Fluorescence-readout as a powerful macromolecular characterisation tool

The last few decades have witnessed significant progress in synthetic macromolecular chemistry, which can provide access to diverse macromolecules with varying structural complexities, topology and functionalities, bringing us closer to the aim of controlling soft matter material properties with molecular precision. To reach this goal, the development of advanced analytical techniques, allowing for micro-, molecular level and real-time investigation, is essential. Due to their appealing features, including high sensitivity, large contrast, fast and real-time response, as well as non-invasive characteristics, fluorescence-based techniques have emerged as a powerful tool for macromolecular characterisation to provide detailed information and give new and deep insights beyond those offered by commonly applied analytical methods. Herein, we critically examine how fluorescence phenomena, principles and techniques can be effectively exploited to characterise macromolecules and soft matter materials and to further unravel their constitution, by highlighting representative examples of recent advances across major areas of polymer and materials science, ranging from polymer molecular weight and conversion, architecture, conformation to polymer self-assembly to surfaces, gels and 3D printing. Finally, we discuss the opportunities for fluorescence-readout to further advance the development of macromolecules, leading to the design of polymers and soft matter materials with pre-determined and adaptable properties.


Introduction
Polymers play an indispensable and ubiquitous role in human society, with applications ranging from clothing to medication to aviation.Polymers are formed through polymerisation processes, which transform monomers into polymeric chains.][3][4][5][6][7][8][9] Historically, the discovery of living anionic polymerisation by Michael Szwarc in 1956 opened a key avenue for the synthesis of well-dened polymers, 10 albeit not with the precision of contemporary sequence-dened polymers.7][28][29][30][31][32][33][34][35][36][37] In order to perform polymerisation under more diverse conditions to address a broader scope of applications, light, [38][39][40][41][42] electricity, [43][44][45] mechanical force 46,47 and chemical triggers 48,49 have been successfully utilised as external regulators in polymerisation reactions.Among them, light is arguably the most attractive regulator due to its unique capability to spatiotemporally control chemical reactions.4][65] To reach this aim, it is critical to develop advanced analytical techniques able to investigate polymers and polymerisation processes at molecular and microscopic levels, in addition to progressing advanced synthetic methods.A plethora of analytical techniques are routinely applied in polymer science, including spectroscopic methods (nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), ultraviolet-visible (UV-Vis) spectroscopy , Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS)), thermal and mechanical methods (differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA)), chromatography (size-exclusion chromatography (SEC), high-performance liquid chromatography (HPLC)), gravimetry, microscopy (optical microscopy, atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM)) as well as coupled techniquesall of which are able to provide valuable information, such as molecular weight, molecular weight distributions, chemical compositions and polymer structures.However, when considering the time scale of polymerisation processes and dynamics in polymer systems, the complexity of polymer architectures and organisation as well as heterogeneities of polymer systems, these techniques exhibit limitations and disadvantages arising from the necessity of sample preparation, invasive and destructive tests, low sensitivity and macroscopic measurements.Thus, it is a considerable challenge for polymer scientists to investigate polymer systems and polymerisation processes at molecular and microscopic levels, especially during the macromolecular growth process.Among the available methodologies, uorescence-based techniques present appealing features, i.e., high sensitivity (e.g., sub-micrometre spatial resolution and below millisecond time resolution), high selectivity, large contrast, fast and realtime response, and non-invasive characteristics.][91][92][93] In fact, uorescence-based techniques have been applied to analyse polymers for several decades.1][102][103] In addition, recent years have also seen exciting advances in exploiting uorescence principles and technologies in synthetic polymer systems, including uorogenic, 104 AIE, 105 FRET, 82 uorescence lifetime imaging microscopy (FLIM), 85 stimulated emission depletion microscopy (STED) 106,107 and single-molecule localisation microscopy (SMLM). 83,92,93Thus, our aim is to provide a contemporary review that critically assesses the recent advances in the eld and the opportunities and challenges that have emerged.
In the current review, we thus focus on recent scientic achievements covering the analysis of wide aspects of synthetic polymer systems based on uorescence techniques (refer to Fig. 1).We provide an overview of the eld via the discussion of representative examples from 2010 onwards.The review is organised as follows: we commence with two essential parameters for polymerisation processesmonomer conversion and molecular weight, describing different uorescence phenomena and their adaptation to various types of polymerisations for the determination of these two parameters.We subsequently discuss uorescence-based techniques for the investigation of polymer architecture, conformation and selfassembly, highlighting the use of a variety of uorescence phenomena and advanced technical methods to access different parameters in these areas, for example the lengths of arms in specic polymer architectures, end-end distances in polymer conformations as well as monomer exchange dynamics during self-assembly.Extending from polymers to so matter materials, next, we discuss how uorescence-based techniques can benet the study of polymer surfaces, gels and 3D-printed structures.Finally, we share our vision for the future of exploiting uorescence-based techniques in polymer and materials science, which we suggest will give further deep and new insights.

Monomer conversion and molecular weight
In polymer chemistry, monomer conversion is dened as the consumption of the initial monomer(s) during macromolecular growth, and molecular weight oen refers to an average molecular weight due to dispersity.Both of these are strongly linked to the progress of polymerisation and have a striking impact on the polymer properties, from the rheological and mechanical properties to morphological characteristics. 108,109herefore, following the reaction kinetics via monomer conversion and determining the molecular weight of a polymer is oen the initial key step in its characterisation.
Monomer conversion and molecular weight can be determined by several analytical methods, including gel-permeation chromatography/size-exclusion chromatography (GPC/SEC), NMR spectroscopy, MS, FTIR and Raman spectroscopy.However, these methods are typically challenged in providing real-time information for ongoing reactions, and they oen require fully soluble polymers, preventing characterisation of cross-linked and high-molecular weight polymers as well as conjugated polymers.Fluorescence-based techniques provide the opportunity to overcome these difficulties.In order to successfully visualise and monitor polymerisation processes, the addition of a uorophore (prouorophore) to a polymer system or, in many cases, the covalent attachment of a uorophore (prouorophore) to a polymer chain is oen required due to the generally very weak intrinsic uorescence of polymers.
In the following, a variety of uorophores will be introduced with regard to different polymerisation systems ranging from conventional polymerisation, conventional photopolymerisation, to controlled polymerisation, then to supramolecular polymerisation.
While different types of polymerisations exhibit different mechanisms and kinetics, the polymerising system always undergoes a transformation from a lower viscosity liquid to a more viscous, even rigid material as monomer conversion and molecular weight increases.][112][113] For example, pyrene can undergo intermolecular interactions to form excimers (dimers that exhibit different uorescence emission from the single molecule, Fig. 2) depending on the lateral diffusion of pyrene in the medium. 114An increase in local viscosity limits the diffusion of the ground state pyrene molecules towards the excited state ones, resulting in a decrease in the ratio between the maximum uorescence intensity of the excimer and the maximum uorescence intensity of pyrene (I excimer /I pyrene ).This has been exploited to follow the conversion of methyl methacrylate (MMA) in situ during miniemulsion polymerisation. 110However, a high concentration of pyrene is required in order to achieve sufficient proximity for two molecules to form excimers, and due to the rapid decrease of I excimer / I pyrene , it is oen challenging to monitor long-term polymerisation processes.
Unlike the excimer-forming compounds, different types of environment-sensitive uorophores including intramolecular charge transfer compounds (Fig. 2) have been developed by the group of Ortyl and others, such as coumarin-based molecules, 111,115 2-amino-4,6-diphenyl-pyridine-3-carbonitrile derivatives, 116 rare earth complex compounds, 117 meta-terphenyls, 118 dansyl hyperbranched uorophores, 119 benzylidene scaffoldbased and 4,4-diuoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based iodonium salts, 112,120 to monitor free-radical photopolymerisation, ring-opening and chain growth cationic photopolymerisation, based on a changing uorescence intensity or uorescence intensity ratio as well as shiing emission wavelengths.We note common principles: (i) when the excited state of the uorophore is more polar than the corresponding ground state, the reduced polarity of the system leads to an increased energy gap between the ground and the excited state of the uorophore.As a result, the uorescence emission wavelength shis toward shorter wavelengths; (ii) the increased microviscosity of the system slows the solvation of the excited uorescent molecules, thus preventing them from reaching their most relaxed conformation before emission of a photon, while the rigidity of the medium inhibits conformational changes of the excited molecules which is one of the deexcitation pathways.Therefore, a hypsochromic shi of the emission wavelength and an increase in uorescence intensity can be observed. 111,121][124] One of the major benets of photopolymerisation is the ability to manipulate the light source to switch reactions between 'ON' and 'OFF' states and to adjust polymerisation rates.In general, the same wavelength is applied for the initiation of the reaction and the excitation of the uorophore.A very elegant approach is to use the existing photoinitiator in the system as a uorophore for monitoring, such as benzylidene scaffold-based and BODIPY-based iodonium salts developed by Ortyl's group or 2,6bis(furan-2-ylmethylidene) cyclohexan-1-one developed by Li et al. 112,120,125 It is worth noting that the design of uorophores for cationic polymerisation is more challenging as they need to resist strong acidic environments.
An alternative type of uorophore is the molecular rotor (Fig. 2), whose uorescence depends on the viscosity of the surrounding medium.Upon excitation, the molecular rotor enables rapid non-radiative de-excitation via intramolecular rotation in low viscosity environments, in contrast, high local viscosity hinders the rotation and thus limits non-radiative pathways, resulting in higher uorescence intensities and quantum yields as well as longer uorescence lifetimes. 126,127olecular rotors, such as BODIPY-C12 (chemical structure refer to Fig. 2), are thus well suited to investigate polymerisation processes. 110,113,128or example, Nolle et al. have successfully used BODIPY-C12 to monitor bulk radical polymerisation of MMA. 113Different from the abovementioned studies, the measurements in their study were based on uorescence lifetime rather than uorescence intensity.Fluorescence lifetime detection is independent of the dye concentration, while this factor strongly affects uorescence intensity measurements. 129,130Importantly, the evolution of heterogeneities during the polymerisation process was elucidated in their study.Meanwhile, the uorescence lifetimes were obtained from an advanced uorescence technique -FLIM. 66,131In addition to the values of uorescence lifetime, FLIM can provide spatially resolved images for additional information, which will be discussed later.Besides, the choice of BODIPY-C12 allows for the measurement of local viscosity.Unlike the previously mentioned uorescent molecules and many other molecular rotors, whose uorescence is strongly inuenced by polarity and temperature, the uorescence of BODIPY-C12 is only very weakly affected by these factors, at least at high viscosities. 129,132n addition to these three types of uorophores, uorogenic molecules, aggregation-induced emission luminogens (AIEgens) and others (Fig. 2) have also been applied to visualise the polymerisation process, [133][134][135] mainly controlled polymerisation.Controlled polymerisationespecially RDRPenables the synthesis of polymers with pre-determined average molar masses, narrow molecular weight distribution, diverse compositions and well-dened architectures.RDRP has driven the rapid advance of polymer science and beneted numerous elds, including biomedicine, energy and nanotechnology. 5,26ecently, the utility of RDRP has been further extended by various externally regulated polymerisations, such as photocontrolled RDRP. 42][19] Allen et al. have synthesised uorogenic monomers to in situ monitor ATRP in aqueous media. 133These monomers are methacrylamide derivatives of polycyclic aromatic hydrocarbon (PAH) probes such as pyrene, anthracene and acridine (Fig. 2).Aer their incorporation into polymer chains, these originally non-uorescent PAH probes will become uorescent, and the uorescence intensity of the polymer system increases as a function of reaction time.A similar working principle has been applied by our group to photoinduced nitrile iminemediated tetrazole-ene cycloaddition (NITEC) step-growth polymerisation, which represents one of the few examples of a photochemically driven step-growth polymerisation. 134The reactions between the non-uorescent tetrazole moiety and the non-uorescent dialkenes produce the uorescent pyrazolinecontaining polymer.Thus, the uorescence emission directly correlates to the number of ligation points in the polymer, forming an ideal self-reporting system.
In photo-controlled RAFT polymerisation, Yeow et al. utilised 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP) as a photocatalyst and uorescence probe to mediate polymerisation and report on monomer conversion. 136The uorescence change of the system is likely due to the incorporation of ZnTPP into the polymer chain.Likewise, to visualise RAFT polymerisation in situ, Liu et al. developed an approach based on AIE (Fig. 3A). 135AIE describes the opposite of aggregation-caused quenching (ACQ), which is a common phenomenon when dyes aggregate.The uorophores used in AIE, known as AIEgens, oen show strong emission in the aggregated state due to the restriction of intramolecular motion, which makes them good candidates for sensing viscosity or other environmental changes. 75In their work, tetraphenylethylene (TPE)-containing dithiocarbamates were designed and synthesised.These compounds play the role of RAFT agents for the control of the polymerisation process.Meanwhile, TPE (a typical AIEgen) can be incorporated into the polymer chain upon light irradiation and thus sense the viscosity change during polymerisation.As noted, AIE may be more suitable for polymer systems with relatively large molecular weights.
In order to track polymerisation reactions on extended time scales, Cavell et al. combined two optically orthogonal readouts, AIE intensity and uorescence polarisation anisotropy, by incorporating TPE-and perylene diimide (PDI)-labelled monomers (very low amounts, ppm and even below) into the ROMP of norbornene within a single microdroplet (Fig. 3B, top). 137luorescence polarisation anisotropy can quantify the rotational time scale of a uorescent molecule, further providing information about the chemical evolution of its environment.As the polymerisation proceeds, anisotropy increases due to the loss of rotational freedom upon the monomers' incorporation into the growing polymer chain.Anisotropy is sensitive at early temporal regimes of the reaction, thus together with AIE can provide complementary information (Fig. 3B, bottom).
Here, ROMP is one type of controlled polymerisation that converts cyclic olens into a polymer material.The key feature of ROMP is that any monomer-associated unsaturation is retained during the conversion of monomers into polymers, which allows ROMP to synthesise polymers with unique architectures and useful functions. 28n order to monitor ROMP, Iv et al. doped the initial reaction solution with a very small amount of uorophore-labelled norbornene monomers, but, in this case, with BODIPY-labelled monomers (Fig. 4A) and applied advanced uorescence microscopy -FLIM. 85As mentioned earlier, uorescence-based techniques have signicant advantages over conventional techniques: in addition to real-time characterisation, they may allow for determination of monomer conversions and molecular weights of insoluble polymers.Their work effectively demonstrates these advantages.The correlation between polymer uorescence lifetime measured by FLIM and molecular weight allows readout of the molecular weight of polydicyclopentadiene (polyDCPD) in ongoing reactions, as well as molecular weight calculations for GPC-solvent insoluble, highmolecular weight, cross-linked polyDCPDs (Fig. 4B).Not only that, FLIM is capable of providing spatiotemporally resolved information on polymer morphology, which plays an important role in the properties of polymers, and the use of uorescence techniques for morphology studies will be discussed in later sections.
Another advantage of uorescence-based techniques is their ability to determine molecular weights of conjugated polymers, which are oen overestimated by GPC.Tian et al. employed single-molecule uorescence spectroscopy to determine the molecular weight of conjugated polymers using a singlemolecule counting method. 138However, spin-casting of the polymer matrix in which these polymers are embedded is required.In order to count molecules in a solution phase, uorescence correlation spectroscopy (FCS), 139,140 a powerful tool for detecting molecular dynamics, may be a good choice.
Notably, the aforementioned uorophores, especially viscosity/rigidity-sensitive uorophores, typically blueshi their emission or merely alter uorescence intensities with the growth of polymer chains.To address the challenge of developing uorophore probes with bathochromic emission shis, Feng et al. developed phenanthridine-fused tri-azatruxene uorophores (PTFs, chemical structure refer to Fig. 2) by judiciously selecting each unit in the uorophore: (i) phenanthridine units to enhance the uorophore/polymer interaction; (ii) a rigid planar conformation by fusing p-rings to suppress nonradiative decay; (iii) large p systems to promote electronic coupling and frontier molecular orbital energy level alignment between the uorophore and the polymer. 141Finally, in the polymerisation of pentauorophenylacrylate (PFPA), a signicant uorescent colour change from blue to red-orange with increasing polymer molecular weight was observed (Fig. 2), which can be ascribed to dipole-dipole and polar-p interactions between PTFs and polymeric matrixes to form chargetransfer complexes.
6][147][148][149][150][151] Due to these non-covalent interactions, supramolecular polymers are endowed with some special properties, including reversibility, recyclability and stimuli responsiveness, which can facilitate the design of responsive, self-healing and environmentally friendly materials. 6,36In order to determine molecular weight of a supramolecular polymer by optical methods, Sessler, Zhu, Ji and others proposed to incorporate two types of uorophores into supramolecular polymer monomers, a J-type dye (its aggregates exhibit bathochromically shied absorption bands) naphthalene diimide (NDI) (Fig. 5A, top) and an AIEgen pyrene benzohydrazonate-based uorophore (Fig. 5B, top), respectively. 152,153The former monomers interact with Zn(OTf) 2 to form supramolecular polymers through terpyridine-Zn 2+ coordination (Fig. 5A, top).With increasing molecular weight resulting from the increasing monomer concentration, a change in the uorescent colour from green to yellow to orange was observed (Fig. 5A, middle).This can be ascribed to the supramolecular assembly-induced aggregation of NDI groups, i.e., the supramolecular polymerisation and polymer assembly process leads to dimeric forms of NDI followed by the formation of J-aggregates (Fig. 5A, bottom).Differently, the latter monomers polymerise through hydrogen bonding (Fig. 5B, top) and the molecular weight was visualised through aggregation-induced emission, i.e., with the increase of molecular weight, a change of the uorescent colour from dark blue to yellow-green was found (Fig. 5B, bottom).Polymer assembly will continue to be discussed in Section 5.
This section focuses on the principles of emission changes of various uorophores, including uorogenic molecules, AIEgens and molecular rotors, as well as their adaptability to the polymerisation process.Although NMR and SEC are the most common techniques for determining monomer conversion and molecular weight, uorescence-based techniques are promising for in situ and real-time characterisation.Currently, conventional uorescence spectrometry is the main adopted uorescence technique, whilenotablyadvanced uorescence techniques are able to access more types of polymers (e.g., insoluble and conjugated polymers) and to provide more useful information, for example, FLIM for spatiotemporal morphological information and FCS for information about diffusion linked to polymerisation kinetics.Monitoring polymerisation processes from early to nal stages and gaining deeper insights into polymerisation kinetics and mechanisms will aid in controlling polymerisation to obtain polymers with tailored properties and uorescence-based techniques can critically assist along the way towards these goals.

Polymer architecture
Inspired by nature's complexity, polymer scientists have made remarkable efforts in exploring synthetic polymers with sophisticated structures.2][163][164][165][166][167][168][169][170] These architectures endow polymers with unique thermal (crystallinity, glass transition temperature), mechanical (density, viscosity, elasticity) and morphological properties, associating them with a plethora of applications, such as viscosity modiers, dispersion stabilisers as well as nanomaterials for lithography and drug delivery. 166,169,171For example, dendrimer-like polymers have fascinating properties, including low intrinsic viscosity and intramolecular topological cavities, rendering such materials potential candidates for sensor and drug delivery applications. 172,173ith ourishing research on synthesis and application of polymers with complex architectures, advanced analytical methods are needed for the characterisation of this complexity, which is essential for the synthesis and the study of properties of these polymers.Conventional methods, including rheometry, SEC and NMR, show signicant drawbacks, especially for complex macromolecular architectures, for example in the quantication of crosslinking points in a network and calculation of arm length in multi-arm star polymers.Taking NMR as an example, the analysis suffers from signal overlaps from different groups.The overlap problem is severe for linear polymers due to broader absorption from similar groups, and becomes worse for more complex polymers.Thus, a direct, facile and robust platform is required to gain quantitative information of polymer architectures, for which uorescencebased techniques are well suited.
Wang et al. successfully synthesised cyclic oligomers based on the intramolecular thiol-maleimide Michael addition reaction and monitored these cyclisation processes in situ. 174They rst synthesised a dimer containing a uorene moiety as a uorescent group, a maleimide group and a thiol group, based on the thiol-maleimide Michael addition reaction (Scheme 1A).In this dimer, the uorescence of uorene is effectively quenched due to photo-induced electron transfer between uorophores and adjacent electron-decient double bonds of the maleimide group.The cyclisation reaction restores the uorescence emission of uorene, which increases with the consumption of maleimide groups.Furthermore, they successfully expanded the cyclic topology to a cyclic-brush-like topology by reducing cyclic oligomers, then graing linear thiol-terminated polymers onto the backbone of cyclic oligomers.The degree of graing can be monitored in a similar way.This work demonstrates the potential of maleimide-based uorogenic probes as an effective method for monitoring topology formation.Here, it is worth mentioning that the thiol-Michael addition reaction, including the thiol-maleimide Michael addition, as a type of click reaction, is a powerful and widely used tool for the generation of polymers, even with complex architectures. 175Besides, the maleimide group is a robust group involved in a plethora of reactions.
7][178] Very recently, our group applied this reaction for the synthesis of three-arm star polymers by employing a trifunctional pentauorobenzyl (3PFB) linker along with a poly(ethylene glycol) bearing a thiol end-group, and determined the number of PFTR events forming the polymer arm using a chemiluminescence (CL) read out (Scheme 1B). 179In PFTR, a thiol is deprotonated by a base and subsequently reacts with the para-carbon of a PFB moiety, releasing a uoride ion.The uoride ion can trigger the CL of Schaap's dioxetane 180 and can thus serve as a quantitative read-out method.The large advantage of the CL method is that it requires neither an external light source, nor decomposition of polymer arms, therefore allowing easy access to star polymers as well as to more complex systems, as will be discussed later.
As one of the most studied polymer architectures, star polymers generally have compact structures and high segment density, making them excellent candidates for storing and delivering drugs. 166,181Among star polymers, miktoarm stars enable forming special morphological structures and supramolecular assemblies due to their chemical asymmetry, and thus have recently caught much attention. 167,169,182,183A crucial issue for miktoarm stars is the ratio/concentrations of different arm components, yet it is challenging to characterise them.
Very recently, Shi et al. introduced a uorescence strategy for the direct quantication of arm species in miktoarm stars. 184A series of miktoarm star copolymers end-labelled with various uorophores, including coumarin, BODIPY and bisindolylmaleimide (BIM), were prepared via an "arm-rst" approach, in which the arm polymers were synthesised rst by RAFT polymerisation, followed by the preparation of stars with the aid of a cross-linker (Fig. 6A).Due to the judicious choice of uorophores, the concentrations and ratios of different arm components in these stars were successfully quantied by measuring uorescence emission intensities of different uorophores.These results reveal the ability of arm polymers with different monomeric units and molecular weight to form stars.To further demonstrate the capability of this uorescence strategy, they applied it to quantify miktoarm stars bearing arms of the same monomeric units but different molecular weights, which cannot be characterised by NMR techniques (Fig. 6B).
Polymer networks, referring to polymer molecules with a high degree of crosslinking, represent another very important category of polymer architectures.6][187][188][189] Controlling the molecular structure of networks is critical for tailoring chemical and mechanical properties to these applications.The advent of controlled polymerisation methods and highly efficient linking reactions, e.g., click reactions, has led to a signicant progress in the synthesis of better-dened polymer networks. 161,190owever, detailed characterisation of networks including the number of crosslinks and structural defects remains challenging.Our group has successfully synthesised well-dened polymer networks by combing RAFT polymerisation and the NITEC reaction (mentioned in Section 2) and introduced a powerful uorescence-based methodology to quantify the number of crosslinking points in the resulting networks. 191RAFT polymerisation allows for the incorporation of pro-uorescent tetrazole moieties at the termini of polystyrene (PS) chains, and their subsequent reaction with trimaleimide via NITEC forms networks with a uorescent pyrazoline ring at each linkage point (Fig. 7A).Aer network disassembly (i.e., cleavage) via aminolysis of the trithiocarbonate moieties, all uorescent crosslinking points are preserved in a soluble system and the number of crosslinking points becomes quantiable via uorescence-readout, based on the correlation between pyrazoline concentration and uorescence intensity (Fig. 7B).Our method is in-principle applicable to all polymers capable of RAFT polymerisation.In addition, photoactivated cycloaddition NITEC offers spatiotemporal control over the crosslinking process.To avoid network degradation, a chemiluminescence read-out method to assess network formation in PFTR systems was proposed by our group, 179 which was carried out similarly to the characterisation of arm formation in star polymers described above.
Mimicking nature's complexity has given rise to polymers with sophisticated architectures like star, gra and multiblock polymers.Currently, the scientic community is developing effective methods to generate polymers with more complex architectures and controllable dispersity.To complement the increasing complexity of polymer architecture, advanced analytical methods are required to study the synthesis and properties of these polymers.Despite the small number of examples, uorescence-based techniques have, by providing information on miktoarm star copolymers and polymer networks, well demonstrated their ability to analyse polymer architectures.We anticipate that exploiting more uorescence principles (e.g., FRET) and advanced techniques (e.g., singlemolecule uorescence microscopy) will give deeper insights into polymeric architectures.

Polymer conformation
Polymer conformation describes the three-dimensional arrangement of the constitutive atomic groups of a polymer.It is perhaps the most important concept in polymer physics, as it has a profound inuence on the polymer's physical properties.5][196] The various conformations of a polymer chain are a consequence of rotations around single bonds within the polymer backbone, depending on chain exibility and interactions between monomeric units as well as with surroundings (e.g., solvent). 197][200][201][202] The important parameters in the study of polymer conformations include the end-end distance of the polymer chain (R ee ), the radius of gyration (R g ) dened as the root of the meansquare distance of a monomer from the centre of mass of the chain averaged over all monomers, 203 and their respective distributions.
Correspondingly, experimental characterisation and data acquisition is of considerable importance to validate and improve different models and theories which describe polymer conformations.5][206][207][208] However, light scattering can only provide size information for polymers with very high molecular weights or polymer ensembles due to the relatively large wavelength of light.X-ray and neutron scattering allow smaller detection scales, but specialised equipment and/ or chemistries are required, such as synchrotron light sources for small-angle X-ray scattering as well as spallation sources and deuteration for neutron scattering. 209In addition, all scattering experiments provide reciprocal space data and must be paired with complex tting models to obtain the size of polymer chains.
Alternatively, uorescence-based approaches may allow the facile and direct measurement of polymer conformations.They offer advantages including fast data acquisition, in situ measurement as well as labelling and location specicity.1][212][213][214][215] In both cases, the initial step is to label individual polymer chains at specic positions.
An excimer, e.g., an excited pyrene dimer (as mentioned before), is formed by collisional association of a ground state molecule with an excited one.Time-resolved uorescence measurements show that only emission of pyrene in the endlabelled monodispersed polymers is observed immediately aer excitation.The broad and structureless emission then grows as excimers are formed by diffusional encounters of the excited pyrene covalently attached to one end of a polymer chain and the ground state pyrene at the other chain end. 211,216he average translational diffusion coefficient (D trans ) of polymer chains within a certain molecular weight can thus be determined via the intramolecular quenching rate constant k calculated from the uorescence decay curve. 211s a comparison, methods based on FRET can provide further information about polymer conformation, such as R ee , with a larger R ee indicating a more extended conformation. 212,214RET describes non-radiative energy transfer occurring via dipole-dipole coupling from an excited state uorophore (donor, D) to a ground state molecule (acceptor, A) under the condition of sufficient spectral overlap between the donor emission and acceptor absorption and close proximity between donor and acceptor (Fig. 8A). 72Due to the inherent inversesixth-power distance dependence of FRET efficiency, it enables nanoscale proximity detection with a typical range of 1-10 nm. 72,73The efficiency can be determined by measurement of uorescence intensity or lifetime.
][219][220] Controlled polymerisations and click chemistry have signicantly advanced the synthesis of well-dened polymer structures containing uorescent end-groups, 212,215,221 which brings unprecedented opportunities for FRET methods to quantify synthetic polymer conformations, unlike previous qualitative analyses. 212,215,222,223ha et al. prepared heterotelechelic PS and PMMA possessing FRET pairs (carbazole as donor and anthracene as acceptor) at chain ends through the combination of ATRP and click chemistry or RAFT polymerisation alone using a difunctionalised RAFT agent (Fig. 8B). 212,223In another work, Simon, Qiang and others also applied RAFT polymerisation for the synthesis of FRET paired-end labelled PS and PMMA, but with anthracene as donor and the phenylthiocarbonylthio part as acceptor (Fig. 8B). 214These polymers have controlled molecular weight, low dispersity and nearly stoichiometric amounts of donor and acceptor molecules, thus enabling the determination of average R ee of polymer chains and their distribution in solution through FRET efficiency, which can be calculated through the relative emission intensities of the donor and acceptor measured by uorescence spectroscopy.Notably, in the work of Simon and Qiang, the average R ee determined through the FRET measurements is in agreement with simulation results obtained from all-atom molecular dynamics (Fig. 8C); the distribution of R ee in Sha's work obeys Gaussian statistics (Fig. 8D).
Solution concentration has a signicant effect on polymer chain conformation.According to the scaling theory of polymer solutions, 224 in diluted solution, polymer chains are isolated and behave as single coils.With increasing concentration, the distance between these coils becomes smaller.At the critical concentration (c*), the polymer coils start to come into contact with each other, causing them to assume a more densely packed state.Following that, the coils overlap and interpenetrate, and the solution is termed as semidilute.Finally, polymer chains are strongly entangled at concentrated solutions.The importance of c* in the study of polymer solutions cannot be overstated, as the chain overlap above c* results in a new conformational scaling behaviour. 224,225FRET has been employed to study the inter-and intra-chain conformation transition and determine c* (Fig. 8E). 213,223,226Moreover, it also represents an efficient and simple tool to determine the inuence of temperature, solvent polarity and viscosity on polymer conformational changes. 215,227espite the signicant potential of FRET spectroscopy in studying polymer chain conformations, several problems may be encountered.For instance, diffusion-enhanced FRET efficiency 228 and interchain FRET may disturb the measurements of R ee , and the applicable distance scale of FRET limits conformational studies of long polymer chains.
0][231][232] It can reveal rare events, allows access to distributions and time-and space- dependent properties, and helps understanding how molecularscale behaviour gives rise to bulk (macroscopic) properties, while ensemble measurements, e.g., FRET-based uorescence spectroscopy, yield information only on average properties.Furthermore, uorescence microscopy is capable of imaging polymer chains in their native environment, which is challenging for other microscopy techniques, such as AFM and TEM.AFM cannot probe polymers in their native environment due to the lack of sufficient contrast between polymer chains, while TEM requires invasive sample preparation and highvacuum environments.
Revealing single polymer chains through SMLM relies on the deactivation of uorophores during image acquisition. 233,234luorophores labelled onto a polymer chain are typically a few nm in size, but, due to the optical diffraction limit, they appear as Airy discs of 200-300 nm diameter under optical microscopy. 235Even so, single, isolated emitters can be localised with nanometre precision by tting their spot images with point-spread functions (PSFs, e.g., 2D Gaussian function). 236hus, one can construct an image of a polymer chain with nanometre resolution and calculate the distance between labels once the positions of all the emitters in a polymer chain are determined.However, the real distance between two positions on the chain is generally smaller than the optical resolution and thus the PSFs of different uorophores will blur together and cannot be individually resolved.7,238 Under suitable conditions, only a small fraction of these molecules is 'ON'.The OFF/ON/OFF switching leads to uorophore 'blinking', their PSFs can thus be spatially separated and be tted to determine their positions.By acquiring thousands of wideeld image frames and subsequent computational processing of these images, the coordinates of a sufficient number of uorophores can be determined and accumulated to generate a pointillistic image.Depending on the experimental parameters, images with features as small as a few nm can be achieved. 233,234ompared to studies of biopolymers, SMLM for the study of synthetic polymers is still in its infancy, but it undeniably has enormous potential for revealing the properties of synthetic polymers, including their conformations.
Aoki et al. have determined the R ee of individual PMMA chains in an ultra-thin lm and in a bulk state and quantitatively examined the distribution of R ee . 230In their system, both ends of PMMA were labelled with PDI.At the beginning, both PDIs emitted the uorescence, but aer a while one of them was photo-bleached and only the emission from the other was observed.Fitting the PSF from one emitter and subsequently from both allows the evaluation of R ee.It is worth noting that the efficiency of these measurements is low, as only a very low number of chains exhibit such emission behaviour.
Very recently, Wang and others provided clear images of individual bottlebrush polymer chains in a polymer melt composed of linear polymers and studied the dependence of conformation (here, the persistent length, quantifying the rigidity of a polymer) on the molecular architecture (i.e., side chain length and graing density) and the features of the surrounding environment (Fig. 9A). 239Photoswitchable diarylethenes were selected to label the polymer chains.Under UV light irradiation, diarylethenes can be activated to an 'ON' state (closed form), which can convert back to the 'OFF' state (open form) by blue light (Fig. 9B). 240Diarylethene-labelled polymer chains combined with two wavelengths enable the use of photoactivated localisation microscopy (PALM)one of the SMLM techniquesto study polymer conformation.
Characterisation of polymer conformation is of great importance for understanding the physical properties of polymers.FRET as a molecular scale ruler has been established in uorescence spectroscopy (or microscopy) to provide quantitative conformational information.Single-molecule uorescence microscopy provides intriguing opportunities to study individual polymer chain conformation.It can reveal not only the distance between two locations, but also orientation and conformation dynamics, even in 3D. 231,241,242The non-invasive characteristic of uorescence-based techniques allows the characterisation of polymer conformation in their native environment and thus gain a better understanding of conformational behaviours.More effort can be devoted to polymer chains with complex architectures in addition to linear chains.

Polymer self-assembly
4][245][246][247][248][249] A precisely designed block copolymer architecture is a key prerequisite for controlling self-assembly processes.1][252][253][254] Self-assembly of block copolymers can be conducted in bulk or in solution.6][257][258][259] The morphology of the resulting structures depends on a variety of parameters, including the volume friction of blocks, the degree of polymerisation and the Flory-Huggins interaction parameters. 255,256Solution self-assembly of block copolymers is a complex process governed by the interactions of polymer segments with the solvent as well as with each other.5][266][267][268][269][270][271][272] In addition to developing more complex morphologies, a current focus of the scientic community is the precise control of polymer selfassembly. 64][275] In their system, for example, the addition of block copolymer unimers containing a crystallisable coreforming block to cylindrical seed micelles with crystalline cores can lead to micelle growth from the crystalline core termini, allowing access to monodisperse cylinders with lengths determined by the unimer-to-seed ratio.This process can be considered analogous to living covalent polymerisation, e.g., RDRP. 273Solution self-assembly is usually carried out in a multistep process and in dilute solutions, which limits the scaling-up of the preparation. 2768][279][280] It combines polymerisation and self-assembly and can be performed at high weight percentages of solids. 97,276,281urning to supramolecular polymers, on the one hand, molecular building blocks can be polymerised via covalent synthesis into polymers with complex architectures, which then self-assemble into various morphologies. 254,270,282On the other hand, molecular building blocks can be non-covalently linked and further undergo self-assembly. 6,143,150,2836][147][148][149][150][151] The reversible nature of these interactions and the structure of the building blocks endow these polymers with unique properties, including the responsiveness to their surrounding environments. 6,284,285][288][289][290][291] Characterisation of the properties of self-assembled structures as well as of the polymer self-assembly process is essential to advance our fundamental understanding of polymer selfassembly and subsequently enables us to optimise performances of structures for desired applications.
Arising from its sensitivity, uorescence spectroscopy has been exploited to provide a wealth of information about changes in the morphology of assemblies (e.g., from assembly to disassembly, change of size) and properties of assemblies, such as their critical aggregation concentration (CAC, the concentration at which aggregates are formed) and the glass transition temperature (T g ) of the core of micelles, based on Chemical Science Review changes in emission intensity, wavelength or lifetime via uorescence quenching, FRET, AIE, etc. 250,251,[292][293][294][295][296] In bulk self-assembled lamella, a large fraction of polymer segments can be located within a few nanometres of an internal interface, a region with properties that can be strongly modied and signicantly different from those of homogeneous and block polymers. 297Due to the strong sensitivity of pyrene's uorescence intensity to the nanoscale medium, the characterisation of T g over different length scales (nm) of the lamella can be achieved via uorescence spectroscopy using uorescent pyrene-bearing monomers precisely placed at specic locations along the polymer chain (Fig. 10A). 298The strong dependency of uorescence intensity and lifetime of BODIPY-C12 on the viscosity of its surrounding environment allows multiparameter characterisation of self-assembled polymers, including the critical micelle concentration (CMC, concentration of block copolymers above which micelles form) and critical micelle temperature (CMT, the transition temperature, above or below which the formation of associated structures becomes appreciable) via steady-state uorescence spectroscopy as well as micelle core phase transition temperatures, microviscosity within the micelles and the sol-gel transition temperature via time-resolved uorescence spectroscopy. 299The use of molecular rotors can thus eliminate tedious multi-instrument processes for the characterisation of polymeric self-assembly.
Robin et al. have synthesised block copolymer micelles with a uorescent dithiomaleimide (DTM) covalently linked to the micelle core or shell. 250,300The transition from micelle to unimer can be detected for the core-labelled micelles by uorescence lifetime measurements, based on solvent collisional quenching, i.e., a good protection of the DTM uorophore from solvent quenching (e.g., the DTM is located within the dehydrated core) results in a longer lifetime of the micelles (Fig. 10B).Furthermore, the core-labelled micelles can self-report on the presence of a uorescent hydrophobic guest as a result of FRET between the DTM uorophore and the guest.
][303] Ghosh and colleagues synthesised block copolymers polyethylene glycol (PEO)-b-PMMA-co-polyhydroxyethylmethacrylate (PHEMA) with either green (donor, 1,8-naphthalimide derivative) or red (acceptor, rhodamine B) uorescent dyes covalently attached to the hydrophobic blocks. 301Mixing the red-and green-labelled polymers results in highly efficient FRET due to the close proximity between the donor and acceptor aer coassembly of these polymers (Fig. 11A).The CAC, the dynamics of micellisation and the tolerance of the formed micelle aggregates to different solvents can also be investigated by the FRET technique.Besides, this technique allows to monitor the exchange rate between the micelle and unimer, aiding to understand the dynamics and stability of these micelles.Similarly, they also applied FRET to study the co-assembly and selfsorted assembly behaviour between different dye-labelled polymers in a molecular interaction-driven self-assembly system; 270 Huang et al. employed FRET to determine the kinetics of coordination-driven self-assembly processes and to investigate the solvent and anion effect in self-assembly, the stability of metallosupramolecular structures and the dynamic ligand exchange between structures. 149lbertazzi et al. presented the synthesis of multicomponent one-dimensional supramolecular polymers via co-assembly of neutral 1,3,5-benzenetricarboxamide (BTA) monomers with cationic species BTA 3+ , labelled with FRET pairs (cyanine Cy3/ Cy5) (Fig. 11B), and demonstrated that a multivalent recruiter (e.g., ssDNA with multi-charged groups) is able to bind selectively to the BTA 3+ monomers (as receptors) and trigger their clustering (Fig. 11C). 82The dynamics of supramolecular polymers synchronised with the multivalency of a binder can afford spatiotemporal control over the monomer sequence.Here, FRET has been exploited to evaluate the dynamics of the multicomponent supramolecular polymers, the clustering kinetics as well as the effect of recruiters and receptors on monomer clustering. 82,284luorescence spectroscopy provides in-depth information of self-assembled systems, but it is of high signicance to enable optical imaging of these structures.Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM), 304,305 has been used extensively for visualisation and analysis of samples. 107,306,307The excitation light in confocal microscopy is focused through an objective lens to a diffraction limited spot.The generated uorescence is collected by the same objective lens, passes through appropriate lters and pinholes blocking out-of-focus light, and nally reaches a point detector.The complete image is then created by scanning across the sample. 305Using living CDSA, Manners, Winnik and co-workers have prepared well-dened cylindrical multiblock micelles with distinct domains and rectangular platelet micelles with

Chemical Science Review
tuneable dimensions (Fig. 12A). 275,307Imaging of each colour by CLSM reveals the distribution of different domains, which are almost indistinguishable by electron microscopy.FLIM, another advanced imaging technique, bases its contrast on the uorescence lifetimes (extrapolated from excited state decay curves) of a uorescent sample, rendering this technique unaffected by the concentration of uorophores, excitation intensity and photo-bleaching. 66,131The high spatial resolution of FLIM combined with the high sensitivity of covalently attached (separately labelling the shell and core) molecular rotors to their microenvironments has been used by Eivgi et al., to reveal the behaviours and mechanisms of block-selective solvent-triggered assembly and disassembly. 308For example, FLIM reveals that DMSOa solvent that causes disassembly of the copolymer in their system -inuences exclusively the polar block (Fig. 12B).Understanding differential block-solvent interactions can further aid in nely tuning block copolymer self-assembly.Notably, FLIM shows superior sensitivity over 1 H NMR spectroscopy, and uorescence microscopy can reveal the properties of specic regions.Optical diffraction limits the imaging resolution of uorescence microscopy, while the emergence of super-resolution uorescence microscopy (SRFM) techniques [67][68][69][70][71]309 has overcome this limit and improved resolutions, allowing the visualisation of behaviour down to the scale of single molecules. SRF techniques have been widely applied in biology, 310 in contrast, its application in polymer science is still in its infancy, nevertheless, polymer scientists have gained new, important and fundamental insights through the implementation of SRFM techniques.311,312 The most well-known methods include structured illumination microscopy (SIM), STED and SMLM.
4][315] With multi-colour imaging, it can reveal different blocks or domains. 91,107,150,316,317][318][319] Furthermore, the high spatiotemporal resolution enables to visualise structure formation processes, study dynamic behaviours and reveal mechanisms and driving forces behind selfassembly. 83,88,319,320arkar et al. introduced a two-component, sequence controlled, supramolecular copolymerisation to access selfsorted, random and block supramolecular polymers. 150The core-substituted naphthalene diimide p-conjugated monomers (Fig. 13A, top le) are not only capable of supramolecular polymerisation, but also allow the use of SIM to visualise the resulting multicomponent structures due to the orthogonal uorescent nature (green or red emission) of monomers (Fig. 13A, bottom).SIM can surpass the optical diffraction limit by illuminating the sample with patterned light and subsequent soware analysis to extract information from Moiré fringes (Fig. 13A, top right). 309,321,322It does not have specic requirements for uorophores. 322][69][70][71] STED is related to conventional confocal microscopy, but uses a second beam to quench the uorescence of undesired molecules to control the excitation volume and thus exceed the optical diffraction limit (Fig. 13B, top le). 323The second beam is usually a doughnut-shaped beam, whose wavelength is red-shied relative to the excitation laser, and the excitation volume can generally be adjusted by tuning the depletion intensity.Dang et al. have used STED to visualise self-assembled helical bres.These authors synthesised an AIEgen, DP-TBT (chemical structure refer to Fig. 13B, top right), which readily forms helixes (Fig. 13B, bottom). 318Meanwhile, DP-TBT exhibits highly emissive features in the aggregate state, a large Stokes shi and excellent photostability, meeting the stringent requirements for uorophores in STED.DP-TBT-based selfassembled structures can thus be visualised by STED (Fig. 13B, bottom).Furthermore, long-time tracking using STED allows to monitor the formation and growth of helical bres during the self-assembly process.

Review Chemical Science
Manners' group applied STED and dual-colour SMLM to determine the lengths and length distributions of cylindrical micelles formed by living CDSA in situ. 91,106Importantly, both techniques enable the investigation of growth kinetics and the effects of various self-assembly parameters on the kinetics, providing guidelines for the future optimisation of a wide variety of living CDSA systems.
5][326] FRET experiments can provide useful information about the time scale of exchange (see above), but are insufficient to unveil its mechanism. 82,83Moreover, these ensemble experiments fail to detect the diversity among selfassembled structures or within an individual structure.
Meijer's group has reported the use of stochastic optical reconstruction microscopy (STORM) to probe the dynamics of supramolecular bres. 83Different dyes (Cy3 and Cy5) attached to BTA allow the use of multicolour STORM to determine the monomer distribution along the bre backbone (Fig. 13C, top le).By following the monomer distribution during the molecular exchange process (Fig. 13C, right), the unexpected homogeneous exchange mechanism was revealed.Using the same method, the exchange mechanism in peptide amphiphiles-based nanobres has been investigated as well. 320TORM belongs to the group of SMLM techniques and uses uorophores that can photoswitch in a suitable buffer.Cy5, for example, can be switched between a uorescent 'bright' and an inactive triplet 'dark' state (Fig. 13C, bottom le).The red light that produces uorescence from Cy5 can also switch it to a longlived dark state, which can be further stabilised using a buffer.Reaction with oxygen and light exposure can convert Cy5 back to its bright state. 234,327,328ater, the same group exploited multicolour interface point accumulation in nanoscale topography (iPAINT) to investigate the structures and dynamics of multicomponent supramolecular bres in situ (Fig. 13D, top right and bottom), which is achievable by simple noncovalent staining without any chemical modication. 319oth STORM and PAINT lie within the category of SMLM techniques, but they differ in their mechanisms of switching uorophores between their 'ON' and 'OFF' states, which affects the choice of uorophores.In contrast to STORM, PAINT relies on immobilisationfast and freely diffusing uorescent molecules ('OFF' state) become visible only when bound to a target ('ON' state) (Fig. 13D, top le).Self-assembly of block copolymers and supramolecular polymerisation leads to the formation of a large variety of complex systems with useful properties and functions.While several synthetic methods have been proposed to generate well-dened self-assembled objects, the construction of assemblies with desired structures in a controlled manner remains a great challenge.Thus, it is of signicant importance to develop advanced analytical methods to characterise and gain further understanding of the effects of various parameters, including the concentration and structure of the initial monomer or block copolymer, temperature and solvent type, on the self-assembled structures and self-assembly processes.Fluorescence spectroscopy, such as FRET-based techniques, enables certain insights into self-assemblies, including morphologies, self-assembly kinetics and dynamics.Fluorescence microscopy, especially SRFM, is able to visualise self-assembled structures, to reveal the diversity of structures even within a single object, as well as to unveil the driving force and mechanism of self-assembly.Establishing structure-property relationships at various conditions and revealing the self-assembly mechanism is essential to advance the eld, in which uorescence-based techniques with their high sensitivity, good spatiotemporal resolution and noninvasive characteristic will continue to play a vital role.

Surfaces, gels and 3D printing
0][331][332][333] The scope of so matter materials is immense and it would be nearly impossible to discuss every aspect of their application.Therefore, three representative examplessurfaces, gels and 3D printingare selected herein to discuss the impact of uorescence-based techniques on their study.

Surfaces
0][341][342][343][344][345][346] Two approaches -'graing to' and 'graing from'are generally used for the preparation of polymer brushes.The former functioning by graing pre-formed polymer chains to the surface, while the latter is a bottom-up approach where polymer chains are grown directly from the substrate via surface-initiated polymerisation. 99][356][357][358][359][360] Advanced synthetic methods combined with various analytical techniques allow for a better understanding of surface features, such as lm thickness, graing density, chemical composition, molecular weight and dispersity of polymer chains, as well as surface reaction kinetics.][363][364] Among these techniques, uorescence-based techniques enable fast and efficient readout and have been widely exploited to visualise surface graing of luminescent (co-)monomers, chemical functionalisation with uorescent dyes as well as the stability of uorescent lms. 345,349,354,360,365,366For example, Borozenko et al. have utilised total internal reection uorescence microscopy (TIRFM) to monitor in real time the detachment of BODIPY-labelled poly(acrylic acid) brushes from glass substrates under varying pH and salt concentrations. 365This is a challenging task for conventional surface characterisation techniques such as AFM and ellipsometry, which are limited by the scanning area, measurement speed and adaptability to various environments.TIRFM uses specic optics to achieve total internal reection of the excitation light at the interface, thus producing an evanescent electromagnetic eld.The intensity of the evanescent eld decays exponentially with distance from the surface, resulting in the excitation of the region in close proximity to the surface (<200 nm), making TIRFM an powerful analytical technique for thin lms. 367n addition to a fast and efficient response, uorescence microscopy is capable of spatial readout, offering the ability to reveal and distinguish different regions.Surfaces with spatially dened chemical and physical properties are of fundamental importance for studying the structure-property relationships of polymer surfaces and of practical relevance for applications such as microarrays, tissue engineering and microelectronics. 340,341,368Hawker and coworkers prepared multifunctional surfaces, nonlinear chemical concentration gradients (Fig. 14A) as well as hierarchical chemical patterns combined photomediated (metal free) SI-ATRP with photomasks. 341,349,352,369Braunschweig and colleagues employed surface-initiated thiol-ene photopolymerisation and a photochemical printer based on a digital micromirror device (DMD) to generate high-resolution patterns, circumventing the need for photomasks. 370Furthermore, they developed a technique combing a DMD, an atmosphere chamber and microuidics, capable of generating hypersurfaces, in which more than three properties of each pixel (x-and y-position, polymer height and monomer composition) can be controlled independently (Fig. 14B). 368,371In these studies, confocal microscopy plays a key role for visualisation of different domains of patterns and determining pattern resolutions, while the uorescence intensity can be used as an indication of the brush height.
3][374][375][376] Polymer brush surfaces with the ability to change the conformation and structure of polymer chains in response to external stimuli hold signicant potential for applications in drug delivery, self-healing coatings, sensors and actuators. 339,3648][379][380][381][382][383] Characterisation of surface conformational changes in response to different environmental stimuli is essential for understanding their effects on surface properties.5][386] Using similar FRET approach, Besford et al. studied the molecular transport from aqueous droplets (containing the FRET acceptor) into PNIPAM copolymer brush lms incorporating the FRET donor using CLSM instead of TIRFM. 387These uorescence-based measurements demonstrate the changes in polymer brush conformation well.In order to quantify the conformational changes of polymer brushes, Besford et al. prepared PNIPAM block copolymer brushes integrating a FRET donor layer followed by a FRET acceptor layer and utilised CLSM to detect the conformational changes of brushes (Fig. 15A, top). 374The conformation changes of PNIPAM brushes in response to varying solvents affects the proximity of the donors to the acceptors, thus the polymer brush heights can be correlated to the ratio of uorescence intensity from donors and acceptors.Alternative method for quantifying polymer brush conformational changes is based on self-quenching of uorophores covalently tethered to polymer brushes (Fig. 15B). 388The collapse of polymer brushes leads to the  quenching of these uorophores, which can be resolved with uorescence intensity-or lifetime-based techniques (e.g., FLIM), allowing for intensity/lifetime correlation with the heights of brushes.In addition to revealing polymer brush heights, the use of CLSM and FLIM enables the spatial examination of the polymer brush conformation across complex interfaces (Fig. 15A, bottom).In addition, it is worth mentioning that the transduction from conformational changes in stimuli-responsive polymer brushes to the uorescence signal is particularly attractive for developing surface-based sensors for sensing environmental stimuli, such as temperature, humidity and mechanical force. 374,375,377,388,3891][392][393] High spatiotemporal resolution and non-invasive characteristic of uorescencebased techniques allow the study of surface graing kinetics as well as polymer chain dynamics, which will lead to a better understanding of the process and mechanism of surface modication and, in turn, will facilitate the development of more complex polymer surfaces with special properties.

Gels
Polymer networks, consisting of network junctions and strands linked via covalent bonds or non-covalent (supramolecular) interactions, are arguably among the most versatile and widely used so matter materials. 394A large family of polymer networks is the gel, dened as a polymer network that is expanded throughout its entire volume by a gas or more oen by a liquid (e.g., hydrogel). 395,396Classic industrial applications of polymer gels include hygiene and medical products, such as diapers and contact lenses.7][418][419][420][421][422] In order to nely control gel materials and exploit the next generation of functional and sustainable gels, a profound understanding of their structures, properties as well as their interaction with the environment is required.
Broadly applicable methods, including rheology, swelling experiments and mechanical tests, provide insights into network structures, but are limited to the macroscopic level, oen relying on assumed structure-property relationships.Scattering methods, including neutron scattering and X-ray scattering, have been successfully used to probe the structure of polymer networks, however, limited access and complex experimental set-ups constrain localisation measurements and efforts to obtain information on molecular-scale structural features. 207,394,4235][426][427][428][429][430] For example, Creton and co-workers have reported the use of mechanophores as cross-linkers, including a dioxetane derivative (bis(adamantyl)-1,2-dioxetane bisacrylate, BADOBA), 431 a spiropyran derivative (SP) 432,433 and a Diels-Alder adduct (p-extended anthracene-maleimide adduct), 434,435 for the spatially resolved visualisation of bond breakage in multi-network elastomers.Under external force, BADOBA cleaves into two adamantanone units, one of which is in the excited state and generates luminescence upon relaxation (Fig. 16(A1)), 431,436,437 while the colourless SP can be converted to the highly coloured merocyanine (MC) (Fig. 16(A2)), 432,438,439 and the non-uorescent p-extended anthracene-maleimide adduct is able to undergo the retro-Diels-Alder reaction, releasing the uorescent p-extended anthracene moiety (Fig. 16(A3)). 434,435,4402][443] These mechanophores are composed of a uorophore-carrying cycle and a dumbbellshaped molecule containing a matching quencher (Fig. 16(A4)).The displacement of the cycle to the periphery of the dumbbell upon extension leads to uorescence turn-on.The uorescence intensity can be further correlated with the applied force.
In a more recent study, Weder and colleagues incorporated a FRET donor and acceptor into the cyclic structure of the rotaxane-based mechanophore, allowing to report the mechanical force applied in polymer hydrogel (Fig. 16B). 444In the force free state, the donor emission is suppressed due to the close proximity of donor and quencher.Upon deformation of the hydrogel, the uorescence emission of the acceptor increases, owing to the separation of the donor from the quencher, thus enabling FRET.Zheng et al. established an elegant method to detect and visualise mechanochemical damages in hydrogels by utilising a radical-trapping pro-uorescent probe (nitroxide radical tethered to coumarinbased luminophore). 445The mechanochemical damages oen lead to the formation of short-lived free radicals that can either react directly with the probe via the radical coupling reaction or undergo an oxygen-relayed radical-transfer reaction rst to increase the probability of the coupling reaction, thereby resulting in enhanced uorescence emission and altered emission colour (Fig. 16C).
Polymer gels, or polymer networks in general, exhibit spatial variations in crosslinking density.These nanoscale structural heterogeneities signicantly affect the dening properties of gels (e.g., elasticity, swellability and permeability), and thus tools enabling high spatial resolution characterisation are required to provide more in-depth structural information. 394,425,446][449][450][451] Wöll and his colleagues introduced a diarylethene-based photoswitchable uorophore as a cross-linker into PNIPAMbased microgels, enabling the visualisation and quantication of cross-linker points using PALM (Fig. 17A). 92Ullal and coworkers utilised a SRFM-based technique to map the crosslinking density in PNIPAM microgels as well (Fig. 17B). 93They designed a specic cross-linker which can react with rhodamine B or Alexa 647, forming molecules that allow the visualisation by entire cell 4Pi single molecule switching nanoscopy (W-4PiSMSN), a type of optical nanoscopy that enables imaging 3D structures at 10-20 nm resolution.Both studies show the cross-linker density within a microgel decreases as a function of the distance from its centre, revealing the heterogeneity of the polymer networks constituting microgels.
Due to the complexity of their structures, gels oen exhibit sophisticated behaviours and properties.Characterisation of gels with molecular precision is challenging, but critical for the in-depth understanding of structure-property relationships and further development of gels with specic functions.Judicious selection of mechanophores enables the observation of forces in polymer networks and powerful SRFM techniques allow the visualisation of crosslinking networks.Both methods can be exploited to shed new light on polymer gels and will undoubtedly be extended to a wide range of gels and provide even more quantitative information in the future.

3D printing
4][455] The ability to translate computeraided design into complex 3D physical objects has revolutionised manufacturing and has had an immeasurable impact on modern society.][462][463][464] Recently, a variety of (photo)chemical reactions opened new opportunities for 3D printing.Photo-RDRP confers the ability to control the nanostructures and mechanical properties of 3D printed objects. 465The living characteristics of polymer chains formed by RDRP allow the post-modication and self-healing of objects. 52,56,57][468] The use of multiple colours of light instead of a single colour can further exploit the vast potential of light-based 3D printing.Two-colour printing is generally based on synergistic, orthogonal or antagonistic photochemistry. 469Synergistic photochemistry requires the simultaneous irradiation with two distinct colours to trigger a reaction, in an orthogonal system two reactions are induced by two wavelengths and proceed independently, while antagonistic implies opposing reaction processes triggered separately by two colours of light.Twocolour systems not only allow access to new and complex materials with unique properties, but also facilitate the development of superior printing approaches and techniques, such as light-sheet printing, 470 xolography, 471 multimaterial printing 53,472 and STED-based nanoprinting. 473,474In addition to multi-colour printing, research on 4D printing is growing substantially.][481] With the ourishing development of 3D printing towards objects with highly complex materials and structures and with high-resolution features, it is of vital importance to characterise them at the micro-and molecular-scale and further understand how the macroscopic properties are inuenced by the molecular-scale polymer structures and possible heterogeneities, thus beneting control of chemical reactions and resulting materials properties in 3D printing with high precision.Several advanced techniques have been applied to characterise the local properties of 3D-printed objects, including AFM quantitative nanomechanics (QNM) and NanoDMA to determine local mechanical properties, 482,483 Micro-FTIR and time-of-ight SIMS (ToF-SIMS) to provide chemical information at microscale 56,484 as well as X-ray micro-computed tomography (XRM) to visualise 3D internal structures. 485However, these techniques are either restricted to surface measurements or have limited spatial resolution.Thus, it is challenging but critical to develop alternative methods to investigate internal polymer structures at the microscopic and molecular scale.Fluorescence-based techniques have demonstrated strong abilities in various aspects of polymers and so matters and are therefore a high-potential candidate for studying 3D printed so matter structures.
Fluorescence techniques, particular CLSM, have been used to demonstrate the living properties of 3D structures generated via photo-RDRP 54,56,486,487 (Fig. 18A and B), to demonstrate the capability of photoenol-based photochemical ligation reactions for spatially resolved surface functionalisation of 3D microstructures 51,488 (Fig. 18C) as well as to highlight the ability of a microuidic-based 3D laser microprinting to integrate multiple materials 489 (Fig. 18D).The successful proof of these three listed concepts in the eld of 3D printing is attributed to the spatially resolved capabilities of uorescence techniques.
Furthermore, uorescence-based techniques allow for the study of polymer structures in 3D objects at the micro-and molecular scale, thus further establishing the relationship between polymer structures and their properties.Our group has introduced an anthracene dimer-containing photoresist for DLW and succeeded to produce microstructures with adaptive mechanical properties by taking advantage of the [4 + 4] photodimerisation of anthracene (Fig. 19A). 482,484Under visible light irradiation, concomitant with the change in mechanical properties, the uorescence intensity of microstructures decreases, indicating an increasing amount of photodimerisation, as the anthracene dimer does not display uorescence.The direct correlation between uorescence and mechanical properties (Fig. 19B) allows the uorescence-read out for mechanical properties of microstructures.As noted, the uorescence intensity decreases before the change in mechanical properties, as uorescence reects changes on the molecular level, indicating the high sensitivity of uorescence-based techniques for the investigation of polymer structures in 3D-printed objects.Very recently, Wu, Belqat et al. have utilised FLIM to investigate the microenvironment in two-photon fabricated microstructures employing the uorescent molecular rotor -BODIPY-C12 as a viscosity probe (Fig. 19C). 490The bi-exponential uorescence decays obtained from uorescence lifetime measurements in the microstructures show that BODIPY-C12 is present in different local micro-viscosities, indicating the heterogeneity in the fabricated microstructures.The dependence of uorescence lifetime on BODIPY-C12 on the local viscosity has been further exploited to distinguish different materials in multimaterial printed objects (Fig. 19C and D)a strategy that opens new perspectives for probing 4D and multi-material characteristics of 3D objects at the micro-and molecular level.
The stimuli-responsiveness of objects is the key feature of 4D printing.Conceivably, the integration of uorescent probes, including viscosity, humidity probes and mechanoluminophores, into 3D-printed objects, allows to reveal the local microenvironment within the objects, which denes their macroscopic properties and their transformation in response to stimuli.Advanced uorescence techniques, such as FLIM, STED and SMLM combined with suitable uorescent probes can provide a wealth of information on polymer structures, including heterogeneity, crosslinking points as well as polymer chain orientation.In the realm of 3D printing, the study of structure-property relationships at the molecular level is still in its infancy, yet is critical to achieve 3D objects with desired and tailored properties, and we recommend that uorescence-based techniques play a signicant future role.Furthermore, the capability of in situ and real-time response afforded by uorescence-based techniques will certainly be exploited in the future to understand polymerisation kinetics during 3D printing.

Summary and outlook
The eld of polymer science has made substantial progress over the past few decades.The fusion of modern polymerisation methods with advanced organic chemistry has allowed us to access diverse polymers with well-dened architectures and functionalities, including sequence-dened polymers.The incorporation of supramolecular chemistry has further expanded the scope of macromolecules regarding their  morphology and topology.These advances have led to signicant developments of so matter materials, including surface-graed polymer brushes, responsive gels, delivery vectors and sensors as well as a vibrant eld -3D printing.To gain an accurate understanding of these complex materials, the development of advanced analytical characterisation methods is undoubtedly critical.Fluorescence-readout has emerged as a powerful characterisation tool, providing new and deep insights into macromolecules and so matter materials, enabling to visualise and monitor various polymerisation processes in situ, to quantify the ratio of different arms in miktoarm star polymers and the crosslinking points in polymer networks, to visualise a single polymer chain in its native environment, to study kinetics and dynamics of polymer selfassembly as well as to spatially resolve the conformation of polymer brushes on surfaces, to observe forces in gels with molecular precision and to reveal the heterogeneity in printed microstructures, attributed to the successful integration of (pro) uorophores and implementation of advanced uorescence techniques.Building on these successes, the eld holds specic key future opportunities, including but not limited to: (i) in situ monitoring of polymerisation processes starting from the initiation stage, and providing detailed spatial information; (ii) exploiting uorescence-based techniques for more complex architectures and assemblies to determine structure-property relationships; (iii) providing quantitative information on polymer conformation in their native environments for the validation and generation of accurate models; (iv) applying advanced uorescence techniques to a wider range of polymer systems in surfaces and gels, not limited to model polymers; (v) associating uorescent probes with advanced uorescence techniques to reveal micro-and molecular structures and their environment in gels and 3D-printed objects; (vi) studying polymerisation kinetics of surface graing and 3D printing in an in situ, real time manner and (vii) exploiting (orthogonal) uorescencereadouts and tracing providing molecular identity information in an efficient and optically readable fashion.Fluorescencereadout fuels the dream of all polymer chemistscontrol of so matter material properties with precision on the molecular level.However, reaching our aspirations will undoubtedly necessitate coordinated endeavours across disciplines including chemistry, physics, engineering and material science.

Fig. 2
Fig. 2 Overview of six categories of fluorescence compounds allowing to monitor the polymerisation process and map monomer conversion and molecular weight.Anti-rigidochromic fluorophores: images reproduced from ref. 141 with permission from Wiley, copyright 2022.

Fig. 3 (
Fig. 3 (A) Reaction scheme of photo-controlled RAFT polymerisation of MMA using TPE-containing dithiocarbamate as a RAFT agent, series of photographs showing the increase of fluorescence intensity with conversion of reaction and plot of conversion and molecular weight against light intensity.Reproduced from ref. 135 with permission from Wiley, copyright 2018.(B) Scheme of ROMP polymerisation with TPE-and PDIlabelled norbornene monomers as fluorescent probes (top) and visualisation of the polymerisation process over time based on AIE-Intensity and anisotropy signals (bottom).Reproduced from ref. 137 with permission from The Royal Society of Chemistry, copyright 2020.

Fig. 4 (
Fig. 4 (A) ROMP polymerisation of dicyclopentadiene (DCPD) containing a small amount of BODIPY-labelled monomers.(B) FLIM images showing the increase in fluorescence lifetime with higher molecular weight during the ROMP reaction (top), plotted correlation between fluorescence lifetime and molecular weight and extrapolation of the molecular weight of insoluble material from the correlation curve (bottom).Reproduced from ref. 85 with permission from The American Chemical Society, copyright 2022.

Fig. 5 (
Fig. 5 (A) Chemical structure of supramolecular monomers (top), plot of fluorescence emission spectra at different monomer concentration (middle) and scheme of supramolecular polymerisation process with bathochromic shifted fluorescent colour (bottom).Reproduced from ref. 152 (https://doi.org/10.1073/pnas.2121746119),under the terms of the CC BY-NC-ND 4.0 license [https://creativecommons.org/licenses/bync-nd/4.0/].(B) Chemical structure of supramolecular monomers and schematic representation of supramolecular polymer formed by hydrogen bonding (top), plot of fluorescence emission spectra for increasing monomer concentration (middle) and visualisation of the fluorescent colour change (bottom).Reproduced from ref. 153 with permission from Wiley, copyright 2022.

Scheme 1 (
Scheme 1 (A) Intramolecular thiol-maleimide cyclisation reaction.The consumption of the maleimide restores the fluorescence of the molecule and allows to monitor the reaction.Reproduced from ref. 174 with permission from The Royal Society of Chemistry, copyright 2017.(B) The PFTR between a linker (3PFB), a base (TBAOH) and a thiol, triggering the CL of Schaap's dioxetane.Reproduced from ref. 179 with permission from The Royal Society of Chemistry, copyright 2020.

Fig. 6 (
Fig. 6 (A) Illustration of the arm-first approach for the synthesis of miktoarm star polymers bearing different fluorescent end-groups.(B) Linear fits of fluorescence intensities of mikto-star arms with the same monomeric units but different molecular weights, as shown in the inset, to quantify miktoarm star composition.Reproduced from ref. 184 with permission from The Royal Society of Chemistry, copyright 2022.

Fig. 7
Fig. 7 (A) UV-induced NITEC crosslinking reaction between tetrazole chain-ends and trimaleimide cross-linkers, forming insoluble and soluble fractions.(B) Network disassembly via aminolysis.Determining the concentration of pyrazoline after aminolysis via fluorescence spectroscopy allows to quantify the number crosslinking points within the former network.Reproduced from ref. 191 with permission from Wiley, copyright 2018.

Fig. 8 (
Fig. 8 (A) Schematic illustration of the FRET mechanism.(B) Chemical structures of three FRET donor-acceptor end-labelled polymers from ref. 214, 212 and 223 synthesised by ATRP or RAFT polymerisation.(C) Plot of R ee of PMMA determined via FRET as a function of degree of polymerisation, corresponding to simulated ones.Reproduced from ref. 214 with permission from The American Chemical Society, copyright 2023.(D) FRET efficiency against R ee (black curve) and probability distribution of R ee (blue curve) of PS.Reproduced from ref. 212 with permission from The American Chemical Society, copyright 2016.(E) Dependence of the ratio of fluorescence intensities between FRET acceptor and donor on the concentration of PBMA.The marked sharp increase indicates the intra-chain conformation transition.Reproduced from ref. 223 with permission from Wiley, copyright 2016.

Fig. 9 (
Fig. 9 (A) Illustration of PALM SMLM for the imaging of individual bottlebrush polymers.Repeated, stochastical activation of a small fraction of fluorophores that are attached to the polymer allows localisation of each fluorophore by fitting to the PSF.Images of the polymers can thus be reconstructed and allow analysis of chain conformation (persistent length).Reproduced from ref. 239 (https://www.pnas.org/doi/abs/10.1073/pnas.2109534118),under the terms of the PNAS License to Publish, copyright 2021.(B) Chemical structure of the diarylethene derivative employed for PALM imaging in ref. 239 and schematic of the photoswitch between open ('OFF') and closed ('ON') form.

Fig. 10 (
Fig. 10 (A) Characterisation of T g in the block copolymers selfassembled lamella via fluorometry, which is enabled by the precise location of pyrene in the block copolymer.Reproduced from ref. 298 with permission from The American Chemical Society, https:// pubs.acs.org/doi/10.1021/acscentsci.8b00043,copyright 2018 (further permissions related to ref. 298 are to be directed to the ACS).(B) Chemical structure of DTM and comparison of the fluorescence lifetime of DTM in a unimer and in a self-assembled micelle.Reproduced from ref. 300 (https://doi.org/10.1021/acs.macromol.5b02152),under the terms of the CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Fig. 11 (
Fig. 11 (A) Co-assembly of block copolymers with donor-or acceptor-labelled hydrophobic blocks into micelles.FRET allows to monitor micelle formation and exchange rate between the micelle and unimer.Reproduced from ref. 301 with permission from The American Chemical Society, copyright 2015.(B) Chemical structures of neutral monomers (BTA), cationic receptors labelled with Cy3 or Cy5 (BTA 3+ ) and multivalent recruiter (ssDNA).(C) Schematic representation of the reversible clustering of receptors along the supramolecular polymer.A recruiter triggers clustering of receptors, leading to high FRET efficiencies.(B and C) Reproduced from ref. 82 (https://doi.org/10.1073/pnas.1303109110),under the terms of the PNAS License to Publish, copyright 2013.

Fig. 12 (
Fig. 12 (A) Schematic representations and CLSM images of self-assembled platelet micelles, selectively functionalised with fluorescent labels.Reproduced from ref. 275 with permission from The American Association for the Advancement of Science, copyright 2016.(B) FLIM images showing block-selective solvation changes during gradual addition of DMSO to core and shell-labelled micelles in CH 2 Cl 2 /toluene.Reproduced from ref. 308 with permission from The American Chemical Society, copyright 2023.

Fig. 13 (
Fig. 13 (A) Chemical structures of core-substituted naphthalene diimide p-conjugated monomers (top left), illustration of the SIM principle (top right), SIM image and schematic representation of self-sorted supramolecular polymers.Reproduced from ref. 150 with permission from The American Chemical Society, copyright 2020.(B) Mechanism of STED (top left), chemical structure of the AIEgen (DP-TBT) (top right), scheme of the helical self-assembly of DP-TBT and a side-by-side comparison of CLSM and STED images (bottom).Optimised STED can resolve individual turns of a helix (bottom right).Reproduced from ref. 318 with permission from The American Chemical Society, copyright 2019.(C) Illustration of the monomer exchange process of supramolecular fibres (top left), mechanism of STORM, upon irradiation a dye can enter a long-lived nonfluorescent triplet state (bottom left), STORM images of Cy3 and Cy5-labelled supramolecular fibres to follow monomer distribution at different times (right).Reproduced from ref. 83 with permission from The American Association for the Advancement of Science, copyright 2014.(D) Mechanism of PAINT, fluorescent dyes become visible when temporarily bound to a target structure (top left), illustration of copolymerisation of two supramolecular fibres bearing red-or green fluorescent labels (top right), iPAINT images at different times, visualising the dynamics of supramolecular fibres (bottom).Reproduced from ref. 319 (https://doi.org/10.1021/acsnano.8b00396),under the terms of the CC BY-NC-ND 4.0 license [https://creativecommons.org/licenses/by-nc-nd/4.0/].

Fig. 14 (
Fig. 14 (A) Reaction scheme for the chemical patterning of surfaces, application of a greyscale photomask for chemical concentration gradients and analysis of fluorescence intensity across the chemical concentration gradients surface.Reproduced from ref. 349 with permission from The American Chemical Society, copyright 2013.(B) Three-colour fluorescence microscopy image of a polymer brush surface pattern (Barcelona skyline).Reproduced from ref. 371 (https:// doi.org/10.1038/s41467-020-14990-x),under the terms of the CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Fig. 15 (
Fig. 15 (A) Illustration of polymer brushes with integrated FRET-donor and acceptor and their conformations including heights in hexane and in water (top), and CLSM images of the polymer brush at the interface between hexane and water (bottom).Reproduced from ref. 374 (https://doi.org/10.1002/anie.202104204),under the terms of the CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].(B) Scheme of polymer brushes with covalently tethered fluorophores in swollen and collapsed state.Chain collapse causes the quenching of fluorophores.Reproduced from ref. 388 with permission from The American Chemical Society, copyright 2022.

Fig. 18 (
Fig. 18 (A) Fluorescence image of a surface pattern (The Great Wave off Kanagawa) on a 3D printed rectangular prism made by type I photoinitiated RAFT polymerisation.Reproduced from ref. 486 with permission from Wiley, copyright 2021.(B) SEM and FLIM images of a surfacemodified 3D 'bridge' microstructure 3D microstructure made by photoiniferter-RAFT polymerisation and surface functionalisation made from monomers without photoinitiators.Reproduced from ref. 56 with permission from Wiley, copyright 2021.(C) 3D CLSM images showing spatially resolved dual surface functionalisation using photo-ligation reactions on a 3D printed scaffold (green parts, based on photoenol ligation).Reproduced from ref. 51 with permission from Wiley, copyright 2016.(D) Design (left) and CLSM image stack (right) of a multimaterial microstructure made from one non-fluorescent and four fluorescent photoresists.Reproduced from ref. 489 (https://doi.org/10.1126/sciadv.aau9160),under the terms of a CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Fig. 19 (
Fig. 19 (A) SEM image of microstructures containing only anthracene dimers as monomers.Reproduced from ref. 482 with permission from Wiley, copyright 2019.(B) Normalised complex modulus (E*) determined by NanoDMA versus normalised fluorescence intensity of the structures shown in (A) determined by CLSM.Reproduced from ref. 482 with permission from Wiley, copyright 2019.(C) FLIM image of a microstructure made from two different photoresists, containing BODIPY-C12.Reproduced from ref. 490 with permission from The Royal Society of Chemistry, copyright 2022.(D) Fluorescence decay curves of BODIPY-C12 in the same microstructure as (C) showing BODIPY-C12 in different viscosity environments.Reproduced from ref. 490 with permission from The Royal Society of Chemistry, copyright 2022.