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Super-resolution microscopy as a powerful tool to study complex synthetic materials

Citation for published version (APA):

Pujals, S., Feiner-Gracia, N., Delcanale, P., Voets, I., & Albertazzi, L. (2019). Super-resolution microscopy as a powerful tool to study complex synthetic materials. Nature Reviews Chemistry, 3(2), 68-84.

https://doi.org/10.1038/s41570-018-0070-2

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10.1038/s41570-018-0070-2

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Published: 01/02/2019

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Super- resolution optical microscopy has been recently introduced and has rapidly revolutionized the way we look at biological systems, enabling us to visualize cell structures with unprecedented resolution. According to Abbe’s criteria, the spatial resolution of fluorescence microscopy (FM) is limited by light diffraction to a few hundred nanometres, hampering the imaging of molec- ular structures on smaller length scales. Super- resolution microscopy (SRM), or nanoscopy, has recently sur- faced as a powerful technique that complements and overcomes the current limitation of existing imaging approaches1,2. The SRM family encompasses a range of far- field optical techniques that exploit a number of chemical and physical principles to overcome the dif- fraction limit and enable nanometric- resolved imaging.

The impact of SRM was acknowledged with the Nobel Prize for Chemistry in 2014 (ref.3).

Recently, SRM technologies have become influen- tial in chemistry and materials science as a means to unveil the structure and dynamics of complex materials.

Importantly, the minimal invasiveness of optical micros- copy facilitates visualization of materials in situ and in operando, for example, in living cells or during catalytic processes. Therefore, the use of SRM is increasing in numerous fields, including supramolecular chemistry, plasmonics, catalysis and biomaterials, complementing existing techniques such as atomic force microscopy

(AFM)4, electron microscopy (EM)5, ensemble scattering methods and optical spectroscopy.

Our Review highlights the advances in the applica- tion of SRM to the study of synthetic materials, with a focus on biomaterials, and describes more broadly the perspectives of these techniques in chemistry.

Why super- resolution for synthetic materials?

Understanding the structure and the function of syn- thetic materials is critical for the development of improved materials for a variety of applications6. This understanding is even more crucial in the case of com- plex molecular systems, as their dimensions, modular composition, hierarchical structure and dynamics pose a serious challenge for their study with spectroscopic and microscopic techniques such as AFM7, EM8 and FM9. The use of multiple complementary techniques is highly recommended, as each method has its advantages and limitations related to operational requirements and contrast. AFM and EM, for example, offer excellent spa- tial resolution and do not require any labelling, but their required invasive sample preparations, which usually involve freezing or drying procedures, hamper imag- ing in native conditions. Furthermore, both techniques have limited permeation through a sample, usually confined to material surfaces or thin sections, and do not enable multicolour imaging, making it difficult or

Super- resolution microscopy as a powerful tool to study complex synthetic materials

Silvia Pujals1, Natalia Feiner- Gracia1, Pietro Delcanale1, Ilja Voets2 and Lorenzo Albertazzi 1,3*

Abstract | Understanding the relations between the formation, structure, dynamics and functionality of complex synthetic materials is one of the great challenges in chemistry and nanotechnology and represents the foundation for the rational design of novel materials for a variety of applications. Initially conceived to study biology below the diffraction limit,

super-resolution microscopy (SRM) is emerging as a powerful tool for studying synthetic

materials owing to its nanometric resolution, multicolour ability and minimal invasiveness. In this Review , we provide an overview of the pioneering studies that use SRM to visualize materials, highlighting exciting recent developments such as experiments in operando, wherein materials, such as biomaterials in a biological environment, are imaged in action. Moreover, the potential and the challenges of the different SRM methods for application in nanotechnology and (bio) materials science are discussed, aiming to guide researchers to select the best SRM approach for their specific purpose.

1Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.

2Laboratory of

Self-Organizing Soft Matter, Laboratory of

Macromolecular and Organic Chemistry, Department of Chemical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, Netherlands.

3Department of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, Netherlands.

*e- mail: lalbertazzi@

ibecbarcelona.eu https://doi.org/10.1038/

s41570-018-0070-2

REvIEwS

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even impossible to identify different molecular species in the sample. By contrast, FM enables the multicolour imaging of materials in native conditions as well as in a biological environment, at the cost of limited spatial res- olution. SRM overcomes this limitation and allows us to access information on the nanometric scale without the need for invasive sample preparations. The three main types of super- resolution method are structured illumi- nation microscopy (SIM), stimulated emission depletion (STED) and single- molecule localization microscopy (SMLM) (Box 1). These three approaches are charac- terized by different and often complementary features;

thus, a critical step is choosing the ideal techniques for the scientific question of interest.

The appeal of SRM to study complex synthetic materials is manifold. First, the nanometric spatial reso- lution down to 5 nm enables us to resolve 3D molecular structures and link them with the functionality they are responsible for. Synthetic molecular architectures are often hierarchical structures with a defined order on different length scales. Nanoscopy can resolve such multiscale structural features with nanometric accu- racy, as an image can cover a rather large field of view (for example, 100 μm × 100 μm)10. Another intrinsic characteristic accessible by nanoscopy is the complex composition of advanced materials. The multiple build- ing blocks that are incorporated in a material in a mod- ular fashion (for example, multiple functionalities on the same scaffold) can be uniquely identified in space as a function of time. Typically, three different sets of mol- ecules can be separately labelled and imaged; however, examples of six- colour11 and ten- colour12 imaging have been proposed using SMLM. The multicolour quanti- tative nature of SMLM allows us to visualize and count different molecules, therefore providing information on the stoichiometry of synthetic assemblies13. The develop- ment of approaches to simulate SMLM images can greatly facilitate the interpretation of quantitative SMLM analy- sis14. Another interesting aspect of complex materials is related to their ability to continuously change structure over time, for which they are called dynamic materials.

This aspect is particularly relevant for supramolecular structures, materials based on dynamic covalent chem- istry and out- of-equilibrium materials. The dynamic nature of these materials is responsible for some of their most intriguing properties, such as responsivity, adaptiv- ity and self- healing. The understanding of such molecular dynamics is as much important as challenging, owing to the multiple timescales and spatial scales involved in this phenomenon. Finally, it is very important for any charac- terization technique to probe the system of interest in the conditions closest to the real operational environment of the material. Spectroscopic and microscopic analyses are usually performed in idealistic conditions (such as pure water) that are poorly representative of the real world (for example, a living cell, the complex matrix of a material or a complicated device). SRM, thanks to its minimal invasiveness, multicolour imaging ability and temporal resolution, is an ideal technique for proving and unveiling molecular structure and dynamics in situ (Box 2).

In this Review, we discuss a selection of the most rele- vant work in which SRM has been used to study various

classes of synthetic materials, including supramolecular fibres, polymer- based materials, lipid- based materials, DNA origami and metal nanoparticles. Electronic devices and catalytic devices have been extensively reviewed elsewhere15,16; here, we focus on the use of super- resolution to image biomaterials in a biological environment. Altogether, this Review aims to provide a comprehensive picture of the use of super- resolution imaging in chemistry and offers a guide towards the choice of the proper imaging methods to address a scientific question in the material arena.

SRM for molecular observations in vitro

In this section, we review the most relevant contributions to the development of SRM for the study of complex molecular architectures. Several families of materials have been studied, demonstrating the generality of nanoscopy to image virtually any chemical structure.

Supramolecular polymers and nanofibres.

Supramolecular polymers are polymers in which the monomers are linked by non- covalent bonds, typically by hydrophobic interactions and hydrogen bonding.

Their dynamic nature, together with their modularity and responsiveness to different stimuli, makes them promising candidates for several applications in biomed- icine, optoelectronics, sensing and catalysis17. Over the past years, SRM has proved to be a powerful method for studying the structure and dynamics of supramolecular polymers, providing complementary information to that attained with ensemble techniques (UV spectroscopy, circular dichroism and X- ray scattering) and label- free imaging techniques (EM and AFM).

Albertazzi and Meijer reported the first example of supramolecular polymer imaging using SRM18. They investigated the monomer exchange mechanism of water- soluble 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers using a particular SMLM approach known as stochastic optical reconstruction microscopy (STORM). The study reported on a two- colour STORM method able to achieve dynamic infor- mation, such as the exchange of monomers between fibres, using a static technique. This is possible by labelling two batches of materials with two spectrally distinct dyes and following green- labelled monomers joining red- labelled fibres and vice versa at specific time points such that temporal information is imprinted into spectral information. Before the use of SMLM, mono- mer exchange along a fibre was usually explained by polymerization/depolymerization occurring at the end of fibres or by a fragmentation/recombination mech- anism on the basis of data acquired from ensemble studies, such as studies of the bulk solution. However, the combination of STORM and stochastic modelling demonstrated that a homogeneous exchange occurs all along the BTA self- assembled fibrillar structures.

This study was followed by several other reports on the use of the two- colour STORM method. In particular, these studies unveiled how the dynamics of supramo- lecular polymers is influenced by hydrophobicity and chirality19 and by the presence of functional groups20. Notably, tracking and locating specific monomers in

Abbe’s criteria

Criteria that state the resolution that can be obtained in principle, considering the diffraction of light. ernst Abbe described in 1873 that by using light with a wavelength λ travelling through a medium of refractive index n and focused with a half- angle θ, the minimum resolution possible is λ/2nsin θ.

Far- field optical techniques Techniques that make use of optical microscopes in which light does not pass through subwavelength features.

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a mixture of different components can be performed only thanks to the combination of nanometric resolu- tion and specific labelling. The studies mentioned above were performed in water to explore the potential use of BTA polymers for biomedical applications. However, methods that enable super- resolution imaging of supra- molecular structures, such as the use of BTA fibres in methyl cyclohexane in organic media, have also been developed21–23.

In addition to BTAs, other families of supramolecular polymers have been studied using nanoscopy, including ureidopyrimidone (Upy)-based polymers and peptide amphiphiles24. In both cases, the mechanism and the kinetics of monomer exchange were unveiled and the structure–dynamics relationships highlighted.

Altogether, these nanoscopy studies highlight that the dynamic properties of a polymer material depend on how ordered the assemblies are. Ordered structures are

Box 1 | Super- resolution microscopy basics Structured illumination microscopy

Structured illumination microscopy (SIm) relies on the illumination of a sample using high- spatial-frequency patterns175. In this case, the excitation light is not homogeneous but has a specific profile, typically parallel lines (as shown in the figure). When a sample containing features smaller than the diffraction limit is illuminated, the combination of the patterned illumination and the sample generates large, detectable interference patterns (moiré fringes). The incident pattern is typically applied in different orientations, and as the illumination profile is known, a super-resolution image can be mathematically deconvolved from the interference signal. This approach leads to a lateral resolution of approximately 125 nm and an axial resolution of approximately 350 nm.

Moreover, subsecond temporal resolution can be obtained without the necessity of high illumination powers, making SIM suitable for live- cell imaging176. more advanced approaches such as nonlinear SIm177 and instant SIm178 enable us to reach spatial and temporal resolutions of 50 nm and 30 ms, respectively.

Stimulated emission depletion

Stimulated emission depletion (STED) achieves super- resolution by reducing the size of the effective point spread function (PSF), for example, the excitation volume179. This reduction is achieved by illuminating the sample with two aligned beams: a classical confocal excitation beam (to excite fluorescence) and a doughnut- shaped beam (that rapidly turns off the emission by means of stimulated emission), as shown in the figure180,181. Basically, only the fluorophores close to the centre of the doughnut will emit, resulting in a reduced PSF and therefore improved resolution. Typically, STED can reach a lateral resolution of 50–80 nm with a temporal resolution of seconds, comparable to that of a confocal

microscope. Depending on the samples, higher resolution can be obtained.

In particular, interferometric approaches such as 4Pi allow an axial resolution of 40 nm (ref.182).

Single- molecule localization microscopy

Single- molecule localization microscopy (Smlm) achieves subdiffraction resolution, with the accurate localization of individual fluorophores under a wide- field illumination149,183,184. In these methods, the dye emission is photocontrolled, and the fluorophores can be switched on and off with laser illumination. This is carried out to have a large majority of the dyes in the off state, and only a few sparsely distributed dyes are emitted. Because of this, the fluorescence of single markers can be detected, and the precise spatial positioning of the emitting molecules can be identified by fitting a Gaussian profile and obtaining the Gaussian centroid. This operation is iterated and for every frame a few tens of molecules are identified until all the dyes in the sample are localized. A super- resolution image is obtained by summing up the positions of all molecules detected. Therefore, depending on the photons detected, a resolution of 20–25 nm can be routinely achieved, and more advanced setups enable a resolution of 5 nm (ref.75) at the price of a lower temporal resolution (typically minutes) and more cumbersome sample preparation (for example, involving the use of special fluorophores and buffers). moreover, novel approaches that improve temporal resolution down to seconds have been reported185. The most- used Smlm methods are photoactivated localization microscopy (PAlm)149,186, (direct) stochastic optical reconstruction microscopy (d(SToRm))184,187,188 and point accumulation for imaging in nanoscale topography (PAINT)12,189. These methods are all based on the same experimental imaging setup and differ mostly in the way a small subset of single molecules is activated.

SIM Confocal

x–y resolution: ~250 nm z resolution: ~550 nm Time resolution: ~ms–s

x–y resolution: ~120 nm z resolution: ~300 nm

Time resolution: ~s

x–y resolution: ~50 nm z resolution: ~150 nm Time resolution: ~s

x–y resolution: ~20 nm z resolution: ~180 nm Time resolution: ~s–min

STED SMLM

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more static than disordered ones, in which structural defects introduce exchange points that make the mate- rials more dynamic. Static and dynamic structures can coexist, as is the case of peptide amphiphiles, in which fully dynamic areas coexist with kinetically inactive areas within the same fibril25. Building upon previous work that enabled photoactivated localization micro- scopy (PALM) in organic solvents21 and interface point accumulation for imaging in nanoscale topography (iPAINT) in aqueous media26, the internal block- like arrangement of dissimilar monomers within supra- molecular nanowires was elucidated by two- colour imaging in organic solvents using iPAINT23. The quasi- permanent adsorption of both dyes by the polymer in methylcyclohexane was exploited to identify the blocks within the supramolecular fibres, which were prepared by mixing prestained homopolymers. The combination of SRM and spectroscopy holds great promise for study- ing the photophysical properties of intrinsically fluores- cent nanofibres. Scheblykin and co- workers27 exploited the blinking behaviour of perylene bisimide supramo- lecular polymers to spatially and temporally map the energy transfer along fibrillar aggregates. This process paves the way towards the use of nanoscopy to unveil the properties of supramolecular materials with relevant applications in energy and electronics.

Among self- assembled materials, small molecular hydrogelators are commonly used for tissue engineer- ing because they are easily prepared, are inexpensive and

exhibit interesting properties (such as self- healing). An exciting possibility is represented by double- network hydrogels obtained by the mixing of two orthogo- nal hydrogelators that self- sort, that is, assemble in independent structures, into an intermixed structure.

However, the self- sorting (and not the co- assembly, that is, a structure formed by two individual components) of the two building blocks cannot be easily elucidated by means of techniques including solid- state NMR, flu- orescence spectroscopy, small- angle X- ray scattering, transmission EM, scanning EM and AFM, as these do not allow us to follow two separate molecules in situ inside a 3D gel structure. Recently, the group led by Hamachi observed self- sorting in supramolecular fibres using confocal laser scanning microscopy and STED imaging28(fig. 1a). The group used two types of hydro- gelator — one peptide- based and one lipid- based with cationic, zwitterionic or anionic head groups — labelled with two spectrally separated dyes. Self- sorting was visible by confocal laser scanning microscopy but was clearly better visualized by higher- resolution STED

(fig. 1b), as the fibre diameter was below the resolution limit (80–100 nm). STED- based colocalization unam- biguously indicated that there was a weak correlation between the two channels, confirming the self- sorting of the two- fibre network. STED, owing to its ability to image through a thick sample, is the ideal technique to image hydrogelators and might be a powerful tool for the investigation of supramolecular scaffolds for tissue engineering.

Altogether, nanoscopy has been demonstrated to be a powerful tool for the characterization of supramolecu- lar fibres. SRM added the multicolour dimension to the imaging of these structures, typically imaged with AFM and EM, allowing us to perform nanometric colocali- zation between different structures and monomers and to follow the fate of individual molecular species into a multicomponent material. Recently, the super- resolution shadow imaging method, based on 3D- STED and the use of diffusible fluorophores to image large fields of view with neither photobleaching nor phototoxicity, has been used to study biological tissues such as the extracel- lular matrix29. This approach could be a source of inspi- ration to further study the complex 3D morphology of supramolecular fibres and hydrogels.

Polymer- based materials. The pivotal role of poly- mers in materials science originates from their unique structure–property relations. SRM has offered new insight into structural features such as (micro)phase separation, association states and morphologies that are often critical for the optimal performance of a pol- ymer material. For example, the photophysical prop- erties of conjugated polymers in light- emitting diodes and organic solar cells are dependent on the confor- mation of the individual macromolecular chains and their organization into microdomains, thus directly impacting the efficiency of the devices. Owing to the large variety of obtainable structures, such as micelles, vesicles and fibres30(fig. 2a), block copolymers are probably the family of materials for which the widest range of nanoscopy methods has been used (STORM, Box 2 | How to choose the most suitable SRM method for synthetic samples

The different super- resolution microscopy (SRm) techniques present different advantages and disadvantages. It is therefore crucial to choose the ideal technique to answer the desired scientific question.

Single- molecule localization microscopy (SMLM) methods are endowed with the best spatial resolution (typically 20 nm, down to 5 nm in special cases) and single- molecule sensitivity and potentially enable quantitative molecular counting. However, their poor temporal resolution does hamper live- cell imaging. Smlm also requires the use of special probes (photoswitchable dyes) and experimental conditions (a special buffer containing a mixture of redox reagents). Therefore, this approach is preferred for static measurements for which the maximum resolution is needed and when information at the single molecule level is desired. moreover, a high density of labels is necessary to achieve the optimal resolution, and this can result in a perturbation of the structure of interest. This is particularly true when small molecules need to be labelled (for example, small molecular building blocks for self- assembly), as the dye structure may largely affect the molecular properties. Therefore, special care has to be taken in the experimental design and sample preparation.

Structured illumination microscopy (SIm), on the contrary, enables easy and

reasonably fast imaging with standard fluorophores. SIM approaches are recommended when the sample preparation and labelling are particularly challenging. Moreover, SIM does require less illumination power than SMLM and stimulated emission depletion (STED), and it is therefore suggested for photosensitive samples. However, the resolution of SIM (typically 120 nm) is substantially worse than that of SMLM and STED.

Therefore, SIM can be useful to image the material morphology but cannot answer questions on the molecular scale.

Finally, STED provides an intermediate spatial and temporal resolution and fewer limitations than Smlm in the sample preparation. For these reasons, it is commonly used for the study of synthetic samples that do not need extreme resolution and single- molecule sensitivity. Notably, the high power required for STED can be an issue for synthetic samples that are not photochemically or thermally stable, and controls for photodamage would be needed. Notably, STED can be easily coupled with other methods such as time- resolved and wavelength- resolved microscopies121, for example, STED–fluorescence correlation spectroscopy.

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PALM, STED, isotropic STED (isoSTED) and SIM) and represent a good example of how to choose the best technique in view of the structural and functional fea- tures of the target materials. Manners and co- workers observed for the first time block copolymers forming fibre- like structures22 and rectangular platelet micelles31 using SIM, STED and PALM (fig. 2b). Remarkably, SIM does not require the aid of any special dye and provides good time resolution, enabling easy sample preparation and time- resolved studies, for example, of the kinet- ics of fibre formation in organic solvents22. This study demonstrates the power of SRM to not only attain high- resolution images but also gain molecular infor- mation from the measurements of polymeric structures of a large variety of sizes and shapes31. Analogously to supramolecular polymers, the two- colour modality of SRM was used to observe the phase separation or colo- calization of different molecular species in the materials.

isoSTED has also been used to study the 3D structure of poly(styrene- block-2-vinylpyridine) (PS- b-P2VP) block copolymer films32. This method relies on a combination of STED and 4Pi microscopy that enables us to non- invasively and rapidly image nanoscale morphologies spanning the whole volume of the sample. The ability to image large samples in 3D is crucial for the under- standing of complex materials exhibiting hierarchical morphologies.

Imaging morphological changes in block copoly- mer assemblies is of great interest for the development of responsive materials. As a relevant example, Yan et al. imaged the transition from cylindrical micelles to polymersomes of poly(styrene- block-ethylene oxide) (PSt- b-PEO)33. To this end, spiropyrans were incorpo- rated in the hydrophobic, glassy- like poly(styrene) core

of the PSt- b-PEO micelle formed in water. Profiting from the stochastic blinking of spiropyrans in hydro- phobic, solid- like environments, the spiropyran- containing microphases were readily imaged, yielding reconstructions of both the cores of cylindrical micelles and the bilayer walls of the polymersomes with nanometre resolution33.

Protein polymers represent an interesting family of semi- synthetic materials that exploit the program- mability and biocompatibility of recombinant proteins. Polyamino acids composed of hydrophobic and hydro- philic blocks have been proposed and tested for biomed- ical applications34. Beun et al. used STORM to investigate whether both ends of micrometre- long protein–polymer nanofibres are living (that is, are not terminated) once polymerization has ceased and whether growth pro- ceeds in one or two directions (for example, from one or both fibre ends)35. First, a well- known fibre- forming triblock protein polymer was selected that comprises a self- assembling silk- like domain and features two water- soluble collagen- like domains at both ends. Next, nanofibres were formed in aqueous solutions of triblocks bearing Alexa 647 (red). Time- lapse SRM imaging on thus synthetized protein–polymer nanofibres revealed that added Alexa 488 (green)-labelled triblock protein polymers attach to only one of the two fibre ends, resulting in the formation of green–red diblock fibres, indicating unidirectional growth.

Complex coacervate core micelles (C3Ms), also known as polyion complex micelles, are a novel class of association colloids composed of polyelectrolytes.

Their assembly is electrostatically driven rather than hydrophobically driven, as for amphiphilic polymers.

Aloi and colleagues used iPAINT to visualize the

a b

Monomers

Bundle diameter

=10–100nm

Fibre diameter

=5–10nm Dynamics= secondstohours Mesh size

=50nm to 50μm

40μm

12μm

16μm

Fig. 1 | STED imaging of self- assembled supramolecular polymers. a | Schematic representation of the structural and functional properties of self- assembled supramolecular polymers. Monomers (left panel) are typically small organic molecules or peptides with amphiphilic character. In water, they can self- assemble into fibrillar structures (right panel).

Depending on the concentration and molecular properties, these structures can be stable isolated fibres in solution or can bundle, forming higher- hierarchy structures or hydrogels. Such materials are relevant for biomedical applications such as drug delivery , gene delivery and regenerative medicine. Therefore, understanding their structural features is crucial for the design of improved materials. Super- resolution microscopy can contribute to understanding these structures at the nanoscale, imaging crucial features such as the fibre length and diameter, their bundling and the 3D features of gels, such as the mesh size. b | Stimulated emission depletion (STED) imaging of supramolecular hydrogels. Two- colour STED is used to unveil the structure of a double network composed of two different self- assembled monomers. STED revealed the fibre structure, the gel mesh size and, most importantly , the relative distribution of the two monomers in the gel. Part b is adapted from ref.28, Springer Nature Limited.

4Pi microscopy A fluorescence technique in which two objective lenses are focused to the same spatial location to achieve improved axial resolution.

Recombinant proteins Translated products of the expression of recombinant DNA non- native to living cells (such as bacteria, mammalian and yeast).

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concentration- induced shape evolution of C3Ms com- posed of polyfluorene and poly(N- methyl-2-vinyl pyrid- inium chloride)-b- poly(ethylene oxide)26. Moreover, Vicent and co- workers measured the size, shape and dynamic exchange of polymers between aggregates of charged polymers using two- colour STORM36. In this case, the self- assembly induced by the attraction of polyions at low salt content was monitored by a com- bination of STORM and a variety of other spectroscopy and microscopy techniques, which unequivocally pro- vided the first experimental evidence of the theoretically predicted, extraordinary, charge- driven aggregation mechanism. Although only a few studies of coacervates using SRM have been reported thus far, they highlight that optical imaging is a potentially powerful tool for visualizing and probing the properties of soft materi- als that are difficult to study with other approaches.

An interesting future perspective is to exploit SRM to address the molecular composition and stoichiometry of the two charged species with single- aggregate reso- lution. The conformation of individual conjugated and non- conjugated macromolecular chains in polymer blends has been captured successfully by SMLM37,38. Aoki and colleagues used PALM to visualize the archi- tecture of poly(butyl methacrylate) (PBMA) labelled

with rhodamine spiroamides and embedded in a thin film of unlabelled PBMA spincast on a glass coverslip39. Gramlich et al. used STORM to image the nanoscale morphology of polystyrene and poly(methyl meth- acrylate) (PMMA) blends40. The group determined the size, shape and abundance of demixed nanodomains in the thin films and found a correlation between these characteristics and film thickness. Not only the inner structure but also the surface properties of polymer materials can be investigated with nanoscopy. Indeed, STORM experiments revealed the surface densities and heterogeneous distributions of dye- labelled −COOH groups on different PMMA surfaces41.

The intrinsic fluorescence of conjugated polymers is typically emitted from localized regions within individ- ual chains. The migration of excitons and its depend- ence on the polymer conformation are critical, yet only partially understood, parameters for the performances of such polymers in optoelectronics (such as in light- emitting diodes and solar cells) as sensors and as fluo- rescence markers15,38,42. Offering unprecedented access to both the single- chain conformation and exciton den- sity, SRM can play a pivotal role in unravelling the link between the structure and photophysical properties of conjugated polymers. Habuchi and co- workers exploited a

b

Feature size ≈ 50 nm to 10 μm Self-blinking

Block copolymer assemblies Conjugated polymers

5,000 nm 5,000 nm 5,000 nm 5,000 nm 5,000 nm

Polymer blends

Fig. 2 | Polymeric materials studied with SRM. a | Schematic representations of the main families of polymeric materials studied with super- resolution microscopy (SRM). Block copolymer assemblies (left panel) can self- assemble into nanostructured materials with different morphologies, such as fibres, vesicles and platelet- like structures. These architectures can incorporate multiple components of sizes ranging from the nanoscale to the microscale, challenging imaging methods. Super- resolution has been used to image such materials over three orders of magnitude (10 nm to 10 μm). Conjugated polymers (centre panel) are important materials for molecular electronics thanks to their conductive properties. Nanoscopy can not only study their structure but also provide functional information because of the correlation between the material’s photophysical properties (such as fluorescence blinking) and electronic features.

Polymer blends (right panel) are commonly used in many materials chemistry applications. Their dense and thick structures make them difficult to access by techniques such as atomic force microscopy or electron microscopy.

Super-resolution imaging has been used to probe the structure and the properties (for example, polymer chain diffusion) inside such materials. b | Structured illumination microscopy (SIM) imaging of platelet- like block copolymer micelles.

Thanks to the ease of imaging and multicolour capability of SIM, the relative positions of multiple polymers inside the final structure can be obtained. Part b is adapted with permission from ref.31, AAAS.

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the blinking of individual poly[2-methoxy-5-(20-ethyl- hexyloxy)-1,4-phenylene vinylene] (MEH- PPV) chains embedded in thin films of Zeonex or polystyrene43 to map the emitting sites within individual chains. The label- free SMLM experiments revealed both the MEH- PPV architecture (rod- like or disc- like) and the den- sity of emitting sites, which were approximately 10 nm apart and distributed in a uniform manner44. Similarly, Park and co- workers resolved closely spaced emission sites after stepwise photobleaching of single emitters using so- called single- molecule high- resolution imag- ing with photobleaching45. Penwell and colleagues resolved the structural features (down to 90 nm) of poly(2,5-di(hexyloxy)cyanoterephthalylidene)-based nanoparticles that carried densely packed endogenous chromophores employing a modified STED scheme that featured an excitation laser modulation46. King and Granick imaged operating organic light- emitting diodes composed of MEH- PPV. They used STED- spectral imaging to map the conformation of the chains in the emissive layer, and electroluminescence- STED to obtain super- resolved (~50 nm resolution) electrolu- minescence maps along the axial direction and in the x–y plane47. These examples beautifully illustrate how well suited SMLM is to disclose the structure–function relations of this special class of polymers. Their intrin- sic fluorescent blinking enables a label- free single- molecule reconstruction of their chain conformation and a direct assessment of the spatial distribution of their functional sites. Accessing this information will help us further understand the physical underpinnings of the performance of conjugated polymers in molecular electronics applications.

Fluorescent dyes widely used for SRM are often suboptimal for the visualization of polymer materials owing to the latter’s size, hydrophobicity and limited exposure to solvent. The first generation of nanoscopy labels for biological structures was designed to covalently bind the hydrophilic surface of proteins. The steadily increasing interest in SRM from the polymer and mate- rials science communities has catalysed the develop- ment of novel dyes tailored for nanoscopy in complex environments such as solvent- free polymer blends and hydrophobic cores of block copolymer micelles48–51. Moreover, one of the main limitations of STORM is the necessity of adding cofactors to the solvent to achieve the desired photoswitching. To overcome this limitation, several groups have proposed alternative dyes for super- resolution imaging that spontaneously photoswitch and can be incorporated into block copolymers. Tian, Li and Hu proposed48 the use of spiropyran derivates to perform SMLM and reconstructed images with a res- olution down to 10–40 nm (ref.49) at the cost of long exposure times (1 second) and poor temporal resolution.

Similarly, Yan and co- workers explored the possibility to use spiropyran derivate dyes to image block copolymers (PSt- b-PEO) self- assembled into cylindrical micelles in aqueous media50. A diarylethene derivative has been investigated by Nevskyi et al.51 as a novel photoswitch for PALM imaging owing to its high photostability and strong fluorescence of one of its conformations. As in the case of spiropyran derivate dyes, two laser wavelengths

are used to convert the diarylethene dye from the non- fluorescent into the fluorescent form and vice versa.

The authors demonstrated the possibility to visualize micelles assembled from PSt- b-PEO block copolymers with SMLM.

Although polymers are usually fluorescently labelled in order to be studied with SRM, some polymers may exhibit intrinsic fluorescence in the visible range52. PMMA, polystyrene and Su-8 films were imaged using STORM without any external labelling, showing inten- sive blinking events that enabled the reconstruction of a well- defined image52. The intrinsic fluorescent prop- erties of several materials represent at the same time an advantage and a drawback, as the spontaneous fluores- cence generates a strong background that prevents high- resolution visualization of the structure through specific labelling. Therefore, the development of novel blinking dyes that are emissive in the polymeric environment and the development of self- blinking materials suitable for label- free SMLM represent two interesting perspectives in SRM.

Lipid- based materials. Liposomes and lipid nanopar- ticles play a crucial role in a wide range of biomedical applications, such as drug delivery53. The typical lipos- ome size range (50–200 nm) can be addressed perfectly by SRM. The hydrophobic nature of the bilayer inte- rior of liposomes makes these assemblies ideally suited for PAINT using lipophilic probes. The first examples of PAINT involved the use of hydrophobic probe Nile Red to image 100-nm- diameter large unilamellar vesi- cles and a supported lipid bilayer54(fig. 3a). Nile Red is an ideal probe in PAINT, because its fluorescence emis- sion is almost negligible in water but strong in apolar environments. Moreover, the Nile Red association time to lipids of approximately 6 ms is suitable for on/off switching, thus enabling single- molecule localization54. In later studies, PAINT was exploited to visualize the lateral phase separation of distinct lipids that resulted in the formation of small domains within membranes.

This phase separation remains challenging to image with other techniques55. Spectrally resolved PAINT (sPAINT) has also been introduced56,57(fig. 3b): Sharonov and col- leagues exploited the environment- dependent emission of Nile Red to map the hydrophobicity of the structures, providing an extra dimension to the super- resolution image. The insertion of a transmission diffraction grating into the optical path enables the acquisition of fluores- cence spectra of individual solvatochromic fluorophores and leads to high- resolution maps of the hydrophobicity of large unilamellar vesicles and other structures, such as β- amyloid(1–42) fibres, α- synuclein fibrils and the cell plasma membrane. This pioneering example highlights how SRM can not only reveal structural information but also probe the chemical properties of supramolecular biomaterials.

In addition to the characterization of morphology and size, insight on the internal structure of micelles and ves- icles provides valuable information. A first quantitative study to assess encapsulation and compartmentalization in nanostructured lipid carriers (NLCs) was performed by Boreham and co- workers58. They combined STORM

Stepwise photobleaching The sequential loss of the fluorescence of individual molecules, resulting in a series of distinguishable steps that provide information about the number of fluorophores present.

Transmission diffraction grating

A device with a periodic structure that diffracts incident light into different directions.

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imaging and single- particle tracking approaches to meas- ure the size and shape of the NLC domains loaded with ATTO- Oxa12 fluorescent dyes with a 6 nm spatial reso- lution. This approach enabled the group to monitor the precise distribution of drugs inside the NLCs, revealing the existence of two types of drug- loaded nanocompart- ments of different size (Ø ≈ 70 nm and 120–130 nm) that fill up to ~50% of the volume of NLCs.

More recently, attention has been dedicated to the study of lipid nanoparticles and their surface functional- ization, which can be considered one of the major mech- anisms of nanoparticle–cell interaction. Van Oijen and co- workers59 recently reported on the molecular count- ing of functional proteins on the surface of liposomes using single- molecule imaging. This study opened the way towards multicolour mapping of the functionality of lipid- based drug nanocarriers.

The unique characteristics of liposomes bring with them both advantages and disadvantages for SRM stud- ies. Although the hydrophobicity of the intramembrane compartment enables performing PAINT with polar probes, difficulties in fixation and a lack of stability of lipid assemblies challenge other methods such as STORM or STED. The development of novel sample labelling and preparation procedures will open new avenues in liposome nanoscopy imaging.

DNA origami. Among nanostructured materials, DNA origami60 has gained a prominent position owing to the versatility and high programmability of the nucleotide assembly61. However, the small size and complexity make DNA origami rather challenging to characterize with nanoscale accuracy; for this reason, super- resolution imaging of DNA origami has gained increasing attention since the first reports of such materials62.

Tinnefeld and co- workers were among the first to use STORM to image DNA origami. They were able to visualize and measure with nanometric resolution the distance between two labelled strands that popu- lated the diagonally opposite corners of a rectangular DNA assembly of known dimensions63. STORM meas- urement of the labelled strands (88 nm) resulted in excellent agreement with the designed diagonal length (89.5 nm). This pioneering work demonstrated that SMLM is an effective tool for studying DNA origami and that DNA origami can be used as the calibration standard for SRM. Indeed, the controllable design of DNA origami at the molecular level enables the crea- tion of nanorulers to test the performances of a specific SRM setup. For this purpose, DNA origami decorated with fluorophores at nanometric distances have been designed and used to challenge STED, SMLM and SIM64,65. The same group extended the use of direct STORM (dSTORM) to attain 3D images of DNA ori- gami, as in the case of DNA nanopillars, demonstrat- ing the ability of this technique to resolve both origami structure and orientation64.

Being composed of DNA strands, origami construc- tions are considered ideal structures to be studied with DNA- PAINT, as shown by Jungmann, Simmel and co- workers66. Extra oligonucleotide sequences acting as docking strands can be included in the origami struc- tures, while complementary dye- labelled sequences are added to the solution as imager strands (fig. 4a). Several origami structures have been imaged using DNA-PAINT, and a particularly elegant example is the DNA rigid, closed, hollow tetrahedron structure featuring 75-nm- long edges67. Authors have succeeded in binding fluorescent molecules at the edges of a tetrahedron, thus validating its potential as a 3D ruler

a b

Liposome diameter=50–200nm

Hydrophobic probe DL SR sPAINT

Polar probes

Bilayer thickness=5nm

Fig. 3 | Super- resolution imaging of lipid- based materials. a | Schematic representations of the structures and features of lipid materials. Liposomes (left panel) are a cornerstone of drug delivery , but owing to their small size, their dynamics (for example, lipid lateral diffusion) cannot be easily studied by classical fluorescence microscopy. Nanoscopy can provide nanoscale information on size and component distribution not accessible before. Similar considerations apply to supported lipid bilayers (right panel) commonly used as model membranes and coatings. Owing to the presence of a hydrophobic pocket, hydrophobic probes can be used to access the interior of liposomes or bilayers, making covalent labelling of the structure of interest unnecessary. Moreover, solvatochromic probes, such as fluorophores that change emission depending on the polarity of the environment, can be used to obtain information on the type of assembly and distribution of the lipids. b | Images of lipid assemblies using spectrally resolved point accumulation for imaging in nanoscale topography (sPAINT) that reveal liposome nanostructure and polarity. In this method, spectra of single solvatochromic fluorophores are recorded during single- molecule localization microscopy , providing a nanoscale map of polarity. Images in the left column are diffraction- limited (DL) images, those in the middle are super- resolution (SR) images, and those on the right are sPAINT hydrophobicity maps. Scale bars are 500 nm and 20 nm in the zoom. Part b is adapted from ref.56, CC- BY-4.0.

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for SRM. DNA- PAINT was also used to look at the structure and orientation of single- stranded tiles that featured labelled docking strands at the four corners

(fig. 4b). Simmel and co- workers succeeded in imaging with DNA- PAINT68 one of the smallest DNA struc- tures: a scaffold- free DNA cube, the edges of which are 32 base pairs long. DNA-PAINT has also been used to identify gene sequences by means of relatively inexpensive short single- stranded DNAs (ssDNAs) with a sequence resolution of 50 nucleotides, which corresponds to a spatial resolution of 30 nm (ref.69). Knudsen and co- workers studied the immobilization of polymers into a DNA origami structure by DNA- PAINT70, as a first step towards the creation of hybrid origami–polymer biomaterials.

Other implementations of DNA- PAINT have been developed with the aim of reducing the image acquisi- tion time and the background level. Schueder and co- workers achieved a faster and less invasive DNA- PAINT imaging of 3D DNA origami using a spinning disk microscope71. An interesting fluorescence resonance energy transfer (FRET)-based version of DNA- PAINT was developed by Auer and colleagues, who introduced specific single- strand docking sequences of programma- ble length to a DNA origami scaffold. In this approach, the single- molecule localizations are related to a FRET event such that the background level and image acqui- sition time of DNA- PAINT are substantially reduced72. Additionally, the counting of densely packed organic fluorophores was demonstrated by analysis of their

photoswitching kinetics during STORM imaging. In this case, a DNA origami scaffold was decorated with two trimers of closely packed organic fluorophores, which are not individually resolvable with STORM but can be discriminated by means of the image analysis73. STED microscopy has also been used to study the photophys- ical behaviour of different fluorescent probes on DNA origami structures74.

The relationship between DNA origami and nano- scopy leads to a clear mutual benefit. On one side, DNA origami is a promising new class of materials with complex features that need SRM as a powerful characterization technique. On the other, DNA origami are molecularly controlled scaffolds that can serve as a benchmark for super- resolution methods and labels, contributing to the advancement of the technique. For example, the use of DNA- PAINT for the study of DNA origami resulted in some of the best advanced perfor- mances currently reported in terms of multiplexing (up to ten colours have been recorded12), 3D imaging70,71 and resolution (being able to solve a densely packed triangu- lar lattice with a 5 nm point- to-point distance with three different colours in multiplexed exchange- PAINT12,75).

Nanoparticles. The power of SRM to resolve subdiffrac- tion structural and functional details of nanoparticles that are often much smaller in size than the diffraction limit is becoming increasingly recognized (fig. 5a,b). Pioneering studies in this area have demonstrated the visualization of the morphology and surface function- alization of individual nanoparticles21,26,76, their dense packing in clusters and colloidal crystals21,77, the plas- monic properties of metallic nanoparticles and the cata- lytic properties of oxides. Nanoparticles can also facilitate SRM imaging of other materials, because they can serve as sensitive markers for drift correction to enhance precise localization and overlay, for example, in correlative SRM and AFM–EM studies78. Bon and colleagues used dSTORM to localize gold nanoparticles in 3D space with nanometric accuracies down to 0.7 nm in the lateral and 2.7 nm in the axial directions by collecting 50 frames per second, even in the presence of micrometre- large fluctuations79.

Large, micrometre- sized colloids have long been studied using scattering methods and quantitative con- focal imaging as experimentally accessible model sys- tems for atoms. The advent of SRM offers the possibility to access time- lapse, real- space information on the phase behaviour and superstructure formation in the entire colloidal space domain (nm–μm). For example, Harke and co- workers used 3D- STED imaging (with a lateral resolution of 43 nm and an axial resolution of 125 nm) to unambiguously identify the structure of densely packed colloidal crystals assembled from latex spheres by confined convective assembly77.

SRM has also contributed to an improved under- standing of the relation between the catalytic activity and morphology, structure and surface functionaliza- tion of nanoparticles80–82. Zhou and colleagues analysed the catalytic activity of single gold nanorods coated with a mesoporous silica, reaching a spatial resolution of ~40 nm and a temporal resolution of a single catalytic a

b

DNA origami Direct imaging

with DNA-PAINT

200nm 200nm

10–200nm

z x

y z

x

y y

x

z x

Fig. 4 | Super- resolution imaging of DNA origami. a | Schematic representations of DNA origami structure and features. DNA origami are obtained by the self- assembly of a large, circular single- strand DNA (template) with multiple small DNA sequences (staple strands). The programmable DNA–DNA hybridization enables us to design sequences to obtain a final structure with molecular precision. Owing to the intrinsic composition of DNA origami, DNA- point accumulation for imaging in nanoscale topography (PAINT) is one of the most- used super- resolution techniques for characterizing these structures.

This method uses small dye- labelled DNA strands to directly probe the origami structure by reversible and specific hybridization. b | 3D- PAINT imaging of DNA origami. Owing to the unbeatable resolution, 3D ability and insensitivity to bleaching of PAINT, nanoscale 3D origami can be resolved. Part b is adapted with permission from ref.190, AAAS.

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event83. Cang and co- workers detected single hot spots as small as 15 nm on silver nanoparticle clusters84. STED and PALM studies at length scales below 10 nm (ref.85) revealed a clear correlation between nanoparticle size

and catalytic activity86–88. Temporal activity fluctua- tions of individual nanocatalysts have been related to surface restructuring85,89, and activity gradients, to surface defects.

Care must be taken when interpreting the SRM results of plasmonic nanoparticles, as the electro- chemical and photophysical properties of the dyes are affected by the nature of the metallic surfaces90,91 when the emission of the dye couples with the surface plas- mon polaritons92,93. The probe brightness and emission are modulated, with important ramifications for the sensitivity of single- molecule surface- enhanced Raman spectroscopy (SM- SERS) and the precision and accu- racy of SRM imaging. A direct comparison between SM- SERS results of physisorbed and tethered Nile Blue revealed variations in the potential- dependent SERS intensity for chemisorbed but not for the physisorbed solvatochromic dye90,91. Direct evidence of the mis- localization of dyes on plasmonic nanostructures was obtained from correlative AFM and SRM studies that revealed smaller- than-expected reconstructed images of gold nanorods94. To overcome such localization errors effectively, the emission of the fluorescent tags and the surface plasmons can be decoupled92,93,95.

By contrast, at optimal dye–metal distances, the emis- sive coupling can be exploited to enhance fluorescence of the probe, to boost the photocatalyst activity and for site- specific functionalization. Highly system- specific sweet spots exist at which dye fluorescence enhancements are notable96. Profiting from this opportunity, Uji-i and co- workers imaged fluorescently tagged proteins on plas- monic nanowires97. Aramendía and co- workers used a similar two- laser activation strategy to convert spi- ropyran into its fluorescent merocyanine form on gold nanorods98. The optimal dye–metal distance is short enough to promote fluorescence enhancement and long enough to limit fluorescence quenching. This offers maximal signal- to-noise ratios and improved localiza- tion precision99. Johlin and colleagues interrogated the near- field optical interactions between quantum emit- ters and nanostructures by directly measuring the mod- ulation of probe brightness and its location relative to a silicon nanowire, aiming to image the strong, extended coupling between dipole- like sources and nanoscale antennas100. Simoncelli and co- workers developed a ver- satile plasmon- induced approach for the in situ surface functionalization of gold nanoparticles. This approach is based on a plasmon- selective photocleavage of surface thiols followed by ligand replacement with, for exam- ple, thiolated DNA oligonucleotides101. The success of the selective surface functionalization was demonstrated by metallic DNA- PAINT (m- PAINT) upon binding to the surface strands of short, complementary strands car- rying fluorophores that exhibit absorption and emission spectra decoupled and blue- shifted with respect to gold plasmonic modes12,66,102(fig. 5b).

Imaging materials in action

In the previous section, we described the current state of the art in the use of SRM to study complex materials.

However, these studies were often performed under conditions (for example, in pure water) that are far a

b i

ii

iii

i

ii

iii

Diameter = 50–100 nm

100 50

– 50 – 100

50 100

–50

50 100

–50

–100–100 –50 0 50 100 1.4 1.5 1.6 1.7 1.8 1.9

0 180

–100 0 0

Y (nm) Scatt. intensity (a.u.)Scatt. intensity (a.u.)Scatt. intensity (a.u.)

Y (nm)Y (nm)

X (nm) Photon energy (eV)

Plasmonic effects

PAINT probes

Catalysis

S P

Gold nanorod Gold

NP

180 180

1.0 0.8 0.6 0.4 0.2 0.1 1.0 0.8 0.6 0.4 0.2

1.0 0.8 0.6 0.4 0.2 0.0 0.1

Fig. 5 | Super- resolution imaging of metal nanoparticles. a | Schematic representations of the structural and functional features of gold nanoparticles (NPs). Gold nanorods and NPs are extensively used in nanotechnology thanks to their intrinsic properties, such as plasmonic effects (left panel) and catalytic activity (right panel). In addition to size and shape, super- resolution microscopy (SRM) has been used to probe these unique features, as they can interfere with fluorescence emission. Plasmonic effects can quench or enhance the fluorescence of fluorophores placed in proximity of the NPs, which act as nanoscale functional probes of gold structure properties. Using substrates that change fluorescence upon conversion, the catalytic efficiency of the gold catalyst can be probed at the single molecule level. In this framework , SRM can not only provide structural information, such as particle size and morphology , but also offer information on the plasmonic and catalytic activities of the NPs. b | DNA- point accumulation for imaging in nanoscale topography (PAINT) imaging of gold NPs. Combining SMLM and plasmonic measurements, information regarding the DNA ligands on the nanorod surface (left panel) and regarding the plasmon energy (right panel) can be obtained. Part b is adapted with permission from ref.102, ACS.

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from the real conditions of application. Notably, SRM is minimally invasive compared with other techniques, such as electron microscopies and probe microscopies, and it can often be used to perform measurements of a material in its operational conditions. For example, SRM has been used to unveil the photophysical and electronic properties of devices made of conductive polymers43 or perovskite103. SRM has also had a dramatic impact in the field of catalysis, in which single- reactivity events have been probed with nanometric accuracy in catalytic reactors such as zeolites83,104–108. Finally, SRM mild imag- ing conditions allow one to study biomaterials in situ, for example, in cellular environments. As SRM was initially developed for the imaging of biological struc- tures, material–cell interactions were the first targets to be studied.

In the next sections, we discuss the latest applica- tions of SRM for the study of chemical biology and biomaterials. In particular, we report on the study of cell uptake and trafficking, the study of stability and material alteration inside cells and the probing of material–biomolecule interactions (fig. 6).

Cellular trafficking and localization. Understanding the exact localization of materials inside cells during cell internalization is crucial for applications such as drug and gene delivery, as different cell locations imply dif- ferent biochemical environments (fig. 6a). To this end, SMLM, SIM and STED have been employed, taking advantage of the peculiarities of the different techniques.

SIM is ideal for performing live- cell studies owing to its good temporal resolution and the minimum illumination power required. The exact intracellular

localization of different nanomaterials has been deter- mined thanks to the high particle localization accuracy of SIM109–112. In addition to the exact localization of the nanomaterial, SIM enabled the monitoring of its deg- radation over time, as fragments of 100–200 nm could be imaged. Another study reported on the internali- zation and trafficking of metal–organic frameworks in live cells, avoiding any fixation aberration using SIM110. Nanoporous polyethylene glycol/poly- l-lysine particles complexed with siRNA targeting survivin have also been studied109. Trafficking of these particles inside the cells was investigated using a combination of deconvolution microscopy, fluorescence lifetime imag- ing microscopy and SIM. The latter provided insight on the final fate of the nanoparticles at long incubation times. Despite the limited special resolution compared with that of STED and SMLM, SIM is an optimal choice for a long- term live- cell study of intracellular traffick- ing owing to its good temporal resolution and mini- mal phototoxicity. Reflected light SIM is an alternative approach that uses nanoparticle scattering to provide contrast. This technique was recently used to follow the internalization of iron and cerium oxide nanoparticles, with 100 nm resolution113.

STED was first used to study nanoparticle–cell interactions to demonstrate the nuclear localization and clustering of 30 nm fluorescent silica nanoparticles in Caco-2 cells114,115. More recently, Peuschel and co- workers used 3D- STED to quantify the internalization of 25 nm and 85 nm silica nanoparticles in A549 cells116 and differentiate between membrane- attached and inter- nalized nanoparticles. A similar approach was also used to study the internalization and colocalization of carbon a Cell uptake and intracellular trafficking b Stability and material alteration c

inside cells Material–biomolecule binding

and interactions

Fig. 6 | Interactions between materials and cells or biomolecules can be studied with nanometric resolution using SRM. a | The cell uptake and the exact intracellular localizations can be studied, providing nanometric information that can be relevant for the material application. Stimulated emission depletion (STED) and single- molecule localization microscopy (SMLM) can be used to study the intracellular localization of nanoparticles in fixed cells, and structured illumination microscopy (SIM) and STED can be used to retrieve the same information in live cells. b | The internal structure and stability of a material can be studied using super- resolution microscopy (SRM) to acquire information about changes in the materials at the nanometric scale. Both SIM and SMLM are used to probe shape and size changes following cell uptake. c | Once a material is introduced into living systems, it can interact with the plethora of biomolecules present in vivo. To design effective materials, it is imperative to understand the binding and the interactions with these

biomolecules, which can affect their functionality. Two- colour SMLM is used to measure bio- nanointeractions at the single molecule level, including ligand–receptor and nanoparticle–protein interactions.

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