanical and electrical properties of PEDOT:PSS.

High-resolution cryo-EM imaging of solid films, which are processed from PEDOT:PSS solutions with the various additives, confirms the formation and growth of crystalline domains and heterostructural fibers in solid samples. Furthermore, by employing a combination of cryo-EM, EELS, and an automated crystal feature extraction framework (called Graph based Analysis of Transmission Electron Microscopy, GRATEv225), we show how exposure to moisture reorganizes the PEDOT:PSS nanostructure leading to smaller crystalline domains while heterostructural fibers swell. The former observation is consistent with the reduction in electrical conductivity of PEDOT:PSS in the presence of moisture26,27, while the latter explains the complex mechanical properties of PEDOT:PSS with ionic and PEGDA additives. Our results also show that additives can enhance the hydrophilicity of PEDOT:PSS, leading to higher moisture absorbance and resulting in higher mechanical compliance and a larger crack onset strain in these samples. Subsequently, the combination of heterostructural fibers swelling and reorganization of crystalline domains leads to the formation of simultaneously conductive and stretchable PEDOT:PSS thin films. Our work provides a framework for the complex interplay between morphological, mechanical, and electrical properties of modern formulations of PEDOT:PSS used in different applications, such as implant electrodes5, wearable electronics9, and organic electrochemical transistors8,28.

Results
Cryo-EM of PEDOT:PSS dispersions
Beam damage is one of the main factors limiting the resolution and detailed analysis of organic structures in real space. Cryo-EM has been extensively used in the study of biological samples29,30 to both minimize electron beam damage31–33 and enable studies of hydrated (vitrified) samples31–33. We used cryo-EM to track PEDOT:PSS self-assembly with salt additives. Previously, we showed that the addition of ionic species lead to the formation and self-assembly of elongated fibers in the solution phase of PEDOT:PSS3. Other recent studies have also reported elongated fibrils in doped PEDOT:PSS17,34,35. Despite these initial observations, the lack of high-resolution structural imaging of PEDOT:PSS in either solution or solid films precludes a complete description of PEDOT:PSS phase formation and microstructure. Specifically, the distribution of crystalline domains and the inner structure of elongated fibers in highly conductive and stretchable PEDOT:PSS remain underexplored.

Solution cryo-EM measurements were performed on vitrified PEDOT:PSS samples at liquid nitrogen temperature (see the Methods section for details). Figure 1a–b shows a schematic of the plunge freezing of TEM grids and the chemical structure of PEDOT:PSS and ionic and PEGDA additives, respectively. Schematics of PEDOT:PSS dispersions containing isolated micelles and aggregated micelles in heterostructural fibers are shown in Fig. 1c–d, respectively. High-resolution cryo-EM shows the presence of two distinct domains in neat PEDOT:PSS solutions, (i) isolated micelles with a size of 4.1 ± 1.3 nm, and (ii) elongated fibers (Fig. 1e–g). On the other hand, PEDOT:PSS solutions containing ionic and PEGDA additives, in addition to the higher density of elongated fibers, also show lattice planes of PEDOT:PSS in solution (Fig. 1h–p).

Previous work has used X-ray scattering measurements to propose that PEDOT:PSS micelles consist of a PEDOT-rich nanocrystalline core and a PSS-rich corona15. The higher mass density of the crystalline PEDOT-rich core when compared to the amorphous PSS-rich corona leads us to assume that electron scattering from the micelles is dominated by the core region. As a result, we associate the ca. 3–4 nm features in cryo-EM micrographs with the PEDOT-rich core of PEDOT:PSS micelles. At this point, the size of the PSS-rich corona region cannot be independently extracted from cryo-EM, mainly due to the small scattering contrast between solvated PSS-rich regions and water.

A comparison of micelle sizes in solutions reveals that ionic additives decrease the diameter of PEDOT-rich cores (3.1 ± 0.5 nm), while solutions with PEGDA exhibit micelle sizes comparable to those of neat PEDOT:PSS (3.9 ± 0.8 nm) (see Figs. S1–S2). A reduction in micelle size upon the addition of salts to PEDOT:PSS has been previously reported, which has been attributed to the screening of interchain charge repulsion15,36. Previously reported micelle sizes extracted from solutions of PEDOT:PSS using measurements of the hydrodynamic radius obtained from dynamic light scattering (DLS) show a dominant size distribution between 3 to 15 nm and a few micelles up to 30 nm37. As shown in Fig. 1, we also observe smaller micelles and larger aggregate structures. In addition, given that solvated PSS chains are likely not visible in cryo-EM, we expect micelles in our micrographs to appear smaller than values reported from DLS or SAXS. The micelle size apparent from our cryo-EM images and previous work37 suggests that PEDOT exists as oligomers with limited chain lengths.

We attribute the chain stacking with 1.4–1.6 nm spacing, observed in solution cryo-EM micrographs, to PEDOT:PSS lamella stacks that have previously been attributed to the presence of alternating PEDOT and PSS chains3,38. Examples of these lamellae lattice fringes (parallel lines in cryo-EM micrographs) are indicated by the orange arrows in Fig. 1i–o. To rule out Fresnel-fringe or defocus artifacts, we measured the lamellar spacing at multiple defocus settings; the spacing (~1.5 nm) remained invariant with defocus (Figs. S3–S4). As mentioned before, cryo-EM micrographs reveal fibers with a complex heterostructure of coalesced polymer micelles and extended stacked chains that we attribute to a crystalline, or “proto-crystalline” phase. Figure 1h–j shows that these heterostructural fibers consist of small micelles (core of about 3 nm) surrounded by a crystalline phase parallel to the long axis of the fibers.

As shown in Fig. S5, and consistent with the Vitrobot-prepared (“blot-based”) micrographs in Fig. 1, samples prepared with Vitroject (non-contact, jet-based)39 also display clear elongated fibers in PEDOT:PSS+Li-TFSI dispersions. The agreement between blot-based and non-contact preparation highlights that these fibers are intrinsic to the PEDOT:PSS formulations, not artifacts of the blotting process. Furthermore, these heterostructural fibers can also be observed in PEDOT:PSS+EMIM-TFSI and PEDOT:PSS+PEGDA solution mixtures (Fig. 1k–m, n–p). Additional examples of micrographs collected at different defocus settings and micrographs highlighting lamella stacks plus micelles in PEDOT:PSS dispersions are shown in Figs. S6 and S7. We observe that increasing the self-assembly time after adding the additives leads to more elongated fibers and fewer isolated micelles in PEDOT:PSS+EMIM-TFSI dispersions (Fig. S8). This rise in fiber density with time, together with the drop in isolated micelles, is consistent with our prior results3 and points to micelle coalescence driving the formation of elongated fibers. As will be discussed in the following sections, the presence of crystalline phases and heterostructural fibers has a significant effect on the physical and electrical properties of PEDOT:PSS thin films.

Given the similar trends across various PEDOT:PSS systems, subsequent sections center on PEDOT:PSS+Li-TFSI as a model system to examine morphological evolution of PEDOT:PSS processed with additives.

Cryo-EM of solid films
To elucidate how films assemble from PEDOT:PSS solutions, we performed cryo-EM on spin-coated thin films of PEDOT:PSS processed with and without additives. At lower magnifications, neat PEDOT:PSS films (Fig. S9) show a uniform morphology, while magnified micrographs also show the presence of both PEDOT:PSS lamella stacks and micelles. The crystalline domains in neat PEDOT:PSS are generally around 10-15 nm long and <5 nm wide. The PEDOT-rich cores of the micelles in neat PEDOT:PSS films have an oval shape with a long axis of ~3.5 nm. Unlike in neat PEDOT:PSS solutions, elongated fibers are not visible in cryo-EM micrographs of solid PEDOT:PSS films. We speculate that fibers in neat PEDOT:PSS strongly coalesce or breakdown, making them challenging to identify in micrographs. On the other hand, PEDOT:PSS+Li-TFSI (see Fig. 2a) and PEDOT:PSS+PEGDA (see Fig. S10) samples show a significantly more heterogeneous structure than neat PEDOT:PSS. This is in line with the presence of more fibers and crystalline domains in solutions of additive-containing PEDOT:PSS formulations. Similarly to the aggregates visible in solution, high magnification of the structure reveals that elongated fibers in PEDOT:PSS+Li-TFSI thin films appear to form through the coalescence of polymer micelles (Fig. 2b). Indeed, lamellar structures are now apparent throughout the film and are not limited to areas surrounding micellar fibers.

Overall, we see a close resemblance between solution and solid domains in PEDOT:PSS+Li-TFSI, with larger crystalline domains (>20 nm thick crystals) observed in the solid samples. Thus, the heterostructural fibers and crystalline domains in solution template the growth of the subsequent structures in solid thin films. We also see a fibril structure in samples containing ethylene glycol as a solvent additive and (3-glycidyloxypropyl)trimethoxysilane (GOPS) cross-linker (Fig. S12). PEDOT:PSS+ethylene glycol+GOPS is perhaps the most common formulation used in organic electrochemical transistor devices. Further analysis of cryo-EM micrographs using frequency cut-off filters (see Fig. S13) reveals that the crystalline domains in PEDOT:PSS+Li-TFSI do not align with the orientation of heterostructured fibers. Therefore, although large fibrils in PEDOT:PSS are assumed to be composed of PEDOT (or PEDOT-rich) crystals11,13,17, our work clearly shows that micelles, likely composed of PEDOT and PSS mixtures, make up these domains. We speculate that this structure, with PEDOT proto-crystals coating the outside of micellar structures, is crucial for establishing highly conducting phases by precluding the need to disrupt an insulating PSS corona that was previously assumed to be present.

Besides the PEDOT:PSS lamellar phase with 1.4–1.6 nm spacing (which could correspond to the PEDOT:PSS (100) crystal planes)40, cryo-EM also captures domains with a lattice spacing of ~0.7 nm. The latter spacing corresponds to the pure PEDOT lamella stacking (see Fig. S14 for cryo-EM micrograph and subsequent FFT of the crystalline domains with ~0.7 nm spacing). Compared to in solution, lamella stacks spacings appear to be slightly larger in solid samples. To further investigate PEDOT:PSS microstructure, we performed grazing incidence wide-angle X-ray scattering (GIWAXS) measurements on PEDOT:PSS samples with and without additives. Figures 2e and S11 show 1D GIWAXS profiles along the in-plane and out-of-plane orientations, respectively, for neat PEDOT:PSS and PEDOT:PSS+Li-TFSI thin films. Figure S11 further shows GIWAXS profiles of additional PEDOT:PSS thin films processed with additives. Previous work has associated the peaks at 0.39 Å−1 (16.1 Å) and 0.28 Å−1 (22.5 Å) with stacking of two different variations of alternating PEDOT and PSS chains18,38,41 (as shown in Fig. S15 and Table S1). The peaks at 0.89 Å−1 (7.1 Å), 1.27 Å−1 (4.9 Å), and 1.8 Å−1 (3.5 Å) are associated with the neat PEDOT lamella stacking7, PSS chain stacking18, and PEDOT π-π stacking, respectively18.

The reported diffraction peak positions and spacings can vary slightly depending on the type of additive and measurement conditions, such as humidity18,42. Because lattice fringes are visible only when the electron beam is approximately parallel to those lattice planes, periodicities oriented out-of-plane, which project along the beam, are not visible in TEM micrographs. Thus, the spacings apparent in in-plane GIWAXS (lamella stacks with 15–16 Å spacing) correspond to visible chain stacking in TEM micrographs. Overall, the combination of solution and solid film cryo-EM provides an opportunity to identify key features of the microstructure and how they evolve from solution to film.

Solid-liquid Interactions
We expect different responses from crystalline domains and heterostructural fibers when exposed to moisture. To investigate this hypothesis, we exposed PEDOT:PSS+Li-TFSI thin films to moisture (100% relative humidity (RH)) before plunge-freezing the samples with liquid ethane (see Methods section for further details). This approach allows us to monitor the morphological evolution in high-conductivity PEDOT:PSS thin films in the presence of water.

As shown in cryo-EM images in Fig. 2c, upon exposure to moisture, PEDOT:PSS+Li-TFSI thin films show a uniform morphology with some inhomogeneity observed in the density of PEDOT:PSS micelles. We also show HRTEM images of PEDOT:PSS+Li-TFSI recorded at 30% and 100% relative humidity under varying defocus conditions in Fig. S16. Moisture exposure improves the contrast of polymer micelles, as can be seen in Fig. 2d. Given the expected thickness increase of PEDOT:PSS films upon hydration42,43, the improvement in scattering contrast cannot be explained by reduced inelastic electron scattering or a reduced number of overlapping features in hydrated samples. Subsequently, we deduce that upon exposure to moisture, the PSS-rich shell of the micelles swells, decreasing the scattering from the shell and improving the contrast between the PEDOT-rich core and the swollen PSS-rich corona. This shows that heterostructural fibers consist of self-assembled core-shell PEDOT:PSS micelles that swell upon exposure to water. Figure 2f shows the 1D small angle X-ray scattering (SAXS) SAXS profiles of drop cast neat PEDOT:PSS and PEDOT:PSS+Li-TFSI at two different RH levels. While the SAXS profiles from neat PEDOT:PSS show minimal differences between 40% and 85% RH, PEDOT:PSS+Li-TFSI samples show a clear change that corresponds to the formation of mesoscale structures upon exposure to higher moisture.

The limited field of view of high-resolution TEM (HRTEM) images often limits analyses to qualitative changes. Automated sampling and large-quantity data collection, developed for the study of biological samples44–47, may promote the extraction of quantitative microstructural information from HRTEM. This approach remains under-explored for the quantitative analysis of the morphology of conjugated polymers. Recently, Gamdha et al.25 developed software for a Graph based Analysis of Transmission Electron Microscopy (GRATEv2) of polymeric samples25,48, which can identify crystalline domains from images at high speed, providing information on domain size, orientation, and area.

We employed GRATEv2 to analyze crystalline domain size and distribution in PEDOT:PSS+Li-TFSI thin films before and after hydration (exposure to 100% RH). HRTEM micrographs of PEDOT:PSS+Li-TFSI thin films at 30% and 100% RH can be seen in Fig. 3a–b, respectively. Figure 3c shows the flow chart of the GRATEv2 analysis. Figure 3d–e shows the subsequent GRATEv2 output figures of micrographs presented in Fig. 3a–b, respectively. The crystalline domains with a lattice spacing of ~1.5-16 nm ((100) PEDOT:PSS lamella spacing) are shown with the purple regions. Additional cryo-EM examples at 30% and 100% RH, together with their GRATEv2 analyses, are shown in Figure S17. A histogram of crystalline domain sizes is presented in Fig. 3f. HRTEM shows that hydration leads to the formation of smaller crystalline domains in PEDOT:PSS+Li-TFSI with an average domain area of 117 nm2 at 30% RH versus 74 nm2 at 100% RH. These results show that crystalline domains respond differently to water absorption compared to heterostructural fibers. While crystalline domains shrink in size, heterostructural fibers swell significantly upon hydration. The distinct difference in microstructural evolution of crystalline domains and heterostructural fibers upon hydration shows that these two domains possess different nanostructures. As will be discussed in the next section, the direct imaging of nanocrystalline domains and heterostructural fibers allows us to explain the mechanical and electrical properties of PEDOT:PSS upon exposure to humidity and moisture.

Electrical and mechanical properties of PEDOT:PSS
The high electrical conductivity and mechanical compliance of PEDOT:PSS, often achieved by the addition of ionic additives, are the main factors driving the widespread use of this mixed ionic-electronic polymer in various applications3,7,9,49. In this section, we explore the correlation between morphology, conductivity, and mechanical properties of PEDOT:PSS in the presence of ionic additives. Consistent with prior studies7,12,13,22, we observe an increase in the electrical conductivity of PEDOT:PSS thin films by two to three orders of magnitude following film processing using ionic additives and water treatment of salt-containing films (Fig. S18). The significant increase in electrical conductivity of PEDOT:PSS is often closely correlated with an increase in crystallinity of PEDOT:PSS processed with different additives7,12,13. This is consistent with the larger number of crystalline domains observed in cryo-EM of PEDOT:PSS+Li-TFSI thin films (See Fig. 3d).

Compared to the mechanisms behind the enhancement of electrical conductivity, the improvement in mechanical properties of PEDOT:PSS in the presence of ionic additives is less understood. To ascertain the role of structure on mechanical properties in PEDOT:PSS, we performed crack onset strain (COS) measurements of neat PEDOT:PSS and PEDOT:PSS+Li-TFSI at two different humidity levels (See Methods section for more details). At 70% RH, cracks appear in neat PEDOT:PSS and PEDOT:PSS+Li-TFSI at 11±5 % and 27±8 % strains, respectively (see Fig. 4a). On the other hand, when the same measurements were performed in a 0% RH glovebox, both neat PEDOT:PSS and the additive-loaded samples cracked at much lower strains (~2% COS), with dry PEDOT:PSS+Li-TFSI showing even slightly less stretchability compared to neat PEDOT:PSS. Upon hydration, a similar increasing trend in COS is also observed in PEDOT:PSS+PEGDA (Fig. S19). These results indicate that moisture uptake plays a substantial role in the mechanical properties of PEDOT:PSS processed with additives, including salts50.

To further investigate the effect of ionic additives and their subsequent removal upon water treatment, we performed thermogravimetric analysis (TGA) on neat PEDOT:PSS and PEDOT:PSS+Li-TFSI; results are shown in Fig. 4b. Neat PEDOT:PSS show two main mass loss transitions: the first transition completes around 100 °C; this transition is due to the evaporation of moisture within the PEDOT:PSS structure. The second transition at ~275 °C is associated with the chemical degradation of PEDOT:PSS. On the other hand, PEDOT:PSS+Li-TFSI shows a significantly different TGA profile. TGA from PEDOT:PSS+Li-TFSI does not show clear thermal transitions, but instead a continuous loss of sample mass, with an overall larger mass loss percentage compared to neat PEDOT:PSS. After water treatment (removing added salts), the thermogram reverts to the two-step profile for neat PEDOT:PSS, confirming that water absorption due to Li-TFSI is responsible for the broadened, continuous loss of sample mass (Fig. S20). The water uptake of films containing Li-TFSI likely leads to the formation of water-salt complexes within the PEDOT:PSS structure51,52.

We also measured the water uptake due to Li-TFSI using scanning transmission electron microscopy-electron energy loss spectroscopy (STEM-EELS) at room temperature and cryogenic conditions (Fig. S21). STEM-EELS allows us to establish a compositional-morphological relationship in PEDOT:PSS thin films processed with hydrophilic additives. Although ionic additives are linked to the improved stretchability of PEDOT:PSS, upon water treatment, the stretchability of PEDOT:PSS films significantly drops due to the removal of these additives13. We associate this behavior with the formation of water-salt complexes in PEDOT:PSS and subsequent elimination of these complexes after water treatment. This is consistent with the increase in oxygen composition (as an indicator for water absorption) when we compare PEDOT:PSS+Li-TFSI thin films before and after salt removal (Fig. 4c and Fig. S22).

Methods
Solution preparation
PEDOT:PSS solutions were purchased from Heraeus (Clevios PH1000) and were used without further modification for neat PEDOT:PSS samples. To minimize batch-to-batch variation, all cryo-EM measurements reported here were performed on a single batch (Batch# 9003838499, received on 07/2019). For measurements that required larger quantities of material (TGA and mechanical testing), we used a second PH1000 lot (Batch# 9008427769, received on 02/2022). To check that batch differences do not affect the main morphological conclusions, we also prepared films from both lots and imaged them by cryo-EM. As shown in Fig. S24, the resulting morphologies are similar in both cases, with fibrillar regions visible in each. No filtration was performed for liquid or solid sample processing. Unless otherwise mentioned, all other chemicals used in this study were purchased from Millipore Sigma. Stock solutions of Li-TFSI (Lithium bis(trifluoromethanesulfonyl)imide and EMIM-TFSI (1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide) were made in water with 320 mg/mL concentration. For PEDOT:PSS+salt samples, an appropriate amount of salt solution was added to the as-received PEDOT:PSS to achieve a dispersion with 60 wt% PEDOT:PSS solid content and 40 wt% salt content (assuming 1.2% solid content of as-received PEDOT:PSS solution). These dispersions were used without further purification. For PEDOT:PSS+EMIM-TFSI dispersions, the salt solution was added to the PEDOT:PSS dispersion in 10–20 µL portions to avoid strong aggregation of PEDOT:PSS upon the addition of EMIM-TFSI. All solutions, after being vortexed for 5 min, were stored in amber vials at room temperature before processing. PEDOT:PSS films were prepared by spin-coating solutions on pre-cleaned substrates with a 4000 revolutions per minute (rpm) spin speed. The coated substrates were further annealed at 125 °C for 10 min. Water-treated films were prepared by exposing the pre-annealed PEDOT:PSS+Li-TFSI thin films to deionized water (DI water) for 60 s. These films were further spun at 4000 rpm to dry the films and were annealed at 125 °C for 10 min.

Cryo-EM sample preparation
2D micrographs of the solution were acquired using cryo-EM. Quantifoil Holey Carbon Grids, 300 mesh, with 1 μm hole size and 2 μm spacings (Quantifoil MicroTools, Jena, Germany) were used for the vitrification of neat PEDOT:PSS and additive-containing PEDOT:PSS solutions. A FEI Vitrobot (FEI Company, Hillsboro, OR) was used for the vitrification of PEDOT:PSS solutions. All solutions were kept at room temperature before plunge freezing in liquid ethane. The Vitrobot chamber was kept at 4 °C at 10% relative humidity (RH). 3.5 µL of the solution was administrated to the TEM grids for liquid cryo-EM sample preparation. The vitrification was done using filter papers mounted to the blotting pads with one or two cycles of blotting based on the viscosity of the samples. PEDOT:PSS+salt solutions usually possess a higher viscosity, which necessitates at least two cycles of blotting to achieve a thin enough specimen for TEM imaging. Blotting cycles each lasted for 3 s, with a total blotting time of 6 s for two cycles. The grids were quickly moved to liquid nitrogen containers after the initial vitrification. High-resolution cryo-EM imaging of liquid samples was performed on the FEI Talos Arctica (Thermo Fisher Scientific, OR, USA) at the Huck Institute of the Life Sciences at the Pennsylvania State University. The measurements were conducted using a 200 kV electron source and a Falcon 4 direct electron detector in counting mode. The nanoprobe mode of the microscope with a 70 μm C2 aperture, with a spot size of 5, was employed to collect the TEM micrographs. All cryo-EM micrographs were collected with an electron dose of <40 e/Å2. Magnifications of 73,000x, 92,000x, 120,000x, and 150,000x with the pixel sizes of 0.206 nm, 0.153 nm, 0.122 nm, and 0.0933 nm, respectively, were used for cryo-EM imaging of vitrified solutions. Binning of 1 (no camera pixel binning) was used for the images with 4096×4096 pixels. Binning of 2 was used for displaying of cryo-EM micrographs. Digital Micrograph software was used for Fast Fourier transform of cryo-EM micrographs. Fiji software was used for azimuthal integration of 2D FFT profiles. Additional PEDOT:PSS+Li-TFSI samples were prepared using the CryoSol VitroJet vitrification system operated at 4 °C and 100% RH. Samples were pin-printed onto Quantifoil grids (R2/1, 200 mesh) under the setting for medium ice thickness (Plasma cleaning, 60 s; Spacing, 90 μm; Standoff, 10 μm; Velocity, 1 mm/s; Pin dewpoint offset, 0.1 °C; Grid dewpoint offset, 0 °C; spiral pattern for deposition), followed by rapid vitrification with a liquid ethane jet. The controlled environment ensured reproducible film thickness and minimized sample evaporation prior to freezing.

Solid PEDOT:PSS thin films for cryo-EM and standard TEM measurements were prepared by spin coating of PEDOT:PSS dispersions on a silicon (Si) substrate with a sacrificial layer of PMMA. Silicon/PMMA samples were exposed to UV-Ozone for a 10 min duration to improve the surface energy of the PMMA layer. This leads to better wettability and coverage of PEDOT:PSS on PMMA. After thermal annealing at 125 °C, the Si/PMMA/PEDOT:PSS samples were scratched with metal tweezers and submerged in chlorobenzene. Due to the preferential solubility of PMMA in chlorobenzene, we achieved free-standing PEDOT:PSS thin films which were subsequently picked up by Lacey carbon TEM grids. In the next step, TEM grids were annealed at 90 °C for 10 min to ensure no residual solvents remained on the film.

Moisture-induced thin films were prepared by exposing solid films on TEM grids to either 30 or 100% RH in an FEI Vitrobot chamber before vitrification of the TEM grids with liquid ethane. The chamber temperature was kept at 25 °C before vitrification to limit the formation of frost on solid films during the vitrification process. TEM grids were stored at each humidity level for at least 20 min to ensure the proper swelling of the thin films. No blotting was employed before thin sample vitrification. TEM grids were stored in liquid nitrogen before clipping and loading in the autosampler.

Unless otherwise mentioned, cryo-EM micrographs of solid samples were collected on an FEI Titan Krios microscope (Thermo Fisher Scientific, OR, USA) at the Huck Institute of the Life Sciences at the Pennsylvania State University. The measurements were conducted using a 300 kV electron source and a Falcon 4 direct electron detector in counting mode. The nanoprobe mode of the microscope with a 70 μm C2 aperture and a spot size of 5 was employed to collect TEM micrographs. All cryo-EM micrographs were collected with an electron dose of <50 e/Å2. High-resolution micrographs and low-resolution micrographs of solid thin films were collected at 150,000x (pixel size of 0.043 nm), and 47,000x magnifications (pixel size of 0.14 nm), respectively. At high magnification, the electron dose rate was set to be less than 30 e/Å2 s. Binning of 1 (no camera pixel binning) was used for the imaging with 4096×4096 pixels. Binning of 2 and 4 were used for displaying of micrographs imaged at 47,000x and 150,000x, respectively.

For cryo-EM of solutions, we use 4 °C with 100% RH to suppress evaporation in the blot-to-plunge interval and to promote formation of uniform vitreous ice. In contrast, for hydrated solid films, cooling the chamber (and therefore the Cu grid) below ambient temperature can lead to water condensation on the film. Upon plunging, these water droplets can convert to crystalline ice, and affect the film morphology and TEM imaging. To avoid condensation while maintaining hydration, we therefore set the chamber to 25 °C for solid films. The differences between Fig. 2b/2d and Fig. 3a/3b arise mainly from imaging magnification. Figure 3a–b were recorded at higher magnification (150,000x) than Fig. 2 (47,000x), giving a smaller pixel size and better sampling of high spatial frequencies; as a result, lattice planes are resolved more clearly. In contrast, the lower-magnification images provide a larger field of view and stronger low-frequency contrast, so micelles and lamellar spacing can be visualized in the same frame. To extract defocus values for both solid and solution cryo-EM micrographs, we used the EMAN2 software package. EMAN2 estimates the defocus values by analyzing the contrast transfer function (CTF) patterns in the Fourier transform (FFT) of micrographs and is more accurate than readings from the microscope that rely on establishing the eucentric height60. In EMAN2 CTF fitting, all parameters were left at their default values except pixel size, accelerating voltage (kV), and spherical aberration (Cs, mm). Cs was set to 0.01 mm for the Krios and 2.7 mm for the Arctica.

STEM-EELS
Standard STEM-EELS results were acquired using a Talos F200X (Thermo Fisher Scientific, OR, USA) at an acceleration voltage of 200 kV equipped with a Gatan Imaging Filter (GIF). EELS results were collected with 0.3 eV/channel dispersion, 16 µs pixel time, and 30 nm step size. Auto zero loss peak detection and auto-focus were performed prior to the data collection. Cryo-STEM-EELS measurements were performed on a FEI Titan Krios microscope equipped with a Gatan energy filter and K2 camera. The micro-probe mode was used for Cryo-STEM-EELS to reduce the local temperature increase during EELS data collection. Cryo-STEM-EELS results were collected using a high signal-to-noise ratio (Hi-SNR) aperture with 0.5 eV/channel dispersion, 15 nm pixel size, and 5 s pixel time with the EELS exposure time of 0.1 s at 130 eV (high loss).

Grazing-incidence wide-angle X-ray scattering
For grazing-incidence wide-angle X-ray scattering (GIWAXS), samples were spin coated on pre-cleaned bare Si substrates at 4000 rpm. GIWAXS was conducted at beamline 7.3.3, Advanced Light Source, Lawerence Berkeley National Laboratory61. X-ray energy, beam current, and sample-detector distance were 10 keV, 500 mA, and 281 mm. AgB was used to calibrate the Pilatus 2M detector. GIWAXS scans of samples were measured at 0.12, 0.14, and 0.16° incidence angle for an exposure time of 10 s. In-plane and out-of-plane sector cuts of 2D scattering images ranged 18° from 0° to 90°, respectively. XI-CAM was used to reduce 2D scattering images to 1D sector cuts62.

Small-angle X-ray scattering
SAXS measurements were conducted at beamline 7.3.3, Advanced Light Source, Lawrence Berkeley National Laboratory62. A 10 keV X-ray beam was used to probe the sample at 0°. A Pilatus 2M detector collected 2D scattering data at a distance 3.5 m away from the sample. 2D scattering images were reduced to 1D profiles using Nika on Igor Pro63. For film samples, films were cast onto 100 nm thick silicon nitride windows (Norcada NX5100C). The drop cast films were left at room temperature overnight to dry. In the next step, the silicon nitride coated samples were annealed at 120 °C for 30 min to remove water residual. Film samples were loaded into a 3D-printed humidity chamber with Kapton windows. Humid air from a LINKAM RH95 humidity controller flowed into the chamber at controlled flow rates to keep the environment static at 40% and 85% RH. SAXS of a blank silicon nitride window was used for background subtraction for each RH. 120-second exposure times were used.

Conductivity
A Micromanipulator Co. 2800 manual probe station and Keithley 4200 SCS Parameter Analyzer were used to conduct independent four-point probe measurements inside an anhydrous, N2 glovebox. Probes were 44-6000-S-NA Probe Holder with Integrated Tip from Micromanipulator Co. Voltage drop was measured while sweeping current for four-point probe measurements, where the Au electrode distance was 100 μm. Resistances were extracted from the slopes of independent sweeps, and film thicknesses measured via profilometry were used to calculate conductivities. Conductivities were averaged from 9, 6, and 16 independent electrode sets for neat PEDOT:PSS, PEDOT:PSS+Li-TFSI, and PEDOT:PSS+Li-TFSI-w, respectively. Au electrode dimensions for four-point probe measurements were 220 μm wide, 100 μm long, and 100 nm thick.

GRATEv2 analysis
We employed the open-source GRATEv2 (Graph-based Analysis of TEM) framework to identify and characterize crystalline domains in the HRTEM images of our polymeric samples. In brief, GRATEv2 executes a parameterized image-processing pipeline that involves (1) morphological operations (e.g., blurring, thresholding, opening and closing), (2) skeletonizing line-like image features, and (3) constructing a graph representation to cluster those skeletal elements into distinct crystalline regions. This approach is particularly effective for complex polymer morphologies, as it robustly separates overlapping or coalesced crystal domains while preserving critical features such as boundaries and orientation angles. Following detection, GRATEv2 also extracts essential microstructural metrics (e.g., crystal area, orientation, aspect ratios, and d-spacing), enabling quantitative comparisons across different samples.

For our dataset, the employed parameters are shown in Table 1 for the GRATEv2 pipeline. These parameters were chosen to balance sensitivity to faint or irregular crystal edges with robustness against small artifacts. The GRATEv2 output includes a segmentation mask of the detected crystals, as well as a table of individual crystal properties (e.g., d-spacing and orientation), providing a comprehensive view of each sample’s microstructure for subsequent analyses.

Crack onset strain
Polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (SEBS) was used as a supporting stretchable layer for crack onset strain measurements. SEBS (Millipore Sigma) was dissolved in toluene with 120 mg/mL concentration. The SEBS films were fabricated by drop casting solution in a 7.5 × 7.5 cm2 mold. The drop cast SEBS solution was left at room temperature overnight and subsequently annealed at 60 °C for 1 h. In the next step 30 mL of neat PEDOT:PSS or PEDOT:PSS+Li-TFSI solution was drop cast on the SEBS layer. The SEBS/PEDOT:PSS or SEBS/PEDOT:PSS+Li-TFSI films were kept at room temperature overnight. This step is crucial in achieving a uniform PEDOT:PSS film on the SEBS layer. After the evaporation of the majority of the water content of PEDOT:PSS films, the films were annealed at 60 °C for 1 hour. The SEBS/PEDOT:PSS films were cut into 1 × 4 cm2 samples for crack onset strain measurements (COS). COS measurements were performed using the manual custom-built stretching stage. 0 % RH measurements were performed inside a nitrogen filled glovebox after annealing PEDOT:PSS or PEDOT:PSS+Li-TFSI samples at 80 °C for 1 hour. This annealing step ensures that residual moisture in PEDOT:PSS samples is removed before acquiring COS results. A minimum of three samples were tested for each doping and humidity condition.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Ghasemi and co-authors presents a thorough cryo-EM structural analyses of PEDOT:PSS in solution and in the solid state with and without secondary dopants.\nThe authors aim to shed some light on the morphology of PEDOT:PSS, which is still needed for understanding and controlling the properties of this benchmark polymer.\nThe manuscript is a beautiful demonstration of the abilities of cryo-EM (used mostly for proteins observation or molecular self-assemblies) and of the new generation of high-end microscopes.\nAlthough the PEDOT:PSS molecular structure has not been fully resolved, the real space images of PEDOT:PSS morphology could help a broad audience (including those interested in organic/wearable electronics, bioelectronics, and the EM community,…) to better understand how properties vary in the presence of a dopant or humidity.\nIn general, the method is well-detailed, but additional data and images should be provided, in particular, to rule out artefacts.\nHowever, the heterostructures (fibers consisting of micelles surrounded by a thin layer of PEDOT:PSS lamella) proposed by the authors is unclear.\nThe coexistence of these heterostructures with crystalline domains (not aligned with the fibers) in the doped samples is also unclear.\nSchematics of these heterostructures (for the non-doped and doped PEDOT:PSS) would help the reader.\nPlease see below for more questions and issues that need to be addressed before considering the manuscript for publication.\nWhile Heraeus keep certifying that the PH1000 dispersion contains micelles of 30 nm size, it is now the third research works (to the best of my knowledge) observing fibrils in the dispersion thanks to the use of cryo-EM. (Nat. Commun., 2020, 11, 1–8 ; J. Mater. Chem. C, 2023, 11, 7802-7816 ; and Matter 2024, 7, 1071-1091 (cited in the manuscript)).\nThe authors are not the first ones to observe fibrils by cryo-EM and the previously reported references should be cited.\nI would advise the authors (and the community) to mention the batch number and date of receipt of the PH1000 they used in their study to find out whether or not these fibrils are an effect of a new synthesis pathways recently developed by the supplier.\n1) Morphology of neat PEDOT:PSS in solution\nThe schematic of Figure1c (neat PEDOT:PSS) does not correspond to what is observed in the corresponding TEM image (Figure 1e) where elongated fibrils are observed. Elongated fibrils may be composed of micelles (a periodic electronic contrast along the main axis of the fibrils is seen, but no isolated micelles are observed on the TEM image). The corresponding description page 5 line 127 should also be adjusted. Otherwise, the authors should provide a more convincing TEM image showing isolated micelles.\nThe enlarge Figures 1f and 1e where the authors have highlighted round shape objects are not convincing. Raw TEM images1f and 1g (without the red dots) could be provided.\nThe magnification at which cryo-TEM images of the frozen solution were taken (as done in the case of solid sample) should be given.\nThe CTF value should be given as the images are taken at high defocus. In SI, some images at low defocus could be provided.\n2) Morphology of doped PEDOT:PSS in solution\nAlthough the images of solid films are more convincing, the term 'lattice planes' used by the authors in Figure 1 is open to question.\nHow can the authors be sure that these ‘lattice planes’ are not Fresnel fringes (particularly in blue squares Figures 1 h, k, n)?\nFresnel fringes (Fukushima et al. J.Phys.D:Appl. Phys., vol 7, 1974, 257-268) are characterized by a first intense signal close to the specimen, followed by an attenuated distribution outside the specimen. The periodicity of Fresnel fringes is also known to evolve with the defocus. Such intensity and periodicity variations are noticeable in the cryo-EM images (from the nearest to the furthest from the fibril).\nThe authors should specify the defocus at which the images were taken, their CTF and provide the same images with a lower defocus value to confirm whether the so-called lattice planes exist.\nWhy are the lattice lines so far away from the fibrils (mostly visible in Figure 1o)?\nThese features can also be seen in Figure 1e (bottom right), for the non-doped sample. How do the authors explain this?\nHow do the authors explain the co-existence of fibers and lattice planes? (A schematic would help the reader.)\nIt looks like the PEGDA and EMIM-TFSI images have not been taken at a same defocus. (which could explain the variation in micelle size and the lattice parameters). Can the authors study the object size at a similar defocus (for non-doped, and Li-TFSI, PEGDA and EMIM-TFSI samples)?\nHow can the authors be sure that Li-TFSI or EMIM-TFSI do not self-assemble (and are not at the origin of the so-called lattice planes)?\np17: The authors conclude that ionic dopant leads to a breakdown of micelles. This is not consistent with what is observed in Figure 1. Elongated fibers are thinner in diameter with ionic dopant. But the fibers still show a periodic electronic contrast along their main axis (which may be micelles). Isolated ‘micelles’ might even be seen in Figure 1n and 1p.\n3) Morphology of doped PEDOT:PSS in solid state\nWhen comparing the cryo-TEM images of the solution and solid film of the neat PEDOT:PSS, the elongated fibers are no longer visible in the solid state. Can the authors explain this? What is the thickness of the film?\nFigure 2a appears to be overfocused (see the fringes around the large contaminant). The other images (2b and c) appear to be underfocused. It is therefore difficult to compare the morphology.\nWhat is the defocus used for the cryo-EM of solid films (as compared to the solution cryo-EM)?\nIn Figure 2b, why are the fringes no longer aligned with the fibers is they are supposedly lattice- planes of the PEDOT:PSS?\nHowever, I agree that what is observed here might not be an artefact. The images are closer to the focus than the images of the solution state.\np14: The authors attribute the variation in the mechanical properties of the PEDOT:PSS + Li-TFSI to the formation of water-salt complexes within the PEDOT:PSS structure. Are these complexes visible in the cryo-EM micrographs?\nHow is the presence of these water-salt complexes compatible with the increase in electrical conductivity?\np11 line241: The cryo-EM images Figures 2b and 2c appear to not have the same defocus (which could impact the contrast). It is therefore not sure that moisture exposure improves the contrast of the micelles.\nThe histogram of the crystalline domain area for the additional images in figure S6 should also be provided. How does it compare with the one reported in Figure 3?\n4) Discussion on the PEDOT:PSS structure.\nPage 10. The authors attribute the 1.5- 1.6 nm spacing to the lamellar phase ‘(which could correspond to the PEDOT:PSS (100) crystal planes).’\nThe reference 36, used, says that ‘the weak hump centered at 0.29 ̊Å-1 is assigned to the lamellar stacking distance [d(100)~22 ̊Å] peak occurring from the alternate ordering of the PEDOT and PSS polymer chains.’\nThe 1.5- 1.6 nm spacing, measured by Ghasemi et al, is much smaller than the 22 Å measured by Massonet et al. Therefore, the attribution of the fringes to the lamella spacing is questionable. Another research group (E. Hosseini, et al, J. Mater. Chem. C, 2020, 8, 3982) has also attributed a spacing of 22 Å to the PEDOT to PEDOT d100 lamellae spacing.\nFig S2. How can the authors be certain that the second spot at 0.7 nm is the PEDOT lamella stack of and not a second-order PEDOT:PSS lamella stack?\nA possible lamellar phase can be seen in the GIWAXS profiles in the out-of-plane orientation of the doped samples (up to the second or even the third order?). The reason why the lamellar phase is observed out-of-plane in GIWAXS while the fringes (attributed to the lamellar phase) observed in cryo-EM (and corresponds to an in-plane orientation, is unclear\nIf I am not mistaken, Figures 2b and 2c ‘zoom in view’ should correspond to Figures 3a and 3b, respectively. However, the structures are different. Figures 2C shows electron-rich and electron poor areas (containing micelles?) while crystalline domains are only visible in Figure 3B. Similarly, the fibers seen in Figure 2b are not visible in 3a. This is inconsistent. Can the authors comment on this?\nA schematic of the structure (PEDOT proto-crystals coating the outside of micellar structure and the co-existence of fibers) proposed by the authors would help the readers.\nSTEM-EELS\n-Is it easy to control precisely the humidity rate in a STEM-EELS?\n-I have missed why the carbon content is decreasing upon humidity.\n-To what correspond the 50-200 nm oxygen -rich regions?\nAs minor comments,\n- The vertical axis and the units are missing in Figure 4a.\n- P10. The assumption that the difference of lamella spacing extracted from cryo-EM and GIWAXS, being due to the temperature measurements, is not valid. (Dubochet et al. Q. Rev. Biophys., 1988, 21, 129–228.)\n- Please add the PH1000 Batch number\n- Has the commercial dispersion been filtered or not (as is usually done by the organic electronics community)?\n- The chamber temperature was kept at 25°C for the solid sample while the solution was kept at 4°C. Was there any particular reason for that?\nSupplementary\n-In Figure S4, is there not also a spot at a greater distance (closer to the direct beam)?\n-Fig S5. A table listing the peak positions and corresponding distances would help the reader to compare the structures with the literature and their own data.", "reviewer_2": "Ghasemi et al report on their characterization of different PEDOT:PSS formulation both in the solution and thin film using electron microscopy. The authors show the hierarchical structure of PEDOT:PSS, with micelles, nanofibers, and semicrystalline and disordered regions. I find the work very exciting. It is incredible to clearly see structural features in the solutions and films that normally must be inferred from indirect measurements. This methodology is certainly the future of polymer microstructural characterization, and I expect this work will be important in the field.\nHowever, the conclusions drawn from the analysis of the images are not entirely clear, and there are questions about the sample preparation and the assumptions made when classifying the features seen in TEM. For this reason, I recommend a revision prior to accepting into Nature Communications. My specific comments are below.\nFirst, the paper needs to explain what exactly is being imaged for the vitrified PEDOT:PSS dispersions. Presumably during blotting, some of the water is absorbed into the paper, concentrating PEDOT:PSS in the dispersion. This likely drives the aggregation into fibril structures that is observed. How representative are these images of the dispersion itself? Is there any evidence for fibers forming in the as is dispersion before the blotting process? And how does the aggregation of PEDOT:PSS micelles during blotting relate to aggregation during other common processing methods (spin-coating, blade-coating, dropcasting, etc.)?\nIt doesn’t appear that there is much variation in particle size within a sample, why is that? DLS characterization of particle sizes in PEDOT:PSS dispersions have shown a wide dispersity [https://doi.org/10.1016/j.jcis.2020.09.070].\nI’m a little confused about the proposed structure in Fig 1c. The cartoons show a micelle with a lot of messy, tangled polymer, with a PEDOT rich core and PSS rich shell. However, the size of the micelle in the images is 2.7 nm up to 4.7 nm, is on the order of 2-3x the PEDOT:PSS lamellar spacing distance when the molecules are tightly packed. How would all of that polymer fit into such a small volume, especially when it isn’t tightly packed? In panel j, the micelle diameter is smaller than two repeated lamellae of the shell of the nanofiber. Is this picture physically feasible?\nAlso, in the cartoons, why are the PEDOT chains drawn as very short segments in the neat film, but very long (spanning several micelles) in the secondary doping case? Shouldn’t the Mw of PEDOT:PSS be constant? What is the image intended to convey?\nIn the FFT insets in Fig 3a and Fig S3, there are several rings all the way out to ~3 nm-1. It is surprising to see so many strong peaks since the GIWAXS ~4 peaks which are fairly weak. What is the physical origin of these rings in the FFT images?\nWhy do the HRTEM images in Fig 3 look so different from images in Fig 2b and 2c? Is the imaging mode different? The scale of the images in Fig 3 appear to be similar to the insets Fig 2. Does this mean some of the contrast in the low-res images is due to imaging artefacts?\nWhile the paper shows several interesting modalities for cryo-EM characterization, there is very little continuity in the sample choice. First, 4 different secondary dopants are investigated for solution imaging, but only two of the four are measured as solid films. The authors then show humidity dependent crystal size but only apply the analysis to one formulation (Li-TFSI secondary dopant) and compare the results to GISAX measured at two different humidities from the TEM experiment humidities. Finally, they show SETM-EELS to explore where moisture is located within solid films, but the sample changes again and the authors introduce PEDOT:PSS + Li-TFSI with salt-removed. A clear demonstration of how these techniques complement one another and can be combined to build up a holistic microstructural picture for a single materials system would be much more convincing than the current variety of samples explored here. I was also surprised that the PEGDA doped PEDOT:PSS was not included in the mechanical analysis since the authors discussed in the introduction that PEGDA is usually added to improve stretchability.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their insightful comments. In response, we have added substantial\nnew cryo-EM data and clarified our interpretation of our results. Below is the point-by-point\nresponse to reviewers’ comments.\nReviewer #1 (Remarks to the Author):\n“The manuscript by Ghasemi and co-authors presents a thorough cryo-EM structural analyses\nof PEDOT:PSS in solution and in the solid state with and without secondary dopants.\nThe authors aim to shed some light on the morphology of PEDOT:PSS, which is still needed for\nunderstanding and controlling the properties of this benchmark polymer.\nThe manuscript is a beautiful demonstration of the abilities of cryo-EM (used mostly for proteins\nobservation or molecular self-assemblies) and of the new generation of high-end microscopes...”\nWe thank the reviewer for the kind comments.\n“In general, the method is well-detailed, but additional data and images should be provided, in\nparticular, to rule out artefacts.”\nWe have added new images, in particular that vary in defocus over the same region, to rule out\nthe possibility of artefacts (Fig S3 and Fig S4)\n“However, the heterostructures (fibers consisting of micelles surrounded by a thin layer of\nPEDOT:PSS lamella) proposed by the authors is unclear.\nThe coexistence of these heterostructures with crystalline domains (not aligned with the fibers) in\nthe doped samples is also unclear.\nSchematics of these heterostructures (for the non-doped and doped PEDOT:PSS) would help the\nreader.”\nWe have now updated our schematic in Fig 1 to clarify the strucutural model that emerges from\nour cryo-EM images.\n“-While Heraeus keep certifying that the PH1000 dispersion contains micelles of 30 nm size, it is\nnow the third research works (to the best of my knowledge) observing fibrils in the dispersion\nthanks to the use of cryo-EM. (Nat. Commun., 2020, 11, 1–8 ; J. Mater. Chem. C, 2023, 11, 7802-\n7816 ; and Matter 2024, 7, 1071-1091 (cited in the manuscript)).\nThe authors are not the first ones to observe fibrils by cryo-EM and the previously reported\nreferences should be cited.”\nWe thank the reviewer for pointing out this important prior work. These references are now\nincluded in our revised manuscript.\n“-I would advise the authors (and the community) to mention the batch number and date of\nreceipt of the PH1000 they used in their study to find out whether or not these fibrils are an\neffect of a new synthesis pathways recently developed by the supplier.”\nWe thank the reviewer for this important suggestion. We have now added the receipt dates and\nbatch numbers of the PEDOT:PSS dispersions to the Methods section. To minimize batch-to-\nbatch variation, all cryo-EM measurements reported here were performed on a single batch. For\nmeasurements that required larger quantities of material (TGA and mechanical testing), we used\na second PH1000 lot.\nTo check that the microstructure does not vary between batches used in this study, we also\nprepared films from both lots and imaged them by cryo-EM. As shown in Fig. S4, the resulting\nmorphologies are similar in both cases, with fibrillar regions visible in each.\n“1) Morphology of neat PEDOT:PSS in solution\n-The schematic of Figure1c (neat PEDOT:PSS) does not correspond to what is observed in the\ncorresponding TEM image (Figure 1e) where elongated fibrils are observed. Elongated fibrils\nmay be composed of micelles (a periodic electronic contrast along the main axis of the fibrils is\nseen, but no isolated micelles are observed on the TEM image). The corresponding description -\npage 5 line 127 should also be adjusted. Otherwise, the authors should provide a more\nconvincing TEM image showing isolated micelles.”\nWe have replaced Fig. 1e–g with micrographs of neat PEDOT:PSS solution that more clearly show\nisolated micelles (blue arrows in Fig. 1g). The presence of isolated micelles in neat PEDOT:PSS\nis also consistent with prior scattering reports on this system1, 2, 3.\nTo visualize isolated micelles more clearly, we also collected cryo-EM images of PEDOT:PSS +\nEMIM-TFSI at a short incubation time (defined as the time the solution is stored after additive is\nintroduced). In our previous work, longer incubation time increased fiber density in PEDOT:PSS.\nFigure S8 presents micrographs acquired from TEM grids prepared 12 hours and 2 days after\nEMIM-TFSI addition; blue arrows in Fig. S8b and d mark representative isolated micelles. The\nmain text has been revised to reference these new cryo-EM micrographs.\nFig. S8. Micelles visible from cryo-EM images. a, Cryo-EM micrograph of PEDOT:PSS+EMIM-TFSI\nsolutions after 12-hours of introducing the ionic additive. The micrograph is collected at 92,000x\nmagnification at a defocus of -2.63 μm. b, 200×200 μm2 zoom-in view of the red box shown in panel a.\nThe blue arrows highlight isolated micelles. c, Cryo-EM micrograph of PEDOT:PSS+EMIM-TFSI\nsolutions after 2 days. The micrograph is collected at 92,000x magnification at a defocus of -2.45 μm. d,\n200×200 μm2 zoom-in view of the red box shown in panel c. The blue arrows point to the isolated micelles.\nConsistent with our prior work, an increase in incubation time increases the fiber density1. We also observe\nfewer isolated micelles together with a higher density of fibers, consistent with micelle coalescence and the\nformation of elongated fibers.\n“-The enlarge Figures 1f and 1e where the authors have highlighted round shape objects are not\nconvincing. Raw TEM images 1f and 1g (without the red dots) could be provided.”\nWe apologize for this confusing aspect of our manuscript. Per the reviewer’s suggestion, we\nremoved the red dots from Fig. 1 for clarity and substituted the marking of micelles with blue\narrows. Since the micrographs with closer defocus values were selected for depiction in Fig. 1, the\noriginal Fig. 1 included in the initial submission was moved to the Supporting Information (Fig\nS6).\n“-The magnification at which cryo-TEM images of the frozen solution were taken (as done in the\ncase of solid sample) should be given.”\nWe apologize for this omission. The magnifications at which micrographs were collected, as well\nas pixel sizes, are now in the caption of figures.\n“-The CTF value should be given as the images are taken at high defocus. In SI, some images at\nlow defocus could be provided.”\nWe apologize for this omission. We now include defocus values for our images, and a defocus\nseries in Fig S3 and Fig S4 for solutions and Fig S7 and S16 for films. As expected, the chain\nstacking outside of micelles is visible regardless of defocus value with no change to the apparent\nspacing.\n“2) Morphology of doped PEDOT:PSS in solution\n-Although the images of solid films are more convincing, the term 'lattice planes' used by the\nauthors in Figure 1 is open to question.\nHow can the authors be sure that these ‘lattice planes’ are not Fresnel fringes (particularly in\nblue squares Figures 1 h, k, n)?”\nWe thank the reviewer for pointing out this potential problem. As mentioned above, we now\ninclude defocus panels in Fig. S3 and Fig. S4. We also perform line scans to confirm that the\nspacing of lattice planes is unaffected by the defocus value, even though the spacing of the Thon\nrings clearly changes (as apparent from FFTs). In addition, some of the fibrils show the lattice\nspacings, while some do not, while Fresnel fringes should appear on the outside of all\naggregates. We believe the inclusion of Fig S3 and S4 substantially enhances the rigor of our\npaper and establishes that Fresnel fringes do not lead to the chain stacking apparent in Fig 1.\nFig. S3. Cryo-EM images at various defocus values. a–c, Cryo-EM micrographs of PEDOT:PSS+EMIM-\nTFSI solutions imaged at three underfocus values (−1.37, −2.20, −2.65 µm). d–f, Magnified views of the\nboxed areas showing persistent lattice fringes (shown by white arrows) at all defocus settings. g, Azimuthal\naverages of FFTs of micrographs shown in panels a-c for the three defocus values (CTF modulation varies\nwith defocus as expected). h, Real-space line profiles across the lattice fringes exhibit a constant peak-to-\npeak spacing of ~1.5 nm, independent of defocus. The invariance of the periodicity across multiple defocus\nvalues rules out Fresnel (defocus-dependent) artifacts and supports an intrinsic structural origin of the\nobserved lattice fringes, which we associate with the lamella stacks of PEDOT:PSS. The a-c micrographs\nshow a 250×250 μm2 area and are acquired at 92,000x magnification with a pixel size of 0.153 nm.\nFig. S4. Cryo-EM images at various defocus values. a–c, Cryo-EM micrographs of PEDOT:PSS+Li-\nTFSI solutions imaged at three underfocus values (−1.1, −1.28, −1.60 µm). d-f, Magnified views of the\nboxed areas showing persistent lattice fringes (shown by white arrows) at all defocus settings. g, Azimuthal\naverages of the FFTs of micrographs shown in panels a-c for the three defocus values (CTF modulation\nvaries with defocus as expected). h, Real-space line profiles across the lattice fringes exhibit a constant\npeak-to-peak spacing of ~1.5 nm, independent of defocus. Similar to PEDOT:PSS+EMIM-TFSI, in images\nof PEDOT:PSS+Li-TFSI, the invariance of the periodicity across multiple defocus values rules out Fresnel\n(defocus-dependent) artifacts and supports an intrinsic structural origin of the observed lattice fringes.\nThese lattice fringes are associated with the lamella stacks of PEDOT:PSS. The a-c micrographs show a\n250×250 μm2 area and are acquired at 92,000x magnification with a pixel size of 0.153 nm.\n“-Why are the lattice lines so far away from the fibrils (mostly visible in Figure 1o)?”\nFigure 1o, now Figure S6k in our revised submission, appears to show some space between the\nfibril and the lattice spacing. We speculate that the high level of defocus (-3.1 µm) may blur the\nedges of the fibrils. The updated Figure 1 now includes images of PEDOT:PSS with and without\nadditives at a more modest level of defocus (ca. -1.6 µm), although we retain the original Figure\n1 images as Figure S6.\n“-These features can also be seen in Figure 1e (bottom right), for the non-doped sample. How do\nthe authors explain this?”\nWe do indeed observe a few lattice fringes in neat PEDOT:PSS. However, the density of these\nfeatures is much less, and can be easily missed (Figure 1e and Figure S6a). We believe the\naddition of salts or PEDGA promotes the formation of fibrils and chain stacking, but that both of\nthese features are present in neat PEDOT:PSS\n“-How do the authors explain the co-existence of fibers and lattice planes? (A schematic would\nhelp the reader.)”\nWe thank the reviewer for this suggestion. We have now modified our schematic in Figure 1 to\nclarify this point. Our work shows that the lattice planes form on the outside of fibrils, and that\nionic additives promote the formation of both fibrils and chain stacking. This is consistent with\nthe observed higher number of crystallites in PEDOT:PSS films and higher conductivity\n(previously reported).\n“-It looks like the PEGDA and EMIM-TFSI images have not been taken at a same defocus.\n(which could explain the variation in micelle size and the lattice parameters). Can the authors\nstudy the object size at a similar defocus (for non-doped, and Li-TFSI, PEGDA and EMIM-TFSI\nsamples)?”\nWe apologize for this oversight. To address this and enable like-for-like evaluation, we replaced\nthe micrographs in Fig. 1 with new cryo-EM micrographs acquired at close defocus values (see\nabove). Fig. S1 also reports micelle-size analysis for PEDOT:PSS+Li-TFSI solutions from\nmicrographs recorded at different magnifications and defocus settings. We can see that the\nextracted micelle sizes are comparable across these conditions.\nFig. S1. Micelle size extraction. a–c, Micrographs recorded at 73,000x (defocus, -2.40 μm),\n120,000x (-1.4 μm), and 150,000x (-1.8 μm), respectively. Insets: 2D FFTs of the corresponding\nimages. d–f, Inverse-FFT reconstructions obtained after applying a low-pass filter to the FFTs,\nshown as the inset, in a–c to enhance micellar contrast (same fields of view as in a–c). g–i, Micelle-\ndiameter distributions extracted from images in d–f, 73,000x magnification (3.2±0.6 nm),\n120,000x (3.1±0.5 nm), and 150,000x (3.3±0.7 nm). Analysis was performed in Fiji using\nthresholded masks and Analyze Particles with a circularity constraint of 0.70–1.00 to select near-\ncircular/oval micelles; identical criteria were used for all magnifications. As can be seen similar\nsize distribution of micelles is extracted for the micrographs acquired at different magnifications,\nalthough, as expected, lower magnification micrographs contain a larger number of micelles.\n“-How can the authors be sure that Li-TFSI or EMIM-TFSI do not self-assemble (and are not at\nthe origin of the so-called lattice planes)?”\nDue to the high solubility of Li-TFSI and EMIM-TFSI in water, we expect a low possibility of\nsalt crystallization in the solution phase. Indeed, Li-TFSI “melts” in ambient air due to its strong\nhygroscopic nature. We also do not capture any crystalline structures in solution or solid cryo-\nEM or through X-ray scattering that can be associated with the salt crystals in these samples.\n“-p17: The authors conclude that ionic dopant leads to a breakdown of micelles. This is not\nconsistent with what is observed in Figure 1. Elongated fibers are thinner in diameter with ionic\ndopant. But the fibers still show a periodic electronic contrast along their main axis (which may\nbe micelles). Isolated ‘micelles’ might even be seen in Figure 1n and 1p.”\nWe apologize for the confusion. We agree that the term “breakdown” could be misleading,\nalthough this is the preferred description of PEDOT:PSS solution-to-film structure formation\nprocess. We have modified our text in the Discussion (pg 16) to now reflect the possibility of\nmicelle coalescence or chain rearrangement, which then leads to fibrils and chain stacking. As\nthe reviewer notes, in many cases evidence of the nearly spherical micelles coming together to\nform fibrils is clearly visible.\n“3) Morphology of doped PEDOT:PSS in solid state\n-When comparing the cryo-TEM images of the solution and solid film of the neat PEDOT:PSS,\nthe elongated fibers are no longer visible in the solid state. Can the authors explain this?”\nWe thank the reviewer for highlighting the difference between solution and solid samples. The\nabsence of elongated fibers in neat PEDOT:PSS solid films is consistent with prior reports from\nour group and others4, 5. During film formation, the fibers in neat PEDOT:PSS appear to\ncoalesce, or perhaps break down into micelles. Another possibility is that overlapping fibrils with\nweak contrast are challenging to ascertain from our cryo-EM images. Detailed investigation into\nthis phenomenon is beyond the scope of this study.\n“What is the thickness of the film?”\nThe thicknesses of the neat PEDOT:PSS and PEDOT:PSS+Li-TFSI are 49.4±3.7 nm, 59.2±8.7\nnm, respectively. These values are now listed in the caption of Fig. S18.\n“-Figure 2a appears to be overfocused (see the fringes around the large contaminant). The other\nimages (2b and c) appear to be underfocused. It is therefore difficult to compare the morphology.\nWhat is the defocus used for the cryo-EM of solid films (as compared to the solution cryo-EM)?”\nWe apologize for this oversight. We substituted Fig. 2a with Fig. S9, which is an underfocused\nmicrograph of neat PEDOT:PSS. The defocus values for micrographs shown in Fig. 2 and 3 are\nnow stated in the caption of each figure. Per reviewer #2’s suggestion, Fig. 2a representing cryo-\nEM of the neat PEDOT:PSS was moved to Supporting Information in the new submission for\nclarity of the main text.\n“-In Figure 2b, why are the fringes no longer aligned with the fibers is they are supposedly\nlattice- planes of the PEDOT:PSS? However, I agree that what is observed here might not be an\nartefact. The images are closer to the focus than the images of the solution state.”\nA benefit of solution cryo-EM is that crystalline features are more likely to be isolated,\nthereby limiting the potential impact of overlapping features. In our cryo-EM images of films, we cannot\nexclude the possibility of overlapping domains. Nevertheless, we believe that the larger extent\nof fibrils not aligned with fibers indicates that the solution structure templates further growth of\ncrystalline domains during film formation and annealing. This is consistent with the dominant in-\nplane lamellar spacing of 1.5–1.6 nm.\n“-p14: The authors attribute the variation in the mechanical properties of the PEDOT:PSS+Li-\nTFSI to the formation of water-salt complexes within the PEDOT:PSS structure. Are these\ncomplexes visible in the cryo-EM micrographs?”\nWe are currently unable to resolve water-salt complexes, which is likely due to their amorphous\nnature. In addition, these complexes would also be challenging to discern in films, due to the\npossibility of overlapping domains.\n“-How is the presence of these water-salt complexes compatible with the increase in electrical\nconductivity?”\nWe do believe that the electrical conductivity is suppressed due to the presence of water. As\nshown in Fig. S18, the electrical conductivity of PEDOT:PSS+Li-TFSI after water treatment and\nremoval of ionic additives shows a close to fourfold increase compared to the PEDOT:PSS+Li-\nTFSI samples. Thus, LiTFSI as a solution additive improves conductivity through modulation of\nthe morphology, but removing LiTFSI after film formation (and thus decreasing the film water\ncontent) also enhances the conductivity.\n“-p11 line241: The cryo-EM images Figures 2b and 2c appear to not have the same defocus\n(which could impact the contrast). It is therefore not sure that moisture exposure improves the\ncontrast of the micelles.”\nWe apologize for this oversight. Fig. 2c in the new submission (PH1000+Li-TFSI at 100% RH)\nwas substituted with a new micrograph with a similar defocus to PH1000+Li-TFSI at 30% RH\nsample. As can be seen in the new micrograph, the micelles in 100% RH samples have a better\ncontrast compared to 30%RH samples, which confirms that the increase in micelle contrast is not\ncaused by variation of microscope parameters and is related to the physical change in sample\nstructure.\n“-The histogram of the crystalline domain area for the additional images in figure S6 should\nalso be provided. How does it compare with the one reported in Figure 3?”\nPer the reviewer’s suggestion, the histogram for crystalline domain area was added to Fig. S17.\nThe crystalline domain distribution in dry vs. hydrated samples shows a very similar distribution\nto the micrograph shown in Fig. 3, with hydrated samples containing a larger number of\ncrystalline domains that are smaller in size compared to non-hydrated sample.\n“4) Discussion on the PEDOT:PSS structure.\n-Page 10. The authors attribute the 1.5- 1.6 nm spacing to the lamellar phase ‘(which could\ncorrespond to the PEDOT:PSS (100) crystal planes).’\nThe reference 36, used, says that ‘the weak hump centered at 0.29 ̊Å-1 is assigned to the lamellar\nstacking distance [d(100)~22 ̊Å] peak occurring from the alternate ordering of the PEDOT and\nPSS polymer chains.’\nThe 1.5- 1.6 nm spacing, measured by Ghasemi et al, is much smaller than the 22 Å measured by\nMassonet et al. Therefore, the attribution of the fringes to the lamella spacing is questionable.\nAnother research group (E. Hosseini, et al, J. Mater. Chem. C, 2020, 8, 3982) has also attributed\na spacing of 22 Å to the PEDOT to PEDOT d100 lamellae spacing.”\nWe thank the reviewer for raising the question about lattice spacing in PEDOT:PSS. In general,\ntwo different lamella structures are proposed for PEDOT:PSS. q ≈ 0.40–0.45 Å⁻¹,\nwhich\ncorresponds to d = 2π/q ≈ 1.4–1.6 nm (14–16 Å) and q ≈ 0.270–0.3 Å⁻¹,\nwhich corresponds to\n21-23 Å4, 6, 7, 8, 9. A current interpretation is that the former lamella corresponds to stacks of\nPEDOT chains with perpendicular PSS chains (one or two chains in between), while the latter\nlamella stack corresponds to alternating PEDOT and PSS stacks, “end-to-end” or “side-to-side”.\nWe also agree with the reviewer that the 15-16 Å spacing observed in cryo-EM micrographs\ncannot be from the 22 Å (100) spacing observed in GIWAXS. As pointed out by the reviewer in\nthe next question, the 15-16 Å spacing is mainly observed in the in-plane orientation (see Fig. 2e,\nFig. S11, and Table S1), while 22 Å lamella stacks can be mainly seen in the out-of-plane\norientation of GIWAXS results (out of plane stacking would not be visible in our cryo-EM\nimages). We conclude that the 15-16 Å lamella stacks in solution also act as a template for the\ngrowth of crystalline domains with similar spacing in the in-plane orientation, and have modified\nour discussion to clarify this point.\n“-Fig S2. How can the authors be certain that the second spot at 0.7 nm is the PEDOT lamella\nstack of and not a second-order PEDOT:PSS lamella stack?”\nThe peak at g = 1.37 nm-1 (corresponding to a real-space spacing d = 1/g = 0.73 nm) is attributed\nto pure PEDOT, because the same periodicity is visible in real space, as confirmed by the line-\nscan profile in Fig. S14 d. In addition, the FFT from the blue-boxed region in Fig. S14a, where\nthe 1.37 nm-1 peak is more pronounced, does not show a second-order peak near 1.3 nm-1, further\nsupporting that the 1.37 nm-1 feature corresponds to a repeat spacing of 0.73 nm. We have\nrevised the caption of Fig. S14 to explain these observations in greater detail.\nFig. S14. Lamella stacking. a, Cryo-EM micrograph of PEDOT:PSS+Li-TFSI. The red box shows\ncrystalline domains with PEDOT:PSS lamella stacks in the center. b, c, FFT of the region of interest\nhighlighted by the red and blue boxes, respectively. The two peaks highlighted in panel b show the inverse\nof direct image spacing observed in cryo-EM micrographs. In addition, the peak detected at ~0.32 nm-1\n(corresponding to 3.1 nm spacing) is associated with PEDOT:PSS micelles. c, d, Line profiles of\nPEDOT:PSS lamella stacks (~1.5-1.6 nm), and neat PEDOT lamella stacks (0.7-0.74 nm). FFT from the\nblue-boxed region in panel c, where the 1.37 nm-1 peak is more pronounced, does not show a second-order\npeak near 1.3 nm-1, further supporting that the 1.37 nm-1 feature corresponds to a repeat spacing of 0.73 nm.\nIn FFTs, the spatial frequency g is defined as g = 1/ d (nm-1), whereas X-ray scattering uses the reciprocal\nspace vector q = 2π/d.\n“-A possible lamellar phase can be seen in the GIWAXS profiles in the out-of-plane orientation\nof the doped samples (up to the second or even the third order?). The reason why the lamellar\nphase is observed out-of-plane in GIWAXS while the fringes (attributed to the lamellar phase)\nobserved in cryo-EM (and corresponds to an in-plane orientation, is unclear”\nThe spacings observed in our cryo-EM micrographs are predominantly 15–16 Å, which is visible\nin the in-plane GIWAXS profiles. In contrast, the 22 Å spacing is mainly observed for the out-of-\nplane orientation, which is absent in our cryo-EM images of PEDOT:PSS thin films, as expected\nfor this orientation.\n“-If I am not mistaken, Figures 2b and 2c ‘zoom in view’ should correspond to Figures 3a and\n3b, respectively. However, the structures are different. Figures 2C shows electron-rich and\nelectron poor areas (containing micelles?) while crystalline domains are only visible in Figure\n3B. Similarly, the fibers seen in Figure 2b are not visible in 3a. This is inconsistent. Can the\nauthors comment on this?”\nThe differences between Fig. 2b/2d and Fig. 3a/3b arise mainly from imaging magnification.\nFigures 3a–b were recorded at higher magnification (150,000x) than Fig. 2 (47,000x), giving a\nsmaller pixel size and better sampling of high spatial frequencies; as a result, lattice planes are\nresolved more clearly. In contrast, the lower-magnification images provide a larger field of view\nand stronger low-frequency contrast, so micelles and lamellar spacing can be visualized in the\nsame frame. We have updated the Methods section to note these acquisition differences.\n“-A schematic of the structure (PEDOT proto-crystals coating the outside of micellar structure\nand the co-existence of fibers) proposed by the authors would help the readers.”\nWe thank the reviewer for this suggestion and have modified the schematic in Fig. 1 to show the\nco-existence of fibers and lamella stacks in PEDOT:PSS solutions.\n“STEM-EELS\n-Is it easy to control precisely the humidity rate in a STEM-EELS?”\nTo preform STEM-EELS on hydrated samples, we first exposed the TEM grids to humidity in a\nVitrobot chamber. In the next step, we plunged the hydrated samples using liquid ethane to\npreserve the morphology of the samples in a hydrated state. Cryo-STEM-EELS data was\nacquired using the Titan-Krios microscope with the capability to keep the sample temperature at\nliquid N2 temperature which allows for imaging of hydrated samples. We modified to the\nMethod section to explain this process in more detail.\n“-I have missed why the carbon content is decreasing upon humidity.”\nFigures 4c–f (Fig. S21 in the revised manuscript) present STEM–EELS elemental maps of\nPEDOT:PSS reported as atomic percent (at.%). After hydration (water exposure), the oxygen\nfraction increases, consistent with water uptake, while the carbon fraction decreases accordingly.\nBecause the compositions are normalized to 100 at% at each pixel, any rise in O% necessarily\nproduces a corresponding drop in the relative C% (and other elements) at the same pixel.\n“-To what correspond the 50-200 nm oxygen -rich regions?”\nWe associate these regions to the PSS-rich regions in PEDOT:PSS. Due to higher ratio of PSS to\nPEDOT in PH1000 (2.5:1 by weight) it is expected for PEDOT:PSS thin films to contain regions\nwith higher PSS content.\n“As minor comments,\n- The vertical axis and the units are missing in Figure 4a.”\nWe apologize for this omission and Fig. 4a of our previously submitted manuscript was modified\n(Fig. S18 in the revised manuscript).\n“- P10. The assumption that the difference of lamella spacing extracted from cryo-EM and\nGIWAXS, being due to the temperature measurements, is not valid. (Dubochet et al. Q. Rev.\nBiophys., 1988, 21, 129–228.)”\nWe thank the reviewer for pointing out this issue and sharing the related paper with us. We agree\nwith the reviewer that the difference in lamella spacing is not due to temperature. As discussed\nearlier ~1.5 nm and ~2.2 nm lattice spacing are associated with two different types of lamella\nstacks in PEDOT:PSS with the former lattice stack dominantly existing in the in-plane\norientation (and thus visible in cryo-EM micrographs).\n“- Please add the PH1000 Batch number”\nWe apologize for this omission. The PH1000 batch number is now provided in the Methods\nsection.\n“- Has the commercial dispersion been filtered or not (as is usually done by the organic\nelectronics community)?”\nWe did not use filtration in the sample process, as filtration of the PEDOT:PSS could remove the\nlong-range fibers, which in turn could cause discrepancies in comparisons between solution and\nsolid cryo-EM results. We subsequently modified the Methods section to state that the filtration\nwas not performed in the processing of PEDOT:PSS thin films.\n“- The chamber temperature was kept at 25°C for the solid sample while the solution was kept at\n4°C. Was there any particular reason for that?”\nFor cryo-EM measurements of vitrified solutions, we use 4 °C with 100% RH to suppress\nevaporation in the blot-to-plunge interval and to promote formation of uniform vitreous ice. In\ncontrast, for hydrated solid films, cooling the chamber (and therefore the Cu grid) below ambient\ntemperature can lead to water condensation on the film. Upon plunging, these water droplets can\nconvert to crystalline ice, and affect the film morphology and TEM imaging. To avoid\ncondensation while maintaining hydration, we therefore set the chamber to 25 °C for solid films.\nWe have added this rationale to the Methods to clarify why the temperatures differ.\n“Supplementary\n-In Figure S4, is there not also a spot at a greater distance (closer to the direct beam)?”\nWe agree with the reviewer that there is a weak feature at lower spatial frequency (closer to the\ndirect beam). In Fig. S14 it appears at 𝑔 ≈ 0.32 𝑛𝑚(cid:2879)(cid:2869) , corresponding to 𝑑 = 1/𝑔 ≈ 3.1 nm. We\nassociate this feature with the characteristic size of PEDOT:PSS micelles, consistent with the\n3.0–3.3 nm diameters measured in our cryo-EM micrographs. We thank the reviewer for\nhighlighting this point; we have labeled the feature and clarified in the caption that the FFT axis\nis plotted in spatial frequency 𝑔 (with 𝑑 = 1/𝑔).\n“-Fig S5. A table listing the peak positions and corresponding distances would help the reader to\ncompare the structures with the literature and their own data.”\nWe thank the reviewer for this suggestion. Table S1 was added to the Supporting Information,\nsummarizing peak position and lattice spacing of different phases observed in cryo-EM\nmicrographs.\nReviewer #2 (Remarks to the Author):\n“Ghasemi et al report on their characterization of different PEDOT:PSS formulation both in the\nsolution and thin film using electron microscopy. The authors show the hierarchical structure of\nPEDOT:PSS, with micelles, nanofibers, and semicrystalline and disordered regions. I find the\nwork very exciting. It is incredible to clearly see structural features in the solutions and films\nthat normally must be inferred from indirect measurements. This methodology is certainly the\nfuture of polymer microstructural characterization, and I expect this work will be important in\nthe field.\nHowever, the conclusions drawn from the analysis of the images are not entirely clear, and there\nare questions about the sample preparation and the assumptions made when classifying the\nfeatures seen in TEM. For this reason, I recommend a revision prior to accepting into Nature\nCommunications. My specific comments are below.”\nWe thank the reviewer for the kind comments. We have edited our manuscript to address the\nreviewer’s concerns.\n“-First, the paper needs to explain what exactly is being imaged for the vitrified PEDOT:PSS\ndispersions. Presumably during blotting, some of the water is absorbed into the paper,\nconcentrating PEDOT:PSS in the dispersion. This likely drives the aggregation into fibril\nstructures that is observed. How representative are these images of the dispersion itself? Is there\nany evidence for fibers forming in the as is dispersion before the blotting process? And how does\nthe aggregation of PEDOT:PSS micelles during blotting relate to aggregation during other\ncommon processing methods (spin-coating, blade-coating, dropcasting, etc.)?”\nWe thank the reviewer for pointing out this important concern. The development, including\nautomation, of liquid film preparation equipment (e.g., the Vitrobot) has led to plunge-freezing\nwith minimal artefacts, as the structural biology community has been working with this approach\nfor many years. Nevertheless, the development of an alternative approach that sparys liquid and\nflash freezes it onto grids (e.g., Vitrojet) allows us to completely remove the blotting process and\nobserve if the structure differs. Our new Fig S5 now shows PEDOT:PSS+Li-TFSI solutions\nvitrified using the Vitroject, and fibers are clearly observed in these images as well. We thus\nconclude that the blotting process of the Vitrobot has a minimal role on the resulting structure\nthat is observable in cryo-EM images.\nFig. S5. Cryo-EM of samples prepared using a Vitrojet. a, b, Liquid cryo-EM micrographs of\nPEDOT:PSS+Li-TFSI dispersion samples prepared using a Vitrojet. a and b micrographs were acquired\nwith defocus values of -0.9 μm and -1.2 μm, respectively. Similarly to samples prepared using a Vitrobot,\nfibril aggregates are apparent, although the thicker films from the Vitrojet limit resolution when compared\nto thinner films from the Vitrobot. Thus, the fibers observed in PEDOT:PSS dispersions are present in the\ndifferent formulations of PEDOT:PSS and are not formed due to the blotting process of the Vitrobot. The\nmicrographs show a 250×250 μm2 area and are acquired at 150,000x magnification with a pixel size of\n0.0944 nm.\n“-It doesn’t appear that there is much variation in particle size within a sample, why is that? DLS\ncharacterization of particle sizes in PEDOT:PSS dispersions have shown a wide dispersity\n[https://doi.org/10.1016/j.jcis.2020.09.070].”\nWe thank the reviewer for highlighting this potential comparison. We believe that the wide\ndispersity in micelle size apparent from DLS could arise from the formation of fibrils.\nFurthermore, we expect that the solvated PSS chains are likely not visible in cryo-EM images,\nbut can contribute to the apparent size in DLS.\nDLS yields the hydrodynamic radius (𝑅 ), which for a rod or stiff fiber can be approximated\n(cid:3035)\nfrom its dimensions using 𝑅 ≈ (𝑎𝑏(cid:2870))(cid:2869)/(cid:2871), where 𝑎and 𝑏are the half-length and half-width of the\n(cid:3035)\nparticle, respectively. The long tail visible in the DLS presented by K. Jain et al. is thus\nconsistent with the presence of elongated fibers, as we observe in cryo-EM. For example, a fiber\n(cid:3117)\n100 nm long and 5 nm wide (𝑎 = 50 nm, 𝑏 = 2.5nm) gives 𝑅\n(cid:3035)\n≈ (50×2.5(cid:2870))(cid:3119) ≈ 6.8 nm, which\nis larger than the micellar core size and consistent with the long-𝑅 shoulder observed in the\n(cid:3035)\nreference cited by the reviewer. We modified the main text to clarify this point.\n“-I’m a little confused about the proposed structure in Fig 1c. The cartoons show a micelle with\na lot of messy, tangled polymer, with a PEDOT rich core and PSS rich shell. However, the size of\nthe micelle in the images is 2.7 nm up to 4.7 nm, is on the order of 2-3x the PEDOT:PSS lamellar\nspacing distance when the molecules are tightly packed. How would all of that polymer fit into\nsuch a small volume, especially when it isn’t tightly packed? In panel j, the micelle diameter is\nsmaller than two repeated lamellae of the shell of the nanofiber. Is this picture physically\nfeasible?\n-Also, in the cartoons, why are the PEDOT chains drawn as very short segments in the neat film,\nbut very long (spanning several micelles) in the secondary doping case? Shouldn’t the Mw of\nPEDOT:PSS be constant? What is the image intended to convey?”\nWe thank the reviewer for pointing out the problems with our previous cartoon in Fig 1.\nFollowing both reviewers’ suggestion, we simplified and rescaled the schematic in Fig. 1 to\nbetter represent PEDOT:PSS morphology in solution. We try to incorporate a conservation of\nMW for PEDOT, as the reviewer suggests.\nThe presence of small micelles, confirmed both by cryo-EM and previous DLS results as pointed\nout by the reviewer, strongly suggests the presence of PEDOT oligomers. This would allow\nmultiple oligomers to fit within each micelle. For example, the volume of a 3 nm micelle is about\n14 nm3 (4/3r3), and the volume of a repeat unit is roughly given by the crystal dimensions (0.71\nnm x 0.4 nm x 1.5 nm = 0.43 nm3). That allows for about 30 repeat units within each micelle, but\ngiven the expected long persistence length of PEDOT, we believe that the chain length of\nPEDOT must be limited to allow packing of the backbones within the micelles. We have\nmodified our discussion when comparing previous DLS measurements and our cryo-EM results\nto clarify this point (pg 7).\n“-In the FFT insets in Fig 3a and Fig S3, there are several rings all the way out to ~3 nm-1. It is\nsurprising to see so many strong peaks since the GIWAXS ~4 peaks which are fairly weak. What\nis the physical origin of these rings in the FFT images?”\nWe apologize for the confusion. The concentric rings observed in the FFT insets of Fig. 3a and\nFig. S3 of the initial submission are Thon rings that arise from the contrast transfer function\n(CTF) of the objective lens, and not from the structure of the film. To clarify this point, we now\ninclude note in the caption of Fig. S9. In addition, in the revised manuscript, we substitute FFTs\nin Fig. 3 with zoomed-in images of PEDOT:PSS lattice fringes as insets. We believe improves\nthe clarity of our manuscript.\n“-Why do the HRTEM images in Fig 3 look so different from images in Fig 2b and 2c? Is the\nimaging mode different? The scale of the images in Fig 3 appear to be similar to the insets Fig 2.\nDoes this mean some of the contrast in the low-res images is due to imaging artefacts?”\nAs mentioned in our response to reviewer 1, the differences between Fig. 2b/2d and Fig. 3a/3b\narise mainly from imaging magnification.\nFigures 3a–b were recorded at higher magnification\n(150,000x) than Fig. 2 (47,000x), giving a smaller pixel size and better sampling of high spatial\nfrequencies; as a result, lattice planes are resolved more clearly. In contrast, the lower-\nmagnification images provide a larger field of view and stronger low-frequency contrast, so\nmicelles and lamellar spacing can be visualized in the same frame (detectors . We have updated\nthe Methods section to note these acquisition differences.\n“-While the paper shows several interesting modalities for cryo-EM characterization, there is\nvery little continuity in the sample choice. First, 4 different secondary dopants are investigated\nfor solution imaging, but only two of the four are measured as solid films. The authors then show\nhumidity dependent crystal size but only apply the analysis to one formulation (Li-TFSI\nsecondary dopant) and compare the results to GISAX measured at two different humidities from\nthe TEM experiment humidities. Finally, they show SETM-EELS to explore where moisture is\nlocated within solid films, but the sample changes again and the authors introduce PEDOT:PSS\n+ Li-TFSI with salt-removed. A clear demonstration of how these techniques complement one\nanother and can be combined to build up a holistic microstructural picture for a single materials\nsystem would be much more convincing than the current variety of samples explored here.”\nWe thank the reviewer for pointing out this shortcoming of our manuscript. In our revised\nmanuscript, we focus the story on PEDOT:PSS+Li-TFSI, given its high performance in terms of\nboth charge mobility and mechanical compliance (stretchability). We now include other systems\nas supporting this main storyline. Thus, Fig. 2 and Fig. 4 now focus on the effects of hydration\nfor PEDOT:PSS+Li-TFSI, with other data in the supporting information. We have also moved\nsome of the data on STEM-EELS and PEDOT:PSS+Li-TFSI after salt removal to the Supporting\nInformation for clarity and conciseness of the main text.\n“I was also surprised that the PEGDA doped PEDOT:PSS was not included in the mechanical\nanalysis since the authors discussed in the introduction that PEGDA is usually added to improve\nstretchability.”\nWe apologize for this omission. We now include mechanical testing data on\nPEDOT:PSS+PEGDA. Fig. S19 shows the crack onset strain (COS) of PEDOT:PSS with\nPEEGDA at different humidity levels. Here, the COS increases with increasing hydration, a trend\nthat is similar to what we observed in PEDOT:PSS+Li-TFSI. This further emphasizes the\nimportance of hydration in the phase evolution of doped PEDOT:PSS samples.\nFig. S19. Mechanical measurements. Crack onset strain (COS) of PEDOT:PSS+PEGDA films\non polystyrene-block-poly(ethylene-ran-butylene)-block-polystyrene (SEBS) at three different\nhumidity levels.\nReviewer #3 (Remarks to the Author):\n“I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.”\nWe sincerely thank the reviewer and their co-reviewer for their thoughtful evaluation of our\nmanuscript. We appreciate the time and effort they invested in providing such constructive and\ndetailed feedback, which has greatly improved the quality and clarity of our work.\nReferences\n1. Takano T, Masunaga H, Fujiwara A, Okuzaki H, Sasaki T. PEDOT Nanocrystal in Highly\nConductive PEDOT:PSS Polymer Films. Macromolecules 45, 3859-3865 (2012).\n2. Liu Z, Li X, Zou R, Zhou Z, Ma Q, Zhang P. Deciphering the quaternary structure of\nPEDOT:PSS aqueous dispersion with small-angle scattering. Polymer 261, 125415\n(2022).\n3. Leaf MA, Muthukumar M. Electrostatic Effect on the Solution Structure and Dynamics\nof PEDOT:PSS. Macromolecules 49, 4286-4294 (2016).\n4. Taussig L, et al. Electrostatic self-assembly yields a structurally stabilized PEDOT:PSS\nwith efficient mixed transport and high-performance OECTs. Matter 7, 1071-1091\n(2024).\n5. Wu X, et al. Ionic-Liquid Induced Morphology Tuning of PEDOT:PSS for High-\nPerformance Organic Electrochemical Transistors. Adv Funct Mater n/a, 2108510.\n6. Bießmann L, Saxena N, Hohn N, Hossain MA, Veinot JGC, Müller-Buschbaum P. Highly\nConducting, Transparent PEDOT:PSS Polymer Electrodes from Post-Treatment with\nWeak and Strong Acids. Advanced Electronic Materials 5, 1800654 (2019).\n7. Kim N, et al. Highly Conductive PEDOT:PSS Nanofibrils Induced by Solution‐\nProcessed Crystallization. Advanced Materials 26, 2268-2272 (2014).\n8. Kee S, et al. Controlling Molecular Ordering in Aqueous Conducting Polymers Using\nIonic Liquids. Adv Mater 28, 8625-8631 (2016).\n9. Kim S-M, et al. Influence of PEDOT:PSS crystallinity and composition on\nelectrochemical transistor performance and long-term stability. Nat Commun 9, 3858\n(2018).\nWe thank the reviewers for their thoughtful comments on our revised manuscript. We are grateful\nfor the positive feedback on the clarity, consistency, and quality of the cryo-EM analysis. We are\npleased that the work is now considered suitable for publication in Nature Communications.\nReviewer #1 (Remarks to the Author):\nIf, this would help to further enhance the quality of the manuscript, please find some additional\nsuggestions below:\nWe thank the reviewer for the suggestions, and we have made a number of modifications to the\nmanuscript to address the reviewer’s suggestions.\n1) The sentence p7 lines 165-167 is not clear. As a suggestion: ‘We attribute the chain stacking\nwith a spacing of 1.48–1.54 nm that is observed in cryo-EM micrographs of neat PEDOT:PSS\nand PEDOT:PSS with additives to PEDOT:PSS crystals. This spacing has previously been\nattributed to the presence of alternating PEDOT and PSS chains. Also, the authors may like to\nrefer to the orange arrows (Figure 1 i-o) and the red parallel lines (Fig 1c-d).\nThe sentence p7 lines 165-167 was modified accordingly to improve clarity. Per the reviewer’s\nsuggestion, we also pointed to the orange arrows and parallel line solution cryo-EM of\nPEDOT:PSS dispersions.\nThe spacing is sometimes given at 1.48- 1.54 and, later at 1.5-1.6 nm, or 1.4-1.6 nm (Fig S15).\nThe authors may like to homogenise the values.\nWe thank the reviewer for this helpful comment. We have now homogenized the reported\nlamellar spacings throughout the manuscript to a consistent range of 1.4–1.6 nm.\n2) Is it possible/easy to represent the coalescence of the micelles (forming the elongated fibers)\non schematic fig 1D (via merging of green PSS-rich corona?)\nWe modified the schematic in Fig. 1d to depict the coalescence of the micelles by merging of\nPSS-rich coronas.\n3) Fig S14, a diffraction peak at 3.1 nm is attributed to the PEDOT:PSS micelles. This is\nsurprising as micelles are inherently amorphous.\nWe apologize for the confusion. This peak is indeed due to amorphous packing (liquid-like\npacking) of micelles. We have clarified this point in the caption of Figure S14.\n4) I have spotted two misspelt words (page 5, line 121 ‘detials’ and page 16, line 390 ‘PEDOT+\nPSS chians’).\nWe corrected the misspellings accordingly.\n5) All cryo-EM images are perfectly readable on a screen. But they appear very dark on a\nprinted version. Can the authors/ editors do something about it or provide some advice for\nprinting?\nWe thank the reviewer for the comment. We increased the brightness of the micrographs in the\nmain text, which should also improve the quality of the images for printing.\nWhile this is beyond the scope of the manuscript, this work opens questions about the\nmechanisms behind the self-assembly of the micelles into elongated fibers and on the packing of\nthe 2D lamellar structure onto 3D fibrillar structures. I hope that the authors will continue to\ninvestigate the structure of PEDOT:PSS dispersions to shed some light on these two mechanisms.\nTilting experiments and working with more dilute systems may be options to explore.\nWe agree that the mechanisms underlying micelle self-assembly into elongated fibers and the\npacking of 2D lamellar structures into 3D fibrillar morphologies are questions of great\nimportance and are beyond the scope of the current study. We thank the reviewer for raising this\npoint and, in future work, we plan to perform large-scale automated cryo-EM data collection on\nthese systems, which we expect will provide deeper insight into detailed packing motifs and fiber\nformation mechanisms in PEDOT:PSS dispersions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided a convincing and well-argued point-by-point response to the reviewers. They have ruled out the\npossible artefacts by providing images with different defocus. The clarity of the manuscript has been greatly enhanced. The\ncryo-EM analysis is a beautiful and consistent work.\nThe manuscript now reaches the standards of Nature Communication journal and I suggest to accept it.\nIf, this would help to further enhance the quality of the manuscript, please find some additional suggestions below:\n1) The sentence p7 lines 165-167 is not clear. As a suggestion: ‘We attribute the chain stacking with a spacing of 1.48–1.54\nnm that is observed in cryo-EM micrographs of neat PEDOT:PSS and PEDOT:PSS with additives to PEDOT:PSS crystals.\nThis spacing has previously been attributed to the presence of alternating PEDOT and PSS chains. Also, the authors may\nlike to refer to the orange arrows (Figure 1 i-o) and the red parallel lines (Fig 1c-d).\nThe spacing is sometimes given at 1.48- 1.54 and, later at 1.5-1.6 nm, or 1.4-1.6 nm (Fig S15). The authors may like to\nhomogenise the values.\n2) Is it possible/easy to represent the coalescence of the micelles (forming the elongated fibers) on schematic fig 1D (via\nmerging of green PSS-rich corona?)\n3) Fig S14, a diffraction peak at 3.1 nm is attributed to the PEDOT:PSS micelles. This is surprising as micelles are inherently\namorphous.\n4) I have spotted two misspelt words (page 5, line 121 ‘detials’ and page 16, line 390 ‘PEDOT+ PSS chians’).\n5) All cryo-EM images are perfectly readable on a screen. But they appear very dark on a printed version. Can the authors/\neditors do something about it or provide some advice for printing?\nWhile this is beyond the scope of the manuscript, this work opens questions about the mechanisms behind the self-\nassembly of the micelles into elongated fibers and on the packing of the 2D lamellar structure onto 3D fibrillar structures. I\nhope that the authors will continue to investigate the structure of PEDOT:PSS dispersions to shed some light on these two\nmechanisms. Tilting experiments and working with more dilute systems may be options to explore.", "reviewer_2": "The authors have addressed all of my concerns. I recommend that the paper is accepted for publication in Nature\nCommunications."}, "author_response": "We thank the reviewers for their thoughtful comments on our revised manuscript. We are grateful\nfor the positive feedback on the clarity, consistency, and quality of the cryo-EM analysis. We are\npleased that the work is now considered suitable for publication in Nature Communications.\nReviewer #1 (Remarks to the Author):\nIf, this would help to further enhance the quality of the manuscript, please find some additional\nsuggestions below:\nWe thank the reviewer for the suggestions, and we have made a number of modifications to the\nmanuscript to address the reviewer’s suggestions.\n1) The sentence p7 lines 165-167 is not clear. As a suggestion: ‘We attribute the chain stacking\nwith a spacing of 1.48–1.54 nm that is observed in cryo-EM micrographs of neat PEDOT:PSS\nand PEDOT:PSS with additives to PEDOT:PSS crystals. This spacing has previously been\nattributed to the presence of alternating PEDOT and PSS chains. Also, the authors may like to\nrefer to the orange arrows (Figure 1 i-o) and the red parallel lines (Fig 1c-d).\nThe sentence p7 lines 165-167 was modified accordingly to improve clarity. Per the reviewer’s\nsuggestion, we also pointed to the orange arrows and parallel line solution cryo-EM of\nPEDOT:PSS dispersions.\nThe spacing is sometimes given at 1.48- 1.54 and, later at 1.5-1.6 nm, or 1.4-1.6 nm (Fig S15).\nThe authors may like to homogenise the values.\nWe thank the reviewer for this helpful comment. We have now homogenized the reported\nlamellar spacings throughout the manuscript to a consistent range of 1.4–1.6 nm.\n2) Is it possible/easy to represent the coalescence of the micelles (forming the elongated fibers)\non schematic fig 1D (via merging of green PSS-rich corona?)\nWe modified the schematic in Fig. 1d to depict the coalescence of the micelles by merging of\nPSS-rich coronas.\n3) Fig S14, a diffraction peak at 3.1 nm is attributed to the PEDOT:PSS micelles. This is\nsurprising as micelles are inherently amorphous.\nWe apologize for the confusion. This peak is indeed due to amorphous packing (liquid-like\npacking) of micelles. We have clarified this point in the caption of Figure S14.\n4) I have spotted two misspelt words (page 5, line 121 ‘detials’ and page 16, line 390 ‘PEDOT+\nPSS chians’).\nWe corrected the misspellings accordingly.\n5) All cryo-EM images are perfectly readable on a screen. But they appear very dark on a\nprinted version. Can the authors/ editors do something about it or provide some advice for\nprinting?\nWe thank the reviewer for the comment. We increased the brightness of the micrographs in the\nmain text, which should also improve the quality of the images for printing.\nWhile this is beyond the scope of the manuscript, this work opens questions about the\nmechanisms behind the self-assembly of the micelles into elongated fibers and on the packing of\nthe 2D lamellar structure onto 3D fibrillar structures. I hope that the authors will continue to\ninvestigate the structure of PEDOT:PSS dispersions to shed some light on these two mechanisms.\nTilting experiments and working with more dilute systems may be options to explore.\nWe agree that the mechanisms underlying micelle self-assembly into elongated fibers and the\npacking of 2D lamellar structures into 3D fibrillar morphologies are questions of great\nimportance and are beyond the scope of the current study. We thank the reviewer for raising this\npoint and, in future work, we plan to perform large-scale automated cryo-EM data collection on\nthese systems, which we expect will provide deeper insight into detailed packing motifs and fiber\nformation mechanisms in PEDOT:PSS dispersions."}]

Decision letter