text:
Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor–acceptor pairs during solution-based smFRET. We use this “caged FRET” methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.

F orster resonance energy transfer (FRET) has become a complementary tool in structural biology. 1−7 FRET can act as a molecular ruler based on a nonradiative energy transfer between two fluorescent probes, a donor and an acceptor, with distinct spectral properties. When designed properly, i.e., the orientation of fluorophore dipole moments does not govern energy transfer, 1 the FRET efficiency depends only on the distance between both fluorophores. In that situation, a direct link between FRET efficiency and biochemical structure can be made by strategic labeling with fluorescent probes. 1 In an intramolecular assay, FRET is then indicative of conformational states or ligand-induced structural changes. 8 It can also visualize mobile parts of proteins that do not crystallize, 9,10 but most importantly, it provides access to structural dynamics. 1,2,11−13 For the latter, FRET is combined with single-molecule detection to allow the observation of unsynchronized reactions. Single-molecule FRET (smFRET) has become the tool of choice for investigating structural dynamics with a spatial resolution of nanometers (dynamic range of 2−10 nm) and a subsecond time resolution. 14 Alternative strategies, which are also compatible with single-molecule detection, provide different dynamic ranges and exploit other photophysical effects [photoinduced electron transfer (PET) 15 or proteininduced fluorescence enhancement (PIFE) 16 ] or molecular properties such as diffusion that can be determined simultaneously with FRET to obtain multidimensional synergetic assays. 17,18 The design of a molecular ruler, which monitors conformational states, requires a structure-guided identification of fluorophore labeling sites. 11 These labeling sites are chosen such that changes in biochemical state result in a measurable photophysical signal, i.e., for the FRET ruler in a change of transfer efficiency E. Second, the structure of interest is modified to allow incorporation of the labels at the desired locations. This typically happens via site-directed mutagenesis of single amino acids in proteins to cysteines (alternatively "clickable" amino acids 19,20 ) or the use of modified nucleic acids that allow labeling with reactive synthetic organic fluorophores. 21−24 Because labeling might interfere with biochemical function, assays are needed that directly compare the protein activity and its degree of labeling as control experiments toward a relevant biophysical study. Ultimately, the quality of the final FRET data is related to not only the functionality of the protein but also the degree of labeling and the percentage of molecules containing both the donor and the acceptor dye, because only those provide FRET information.
Especially for smFRET studies, these two requirements, i.e., high labeling efficiency and retained biochemical functionality, are challenging hurdles. Unfortunately, no established quality criteria exist. Optimized labeling protocols 25 and using a biasfree diffusion-based method can prevent "cherry picking" when individual immobilized molecules are being studied at later stages.
It becomes clear that labeling is a crucial step in biophysical smFRET studies and is inherently complex when oligomeric or multisubunit proteins are studied. In this paper, we exploit reductive caging of cyanine fluorophores and photoactivatable rhodamine fluorophores for smFRET studies of exactly such complex biochemical systems. We present proof-of-principle experiments and a characterization of the photochemical uncaging process of dye-labeled oligonucleotides and proteins during their transit through a confocal excitation volume. Using a method dubbed "caged FRET", we show that chemical caging and ultraviolet (UV) reactivation allows temporal uncoupling of convoluted fluorescence signals from, e.g., multiple donor or acceptor molecules. We use fluorescently labeled oligonucleotides, i.e., ruler structures, and the trimeric membrane transporter BetP as examples, demonstrating how caged FRET removes unwanted molecular species with more than two identical labels and hence allows proper interpretation of solution-based smFRET data. We finally outline further potential applications of the "caged FRET" methodology for studying weak biochemical interactions that are yet impossible to study with diffusion-based smFRET because of requirements for low concentrations of fluorescently labeled molecules.
Transport Measurements of BetP-Cysteine Mutants in Cells. The uptake of the 14 C-labeled substrate by Escherichia coli cells was performed as described in ref 58. E. coli MKH13 cells expressing the strep-BetP mutant were cultivated at 37°C in LB medium containing carbenicillin (50 μg/mL) and induced at an OD 600 of 0.5 by adding anhydrotetracycline (200 μg/L) to the growth medium. After growing for an additional 2 h, the cells were harvested and washed with a buffer containing 25 mM KP i (pH 7.5) and 100 mM NaCl and then resuspended in the same buffer containing 20 mM glucose. For uptake measurements of radiolabeled substrates, the external osmolality was adjusted with KCl. Cells were incubated for 3 min at 37°C before the addition of 250 μM 14 C-labeled substrate for osmotic regulation profiles. Uptake was measured at various time intervals after the cell samples were passed through glass fiber filters and washed twice with Labeling of BetP Derivatives with Thiol-Specific Reagents. BetP cysteine-containing derivatives were obtained as described previously 30,59 and stored at −20°C in 500 μL aliquots (1−6 mg/mL) in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 8.6% glycerol, and 0.1% DDM. Stochastic labeling with maleimide derivatives of donor and acceptor fluorophores was performed on ∼5 nmol of protein. Proteins were labeled with Alexa 555-maleimide (donor) and Alexa647-maleimide (acceptor) in a protein:donor:acceptor ratio of 1:4:3. Briefly, purified proteins were diluted and treated with 10 mM DTT for 60 min in a deoxygenated buffer that consisted of 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 8.6% glycerol, and 0.1% DDM (buffer A), to fully reduce oxidized cysteines. The protein mix was further diluted to a DTT concentration of 1 mM and loaded into an equilibrated desalting column (ZEBA, 2 mL) with a 7 kDa molecular weight cut-off to remove the DTT from the protein solution. The protein was washed with deoxygenated buffer that consisted of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% DDM (buffer B). Simultaneously, the applied fluorophore stocks (50 nmol dissolved in 5 μL of anhydrous dimethyl sulfoxide) were added to appropriate amounts of buffer B, immediately applied to the protein solution, and incubated for 3 h at 4°C (under mild agitation). After labeling, unreacted dyes was removed when the samples were sequentially washed with buffer B and a ZEBA desalting column. The protein was eluted in 500 μL of buffer B and analyzed with a size exclusion column (Superose 6 10/300 GL) equilibrated with 50 mM Tris-HCl (pH 7) and 200 mM NaCl with 0.1% DDM.
Steady-State Fluorescence Anisotropy. Free fluorophore rotation and hence the correlation between FRET efficiency and distance were validated by steady-state anisotropy measurements of BetP with Alexa dyes showing R values of ≤0.22 (Table 1). The values for the dyes on protein were even lower than those found on established doublestranded ruler DNAs with a known donor−acceptor separation of 13 bp, where we established before that FRET indeed serves as a molecular ruler. 17 We used a published theory 60 to estimate the relative error associated with distance determination in both dsDNA and BetP when erroneously assuming a fixed dye orientation (κ ∼ 2 / 3 ). Haas and co-workers provide this error as ratio r/r′ of true distance r to apparent distance r′; this ratio (=uncertainty) is moderate for anisotropies R < 0.3 of both dyes. 60 We found r/r′ < 20% for dsDNA and BetP variants. The experimental procedure for determining anisotropy values R can be summarized as follows. Fluorescence spectra were derived on a standard scanning spectrofluorometer (Jasco FP-8300, 20 nm excitation and emission bandwidth, 8 s integration time) and calculated at the emission maxima of the fluorophores (for donor, λ ex = 535 nm and λ em = 580 nm; for acceptor, λ ex = 635 nm and λ em = 660 nm), according to the relationship R = (I VV − GI VH )/(I VV + 2GI VH ), where I VV and I VH describe the emission components relative to the vertical (V) or horizontal (H) orientation of the excitation and emission polarizers, respectively. The sensitivity of the spectrometer to different polarizations was corrected using horizontal excitation to obtain G = I HV /I HH . Typical G values for donor and acceptor dyes were 0.47 and 0.49, respectively. We used 50 mM Tris-HCl, 200 mM NaCl, and 0.1% DDM (pH 7.5) as a buffer and analyzed the anisotropy of the labeled protein and DNA samples in a concentration range of 50−2000 nM. The determined anisotropy values are summarized in Table 1.
Sample Preparation for Single-Molecule Experiments. ALEX experiments were performed at room temperature with a 25−50 pM solution of protein and DNA samples. For the DNA sample, we used imaging buffer phosphate-buffered saline (PBS) at pH 9.0, containing 2 mM Trolox and 2 mM MV with varying concentrations of TCEP; the pH value of the respective buffer was adjusted after addition of TCEP. Protein samples were also analyzed at 25−50 pM in an imaging buffer containing 50 mM Tris, 150 mM NaCl, 2 mM Trolox, 2 mM MV, 0.1% DDM, and varying concentrations of TCEP; the pH value was either 7.4 or 9 (see the text for details). In typical single-molecule experiments, sample solutions were transferred to coverslips that were previously incubated with 1 mg/mL BSA for 5 min for surface passivation.
Single-Molecule FRET and ALEX Spectroscopy. We used a custom-built confocal microscope for μs-ALEX, which we described in detail previously. 17,61 In brief, the setup was extended by a single-line 375 nm UV laser (Coherent, Obis) that was employed at power densities of ≤500 kW/cm 2 at the confocal volume. A 60× oil-immersion objective with NA = 1.35 (Olympus, UPLSAPO 60XO) or a water-immersion objective with NA = 1.2 was used to generate a diffractionlimited excitation spot. The excitation intensity was typically set to 30−60 μW at 532 nm and 15−25 μW at 640 nm with an alternation period of 50 μs. Fluorescence emission was collected in epi-fluorescence mode, spatially filtered by a 50 μm pinhole, matching bandpass filters, and registered by two avalanche photodiode detectors (τ-Spad, Picoquant).
In this mode, three photon streams were extracted from the data corresponding to donor-based donor emission DD, donorbased acceptor emission DA, and acceptor-based acceptor emission AA. S and apparent FRET efficiencies E* were calculated for each fluorescent burst during their diffusion time trough confocal spot above a certain threshold, yielding a twodimensional (2D) histogram. Uncorrected FRET efficiency E* is calculated according to the equation E* = DA/[DD + DD]. Stoichiometry S is defined as the ratio between the overall green fluorescence intensity to the total green and red fluorescence intensity during the green excitation period and describes the ratio of donor to acceptor fluorophores in the sample {S = DA + DD/[DD + DA + AA]}.
Using published procedures to identify fluorescent bursts corresponding to single molecules, we obtained bursts characterized by three parameters (M, T, and L). 17

■ RESULTS
Various methods and approaches that allow specific incorporation of fluorescent labels into nucleic acids and proteins for in vitro biophysical studies exist. 19,21,26 While synthetic oligonucleotides with labels or reactive groups can be purchased, proteins are a more challenging target. The most straightforward approach uses incorporation of cysteines at strategic positions, which allows stochastic labeling with two distinct fluorophores, e.g., for a FRET assay. In multimeric proteins or multisubunit proteins, however, this approach is complicated by ambiguous interactions of the fluorescent labels. Although site-

Biochemistry
Article specific labeling using unnatural amino acids 20 allows for selective tagging of more than two positions in protein complexes, labeling of multimeric proteins for smFRET studies remains challenging. Our group has recently started to explore the structure−function relationship and molecular mechanisms of active membrane transporters with single-molecule FRET. 8 In this study, we describe the first smFRET studies on the homotrimeric osmoregulated transporter BetP (Figure 2A) that serves as a good example of complications encountered when labeling multimeric proteins. The sodium-coupled betaine symporter BetP from Corynebacterium glutamicum is a well-characterized member of the betaine−choline−carnitine transporter (BCCT) family. 27 Several crystal structures show BetP as an asymmetric trimer, in which each protomer can adopt distinct conformations. These were assigned as individual transport states in the alternating access cycle 28 that allow uphill substrate transport driven by the electrochemical Na + potential, i.e., accumulation of betaine, which is the exclusive substrate for BetP, to molar amounts in the cytosol under hyperosmotic conditions. 28 Thus, BetP has two major biochemical functions: sensing of osmotic stress and regulated transport of betaine. The 45-amino acid α-helical Cterminal domain of BetP binds cytoplasmic K + , which is a prerequisite for activation of BetP during hyperosmotic stress. 29 The catalytic domain of BetP consists of 12 transmembrane helices (TMs) and is divided into a transporter core of two inverted five-helix repeats (TM3−TM12) and the two Nterminal helices, TM1 and TM2, which contribute to the trimer contacts. The symmetry between the two repeats (TM3−TM7 and TM8−TM12) is a key to the alternating access mechanism in BetP. For this study, cysteine mutants of BetP were designed to establish a dynamic picture of its structure−function relationship. Mutants were constructed on a cysteine-less BetP (C252T, TM5) containing an engineered cysteine at periplasmic position 516 in transmembrane domain 12, TM12 (Figure 2A). This position is part of the periplasmic gate and undergoes subtle conformational changes in the range of 3 Å during the isomerization from outward-to inward-facing states. Thus we expect only small changes in BetP structure that can be read out with this mutant via FRET. It serves, however, as a relevant example of the type of problems encountered with labeling during smFRET studies of multimeric proteins. The mutant protein was purified and solubilized in a detergent solution according to published procedures 30 as described in Materials and Methods. It shows slightly reduced uptake activity but an activation profile and potassium dependence comparable to those of wild-type BetP 31 ( Figure 2B).
The top view of the BetP crystal structure (Figure 2A) reveals the problems of the FRET approach of a multimeric protein. Because the protein is expressed and purified as a stable homotrimer, the cysteine residue appears in each subunit. Stochastic labeling with the donor and acceptor fluorophore results in a mixture of different subpopulations, comprising various donor-only, acceptor-only, and donor−acceptor species with distinct degeneracy ( Figure 2C).
We used μs-ALEX (microsecond alternating-laser excitation 32 ), in which fluorescently labeled biomolecules diffuse through the excitation volume of a confocal microscope, for smFRET studies of BetP. During its diffusional transit, the labeled protein produces fluorescent bursts in two distinct detection channels that are chosen to selectively monitor donor and acceptor emission. In ALEX, green excitation of the sample generates fluorescent signals that allow calculation of apparent FRET E* and Stoichiometry S. While E* is indicative of the donor−acceptor separation, S distinguishes molecular species by their relative labeling ratio of green to red fluorophores. A low S of <0.2 is indicative of acceptor-only labeled protein, while a high S of >0.8 corresponds to a donor-only species. Macromolecules containing both dyes are found at S values between these two boundaries (0.2 > S > 0.8) (see Figure 2D).
A two-dimensional ALEX histogram of BetP reveals five different subpopulations, which cannot be used for further structural analysis of BetP without additional information and refinement of the experimental conditions ( Figure 2D; labels donor Alexa555 and acceptor Alexa647). While donor-and acceptor-only species can be excluded from the analysis easily by considering only bursts within the S range of 0.2−0.8, Figure  2C suggests the existence of three possible species that contain both fluorophores: donor−donor−acceptor, donor−acceptor, and donor−acceptor−acceptor. To establish a direct link between S range and molecular composition, we analyzed the frequency distribution of photon count rates within single-  Figure  2E clearly reveals that the low-S regime corresponds to a donor−acceptor−acceptor species, the high-S regime is related to donor−donor−acceptor molecules, and only intermediate S values contain donor−acceptor molecules.
To understand which populations contain meaningful structural information in the form of E* distributions that are related to the donor−acceptor distances, we compared the E* histograms in the three regions with that of a double-stranded dsDNA with a 13 bp fluorophore separation, i.e., a distance similar to that of S516C label positions. We found that only the intermediate-S population provides the correct FRET measure while low-S and high-S populations show unexpected E* values. In species with more than two fluorophores, the relation of FRET efficiency and interprobe distance R seems to be lost because of the ambiguous interaction of, e.g., multiple donor with multiple acceptor fluorophores or signal loss via homo-FRET and energy dissipation. A change in the labeling ratio of donor to acceptor allows the relative abundance of the populations (data not shown) to shift; it remains, however, difficult to isolate a single donor−acceptor species.
To solve these problems and to allow smFRET studies of BetP and other complex protein systems, where subpopulations can be assigned clearly, we developed a novel experimental concept that we dub caged FRET. Here, unwanted fluorophore interactions are prohibited via use of reductive caging of synthetic organic fluorophores. This approach is so far typically used in localization-based super-resolution microscopy 33,34 and for FRET studies of surface-immobilized molecules using stochastic photoswitching. 35,36 In caged FRET, a fluorescent dye is treated with reducing chemicals to disable fluorescence; the photoactivation and hence recovery of the fluorescent signal are achieved with UV light ( Figure 3A). Cyanine dyes such as Cy5 are ideal for this because they undergo caging even with mild reducing agents such as TCEP ( Figure 3B). As an example of the caging process, the concentration-dependent reaction of Cy5 was monitored via changes in the UV/vis absorption spectrum of the fluorophore ( Figure 3C). The spectra also reveal that photoactivation ("uncaging") by UV light is achieved efficiently for wavelengths of <375 nm. Both the efficiency of caging and photoactivation heavily depend on fluorophore structure, redox potential, reducing agent, and the

Biochemistry
Article presence of oxidizing compounds in the imaging buffer as described in the literature. 33,34 Caged FRET is implemented in this study using μs-ALEXbased smFRET 32 ( Figure 4A) with diffusing biomolecules. We tested the concept with donor−acceptor-labeled doublestranded DNA (donor fluorophore TMR, acceptor Cy5). In a reducing buffer with 50 mM TCEP (pH 9), only "green" DD signals are observed at ∼50 pM dsDNA. As soon as an additional continuous-wave UV laser (375 nm) illuminates the sample also the sensitized acceptor signal via FRET can be observed ( Figure 4B/C). Doubly labeled FRET species can hence be "switched" off by TCEP and activated with UV light as seen in the corresponding ALEX histograms in Figure 4D. The data shows a reduction of FRET bursts to less than 20% ( Figure 4D, PBS vs PBS + TCEP). Caged molecules can be reactivated with an efficiency of 83%, a value that is close to the original level ( Figure 4D, PBS + TCEP + UV). The achievable photon counts of both donor and acceptor are altered in systematic fashion when TCEP is added or UV illumination is applied ( Figure 4E).
The analysis of photon count rates reveals that mostly the number of fluorescent molecules is decreased in all three channels when TCEP is added ( Figure 4E), but both a high number of fluorescent molecules and the average brightness of donor and acceptor are restored after UV activation ( Figure  4E). As seen in both Figure 4D/E the quality of the FRET histograms and the statistics are reduced insignificantly in caged FRET. The same results as presented for a high FRET sample with 8 bp separation between donor and acceptor fluorophore ( Figure 4D/E) are also observed for intermediate or low FRET samples with 18 and 33 bp separation (data not shown).
It should be noted that the concrete distributions of photon count rates and ALEX histograms depend on the (subjective) choice of burst search parameters and per-bin thresholds applied, which has to be done in a consistent fashion for a set of data. Weighing algorithms that consider the statistical significance of a burst from obtained photon counts or other burst-related parameters 40−44 would be preferential for data analysis instead of plotting each burst with a unity signal in the plot. While the data presented here show the working principle of caged FRET for caging of Cy5 with TCEP, it raises the question of how the quality of the data, the dye photophysics, and reactivation properties depend on the settings of the ALEX (green/red excitation power) and UV laser. An excellent analysis of caging and photoactivation properties of various fluorophores in TIRF-based super-resolution microscopy is given in refs 37, 39, and 45.
To understand the interrelation of setup parameters and fluorophore properties in caged FRET, we studied a DNA labeled with caged rhodamine Abberior Cage552. This nonfluorescent chromophore efficiently photoconverts into a structural analogue of the fluorophore TMR upon UV absorption. 37−39 As seen in Figure 5A, Cage552 is nonfluorescent before UV activation, as indicated by the small amount of coincidence between the red and green signal ( Figure 5A, DA species).
The successful activation of Cage552 is also seen in a frequency histogram of photon count rates of DD of singlestranded DNA containing two Cage552 fluorophores ( Figure  5B); we note that the bright fraction of molecules before photoactivation seen in Figure 5A−C could not be determined accurately because we also found that non-photoinduced uncaging occurs slowly on a time scale of weeks. We hence performed the set of experiments presented in Figure 5 within a short time interval. Thus, the calculated contrast between nonfluorescent and UV-induced bright molecules for Cage552 ( Figure 5C) represents only a lower threshold. It can be improved by use of fresh Cage552 and, e.g., protein/DNA labeling only just before the respective experiment. The experiments reveal, however, that contrast values of >10 can be achieved with caged FRET using the two approaches with a caged donor or acceptor fluorophore (Figures 4 and 5). As seen by a comparison of relative acceptor− to donor−acceptorcontaining molecules [via inspection of S distributions as a function of UV activation intensity ( Figure 5D)], a linear dependence is observed for increasing levels of UV activation. Contrast and activation efficiency depend on UV laser power and on applied green/red excitation intensity and choice of

Biochemistry
Article burst search parameters ( Figure 5C). At UV powers of >0.4 mW (500 kW/cm 2 ), both elevated background signals in the green detection channel and increased acceptor photophysics were observed and are not recommended for caged FRET experiments.
As a next step, we tested caged FRET in DNA constructs with two acceptor ( Figure 6A) or donor fluorophores ( Figure  6B). Here, information about FRET processes is typically convoluted as for BetP and does not permit extraction of the desired information, i.e., donor−acceptor separation. This is shown in Figure 6A, where three different labeled DNAs are compared to each other. Two dsDNA with a TMR−Cy5 donor−acceptor pair show intermediate and high FRET according to interprobe separations of 17 and 9 bp, respectively ( Figure 6A, panels 1 and 2). As soon as two acceptor dyes are simultaneously adjacent to the donor fluorophore, only a single FRET distribution with a high mean value is observed ( Figure  6A, panel 3). This distribution does not contain information about the two molecular distances. Instead, the convoluted signal does not even allow the proper determination of one of the two distances. Upon application of reductive caging of the acceptor fluorophores by TCEP, the convoluted population is reduced ( Figure 6A, panel 4). Subsequent UV activation leads to a stochastic mixture of uncaged molecules with one donor and one acceptor, where the latter has two distinct distances to the donor fluorophore ( Figure 6A, panel 5). While the efficiency of the uncaging process is imbalanced, the information about the two donor−acceptor distances can be restored. Such behavior with more efficient activation of high-FRET species was also described previously. 36 Although different high-and low-FRET samples could be uncaged with similar efficiency in the presence of only one donor and acceptor molecule (see Figure 4 for the high-FRET sample; low-and intermediate-FRET data not shown), the interactions are apparently more complex for the combined construct where two acceptor dyes are present.
To optimize the photoactivation process, we performed a similar experiment with two Cage552 donor fluorophores in combination with one ATTO647N acceptor fluorophore. As a reference, we analyzed a DNA-based ladder with 8, 13, and 18 bp separations for TMR-ATTO647N ( Figure 6B, panels 1−3) showing the FRET ruler character. The silent as well as photoactivated DNA with a Cage552 donor show two different FRET species that can be distinguished clearly. However, much better statistics are obtained with additional UV illumination. As indicated above, the contrast with and without UV can be optimized further by fresh labeling. The results presented in Figure 6 suggest that donor-based activation with Cage552 is a practically more relevant method compared to use of TCEP caging with Cy5, because the activation efficiency of the Cage552 donor does not depend on FRET interactions with the acceptor fluorophore.
Finally, we tested caged FRET on the S516C mutant with the goal of fully isolating a donor−acceptor species. First, we used an excess of acceptor dye for labeling to bias the formation of donor−acceptor and donor−acceptor−acceptor species ( Figure 7A). Under these conditions, we obtain only species in the low-and intermediate-S regime (<0.65), in agreement with data shown in Figure 2. Next, we applied caged FRET to remove the unwanted donor−acceptor−acceptor population at low S values via simple addition of low concentrations of TCEP to the buffer. This stochastically reduces the size of the active acceptor population ( Figure 7B) and thereby allows us to obtain histograms with only one DA species related to a single (and relevant) distance between both probes.
We found that for this specific BetP mutant, pH 9 and 1.5 mM TCEP resulted in a significant decrease in the amount of unwanted DAA species (Figure 7C,D). When the number of

Biochemistry
Article molecules in D-only, A-only, and donor−acceptor-containing fractions (including both DA and DAA) is plotted as a function of TCEP concentration, it is apparent that caged FRET without photoactivation allows improvement of the clarity of the histogram. The latter of course has to be balanced with measurement time and overall data quality because the available mean photon counts ( Figure 7D) of both the donor and the acceptor fluorophore are also reduced when TCEP is applied at increasing concentrations.

■ DISCUSSION AND CONCLUSION
We herein establish the use of caged fluorophores for smFRET studies of diffusing biomolecules. For the "caged FRET" methodology with photoactivation, we suggest the simple addition of an UV laser to a confocal microscope for photoactivation during diffusion. The applied laser wavelength needs to be chosen according to the absorbance properties of the caged species that is often found in a range below 400 nm. 33,34,37 Using this approach, we could remove ambiguous interactions of fluorophores that appear in FRET assays of oligonucleotides and multisubunit proteins with more than two fluorescent labels. For this, we used caged rhodamines and reductive caging of cyanines with subsequent photoactivation. In an even simpler approach, reductive caging can be used to remove overlabeled protein (more than one donor or acceptor) without any UV activation as demonstrated in detergentsolubilized membrane transporter BetP. Caged FRET is also distinct from other established approaches such as photoswitchable FRET, 35,36 which relies on surface-immobilized molecules and stochastic activation of, e.g., acceptor dyes. Stochastic switching would be compatible with caged FRET only if the photoswitching could be made substantially faster as is now to allow photoactivation during diffusion through the confocal volume. 46,47 In the future, we envision that caged FRET not only will be useful for improvement of labeling properties but also might allow solution-based smFRET at elevated concentrations. 48 This would allow studies of two interacting biomolecules with nanomolar to micromolar affinity. 49 For such experiments, the respective biochemical partners would be labeled with a caged donor and caged acceptor. To allow smFRET observation, simultaneous photoactivation of both labels is needed during diffusion through the confocal volume. A strict requirement for such an assay is that both donor and acceptor fluorophore be caged and activated similarly well in the same buffer and for the same UV intensity. In this respect, a combination of caged FRET with local activation of dye, 50 where a FRET acceptor is photoactivated (more) efficiently whenever it is close to the donor fluorophore, could be useful. Out of curiosity, we explored the practical limits of the general idea. When incubating a 1 μM solution of Cy5-COOH with 100 mM NaBH 4 for 48 h, we observed <1 burst/s under standard ALEX conditions (data not shown), indicating that micromolar concentrations are indeed accessible. While the Cy5 fluorophore was caged effectively, the photoactivation reaction was extremely inefficient and has to be optimized for practical future use. The low activation efficiency of Cy5 in the presence of strong reducing agents is in accordance with published studies and relates to the need for strong oxidants. Ultimately, the achievable concentration of caged smFRET will be a compromise of different factors because effective caging is often linked to inefficient photoactivation. Thus, a potent donor and acceptor pair must be identified, where the two requirements, i.e., efficient caging and photoactivation, are fulfilled.

Article
While the experiments presented here are of proof-ofprinciple character, they demonstrate the possibilities of temporal separation of fluorescent signals for FRET-based assays. Such a strategy is already widely used in localizationbased super-resolution microscopy (PALM, 51 STORM, 52 and PAINT 53 ). We consequently think that the caged FRET methodology relates to other multiruler techniques in a manner like stochastic super-resolution techniques (STORM and PALM) compare to targeted nanoscopy (STED and RESOLFT 54,55 ). This idea might be useful for distinguishing multidimensional smFRET-based approaches such as photoswitchable FRET and caged FRET (temporal signal separation) from combinations of different rulers, e.g., PIFE-FRET, 17,18 PET-FRET, 56 or farFRET 57 (spatial signal separation).


s2fieldsofstudy:
Chemistry
Materials Science

text:
Phenylacrylonitrile-bridging triphenylene dimers: the columnar liquid crystals with high ﬂ uorescence in both solid state and solution †

Herein, three phenylacrylonitrile-bridging triphenylene dimers were prepared by condensation and etheri ﬁ cation procedure in moderate yields. They exhibited the ordered hexagonal columnar mesophase based on the strong columnar stacking-induced action of two triphenylene units. A subtle balance between ﬂ uorescence decrease and AIE e ﬀ ect existed upon adding H 2 O to the THF solution, resulting in high ﬂ uorescence for these novel AIE liquid crystals in both solid state and solution for the ﬁ rst time.

Introduction
Organic molecules with columnar mesophases have been paid signicant attention in the past few decades. 1,2 Based on their extended p-stacking arrays for favourable transport of charge carriers along the columnar axis, they have shown broad application prospects in the elds of organic eld-effect transistors, organic light-emitting diodes, organic semiconductors, organic photovoltaic cells, and gas sensors. [3][4][5][6][7] Among all kinds of columnar liquid crystals, the luminescent columnar liquid crystals have attracted signicant research interest due to the effective combination of self-healing characteristics of mesophase and intrinsic luminescence capability. 8,9 For example, using the dyes such as perylene or Bodipy as cores, the luminescent columnar liquid crystals have been prepared successfully. [9][10][11][12][13][14][15][16][17][18][19][20][21] However, although these luminescent liquid crystals exhibited strong emission in solution, they showed weak uorescence in the solid state because of the aggregation-caused quenching (ACQ) effect.
Recently, aggregation-induced emission (AIE) discovered by Tang's group supplied a new strategy to prepare the uorescent liquid crystals. [22][23][24] As a result, some liquid crystalline molecules bearing AIE groups were prepared. [25][26][27][28][29][30][31][32][33][34][35][36] They exhibited good uorescence in the solid state, but they were practically non luminescent in solution, and it was difficult to form columnar mesophase due to the non-coplanar structures of AIE cores against the effective p-p packing in column. Since triphenylene derivatives possess strong columnar-stacking abilities, [37][38][39][40][41][42][43][44][45][46][47] lately, we have prepared the AIE liquid crystals of triphenylenediphenylacrylonitrile derivatives with the ordered hexagonal columnar mesophase based on the columnar stacking-induced action of triphenylene units. 48 Although the strong uorescence in the solid state was observed based on the AIE effect of diphenylacrylonitrile units, the crystals still emitted weakly in good solvents such as THF and CH 2 Cl 2 . Most recently, to attain efficient uorescence in both the solution and solid state, Tang's group has prepared a triphenylamine derivative that possesses not only good emission in solution based on considerable rigidity with limited intramolecular rotation, but also excellent uorescence in the solid state due to the substantial twisting conformation. 49 Inspired by this successful strategy, herein, we report the rst columnar AIE liquid crystals with high uorescence in both the solution and solid state. The columnar mesophase was induced by two triphenylene units, and the uorescence was emitted by the phenylacrylonitrile group. The subtle balance between uorescence decrease and AIE effect was responsible for the high uorescence in both the solution and solid state.

Synthesis
Because triphenylene-diphenylacrylonitrile derivatives have a good AIE effect but show weak uorescence in solution, 48 in this research, two diphenylacrylonitrile units were integrated into one conjugated system as a luminogen. Due to the extended rigidity of the conjugated structure, the crystals can emit effectively in solution, and the restriction of intramolecular rotation would produce the AIE effect; on the other hand, due to the large rigidity structure of this kind of phenylacrylonitrile moiety, two triphenylene units with strong columnar-induced abilities were bridged on it to ensure the formation of the columnar mesophase. The synthetic routes are illustrated in Scheme 1. By the condensation reaction of 4-hydroxyphenyl acetonitrile with terephthalaldehyde in a NaOH/EtOH system, the phenylacrylonitrile derivative 1 with two terminal hydroxyl groups was prepared in a yield of 78%. Treatment of monohydroxytriphenylene 2 with excess dibromoalkane in a K 2 CO 3 /MeCN system afforded triphenylene derivatives 3a, 3b, and 3c in the yields of 66-80% aer column chromatography. Furthermore, the target compounds 4a, 4b, and 4c were obtained conveniently by the etherication of the compound 1 with the compounds 3a, 3b, and 3c in the K 2 CO 3 / MeCN system, respectively. The yields were as high as 72-84% aer column chromatography.
The structures of the compounds 4a, 4b, and 4c were characterized by the 1 H NMR spectra, 13 C NMR spectra, MALDI-TOF-MS spectra, and HR-MS spectra. They exhibited the corresponding molecular ion peaks in mass spectrometry spectra. The deviations of molecular weights were less than 5 ppm, indicating the accomplishment of the condensation on two hydroxyl groups of compound 1. In the 1 H NMR spectra, the proton signals agreed well with the structures. Especially, the splits of ArH were observed clearly (see ESI †). The 13 C NMR spectra also supported these structures unambiguously.

Mesomorphic properties
The mesomorphic properties of the compounds 4a, 4b, and 4c were investigated by differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X-ray diffraction (XRD). The changes of DSC traces for second heating and cooling are presented in Fig. 1. The corresponding phase transition temperatures and enthalpy changes are listed in Table 1. All these samples exhibited the reversible phase transition with two peaks upon cooling and second heating. The phase transition peaks for the sample 4a appeared at 55.2 C and 95.8 C upon cooling and at 64.5 C and 119.2 C upon second heating. Sample 4b showed the phase transition at 18.0 C and 73.2 C on cooling and at 25.9 C and 81.8 C upon second heating. For the sample 4c, the broad thermal peaks at 10.1 C on cooling and at 12.0 C on second heating were observed, and the small peaks at 61.2 C on cooling and at 65.7 C on second heating emerged. These data suggested the thermal process of crystalline phase-mesophase-isotropic phase for samples 4a, 4b, and 4c. Along with an increase in the length of the alkyl bridging chains, the phase transition temperatures decreased orderly with the trend of 4c < 4b < 4a. The broad peaks and the hysteresis phenomena could be ascribed to the viscous morphology of these samples with high molecular weights. All the DSC analyses indicated that although the large rigid structure of two phenylacrylonitrile units were used as bridging spacers, the compounds 4a, 4b, and 4c possessed good liquid crystalline behaviors due to the strong columnar stackinginduced action of two triphenylene units. The longer alkyl bridging chains resulted in the lower phase transition temperatures.
The observation using POM also supported the presence of the mesophase for the samples 4a, 4b, and 4c. At the phase transition temperatures shown in the DSC curves, the corresponding phase transition processes were observed. Just like the compounds 4b and 4c, compound 4a also exhibited the clear uid in mesophase via microscopic observation although its enthalpy of the Col-Iso transition was obviously larger than that of the compounds 4b and 4c; this might be ascribed to the inuence of the short spacer. By cooling slowly from the isotropic phase, the clear textures appeared gradually. Fig. 2 presents the textures of the sample 4a at 75 C and those of the samples 4b and 4c at 40 C. The typical pseudo-confocal conic textures indicated the columnar mesophase for these samples, which were further conrmed by the XRD analysis. Scheme 1 Synthetic routes of target compounds 4a, 4b, and 4c. Fig. 1 The DSC traces of the compounds 4a, 4b, and 4c upon second heating and cooling (scan rate 10 C min À1 ).
The XRD traces for the samples 4a, 4b, and 4c at mesophase were investigated to study the liquid crystalline molecular stacking behavior. The results are illustrated in .74 for the samples 4a, 4b, and 4c, indicating the d-spacings of 3.68Å, 3.66Å, and 3.60Å, respectively, were the typical reections for the intracolumnar p-p interaction of the columnar liquid crystals. All these data suggested that the compounds 4a, 4b, and 4c possessed an ordered hexagonal columnar mesophase, which were in accordance with the POM observation. Furthermore, based on these XRD data, the lattice parameter a for the compounds 4a, 4b, and 4c could be calculated as 35.02Å, 34.56Å, and 34.12Å, respectively. These values were approximately in accordance with the diameter of one triphenylene column. Thus, the possible schematic of the columnar layered molecular arrangement was proposed as the square inserted in Fig. 3, in which the discs resided in separate columns, with the bridge spanning the distance between columns. Combining all these studies of DSC, POM, and XRD analyses, it could be concluded that the unfavorable inuence of the large rigid structure of phenylacrylonitrile for columnar mesophase was compensated by the strong columnar stacking-induced action of two triphenylene units. The compounds 4a, 4b, and 4c possessed a good reversible liquid crystalline behavior with an ordered hexagonal columnar mesophase.

Photophysical properties
The photophysical behaviors of the samples 4a, 4b, and 4c were studied to explore their luminescence properties. Their UV-vis absorption spectra in series of solvents with increased polarities are shown in Fig. S13-S15. † It can be seen that the l abs of the samples 4a, 4b, and 4c were at about 390 nm in toluene and exhibited a small blue shi with an increase of solvent polarities to 384 nm in DMF nally. The uorescence spectra of the samples 4a, 4b, and 4c in toluene, CH 2 Cl 2 , CHCl 3 , THF, and DMF are illustrated in Fig. S16-S18 † and 4 for 4b as the representative sample. With an increase of solvent polarity, their emission peaks presented obvious red shis and the uorescence intensity decreased correspondingly. However, the spectral changes were small in toluene, CH 2 Cl 2 , CHCl 3 , and THF, except for the DMF solution. These spectral changes might be ascribed to the intramolecular charge transfer effect and/or low solubility in highly polar solvents.
Furthermore, the uorescence behaviors of the samples 4a, 4b, and 4c in THF/H 2 O mixtures with different fractions of H 2 O were investigated. All these samples have good solubility in THF and are sparingly solubility in the THF/H 2 O mixture. Their uorescence spectra in the THF/H 2 O mixture and the plots of changes for emission peak intensity are exhibited in Fig. S19-S24. † Fig. 5 illustrates the spectra and plot for sample 4b as the representative sample. It can be observed that being different from the typical AIE molecules with little emission in pure THF solution, samples 4a, 4b, and 4c possess strong emission due to considerable rigidity with limited intramolecular rotation of large conjugated phenylacrylonitrile moiety. With the addition Fig. 2 The mesophase textures observed using POM on cooling at 75 C for 4a and 40 C for 4b and 4c (Â400).  of water to the THF solution, the emission intensity reduced rapidly. As the water contents attained 20-30%, the samples 4a, 4b, and 4c still maintained moderate uorescence although the emission intensities were at the lowest values among various contents of water. Combining the abovementioned absorption and uorescence investigation in various solvents, the decrease of uorescence in the THF/H 2 O mixture might be attributed to the intramolecular charge transfer effect and/or the low solubility in highly polar solvents. The addition of water increased the polarity of solvents and led to both stronger charge separation and dipole moment for the samples 4a, 4b, and 4c and low solubility in the THF/H 2 O mixture; this resulted in the decrease of uorescence intensity correspondingly. On the other hand, the phenylacrylonitrile unit is a typical AIE unit, which produces gradually enhanced uorescence with the addition of water to the THF/H 2 O mixture. Thus, when the contents of water were higher than 50% for 4a and 30% for 4b and 4c, the uorescence intensities increased dramatically. When the contents of water approached 90%, the emissions were even stronger than those in the pure THF solution. Moreover, with the addition of water, the uorescence peaks of the samples 4a, 4b, and 4c became broader and exhibited a slight red shi. These phenomena suggested that the strong AIE process occurred when water was added to the solutions. The increased uorescence based on the AIE process made up well for the previous uorescence decrease. These results also implied a subtle balance between uorescence decrease and AIE effect while adding water to the THF solution. This balance resulted in the strong uorescence for the samples 4a, 4b, and 4c in both the pure THF solution and THF/H 2 O mixture with high contents of water although a relatively low uorescence emerged at moderate contents of water. This kind of balance between uorescence decrease and AIE phenomenon was similar to the recently reported previous studies on uorescent probe, fumaronitrile-based uorogen, and piezouorochromic molecule. [50][51][52] Moreover, the uorescence spectra in powder were obtained (Fig. S25 †), and the uorescence quantum yields (F F ) are summarized in Table 2. It can be seen that the samples 4a, 4b, and 4c possess high uorescence with the F F values around 20% in both the pure THF solution and THF/H 2 O mixture with 90% of water. The F F values in powder also attained around 9%, indicating that the samples could emit uorescence well in the solid state. Furthermore, the uorescence emission in the solid state and mesophase of the compound 4a were observed using a handheld 365 nm UV lamp as the excitation source. The uorescence images in the solid state (26 C before heating), mesophase (90 C), and solid state (30 C aer cooling) are exhibited in Fig. S26. † It can be seen that the compound 4a emits good uorescence in both the solid state and mesophase. The uorescence wavelength in the mesophase showed obvious blue-shi in comparison with that in the solid state; this is a normal phenomenon for the thermochromism of uorescent liquid crystals. 34 Based on all the abovementioned analysis of photophysical data, it could be summarized that the samples 4a, 4b, and 4c possessed good uorescence emission in pure THF solution, THF/H 2 O mixture, and solid state.

Conclusions
In conclusion, three phenylacrylonitrile-bridging triphenylene dimers 4a, 4b, and 4c were designed and synthesized in moderate yields. The studies on mesomorphic property explored that although the large rigid structure of diphenylacrylonitrile units was introduced, the compounds 4a, 4b, and 4c exhibited the ordered hexagonal columnar mesophase based on the strong columnar stacking-induced action of two triphenylene units. Moreover, they exhibited high uorescence in both the solid state and solution. The subtle balance between uorescence decrease and AIE effect was proposed to be responsible for these interesting uorescence phenomena. The compounds 4a, 4b, and 4c were the rst columnar AIE liquid crystals with high uorescence in both the solution and solid state. This  study supplies a new method for the design and synthesis of the novel uorescent liquid crystals.

General
All chemical reagents, organic solvents, and inorganic reagents were purchased from Aladdin Reagent Co. Ltd. They were puri-ed by the standard anhydrous methods before use. TLC analysis was conducted on pre-coated glass plates. Column chromatography was carried out using silica gel (200-300 mesh). NMR spectra were obtained in CDCl 3 at 26 C using a Bruker-ARX 400 instrument. Chemical shis were obtained in ppm with tetramethylsilane (TMS) as the internal standard. MS spectra were obtained using a Bruker mass spectrometer. The compounds 3a and 3b were synthesized according to the reported literature. 53 Compound 3c was prepared by the literature method. 38 UV-vis spectra were obtained using the Varian UV-vis spectrometer. Fluorescence spectra were obtained using a conventional quartz cell (10 Â 10 Â 45 mm) via a Hitachi F-4500 spectrometer. The excitation and emission slits were 5 nm wide. The uorescence absolute F F values were obtained using an Edinburgh Instruments FLS920 Fluorescence Spectrometer with a 6-inch integrating sphere. A polarized optical microscopy (Leica DMRX) image was obtained using a hot stage (Linkam THMSE 600) to investigate the phase transitions. Thermal analysis of the materials was carried out using a differential scanning calorimeter (DSC) (Thermal Analysis Q100) under a N 2 atmosphere at a scanning rate of 10 C min À1 . XRD experiments were conducted by SEIFERT-FPM (XRD7) using Cu Ka 1.5406Å as the radiation source with 40 kV and 30 mA power.
Synthetic procedure for the compound 1

Conflicts of interest
There are no conicts to declare.


text:
Organic electrolyte solutions as versatile media for the dissolution and regeneration of cellulose

Organic electrolyte solutions – mixtures of a (room-temperature) ionic liquid with a neutral, organic, polar co-solvent – are attracting increasing attention as solvents for the regeneration and derivatisation of cellulose. Despite advantages (in comparison to simple ionic liquid analogues) associated with rapid or instantaneous dissolution, reduced viscosity, enhanced thermal stability and fine-tunable physicochemical properties, a firm understanding of the precise solvent–solute interactions and the relative kinetic versus thermodynamic contributions to dissolution remains elusive. The incorporation of a co-solvent introduces an additional layer of complexity, therefore an informed choice of both ionic liquid and co-solvent is necessary in order to achieve the desired properties. This article first provides an overview of the structure and bonding properties of native and non-native cellulose allomorphs, and a brief history of strategies for cellulose dissolution. Subsequently, organic electrolyte solutions are introduced as versatile solvents for cellulose, and the underpinning thermodynamic, kinetic and mechanistic phenomena behind cellulose solubility are critically discussed. The final sections summarize recent advances in the development of organic electrolyte technologies for derivatisation and regeneration of cellulose and critically discuss whether organic electrolytes can, or could be, rightly regarded as ‘green solvents’.

Introduction
Motivated by the pressing need for sustainable alternatives to fossil fuel resources in order to meet mankind's burgeoning material demands, lignocellulosic biomass has received widespread and growing interest in recent years. Biomass is primarily composed of three tightly-interwoven biopolymers: (i) cellulose, a linear and regular carbohydrate polymer that offers important structural integrity to the cell walls of plant species; 1,2 (ii) lignin, a complex and irregular aromatic polymer constructed from para-( propenylated) phenol units that contributes toward cell wall rigidity; 3,4 and (iii) hemicellulose, a comparatively low-molecular-weight branched polymer of (functionalised) pentose and hexose sugars, serving as a cross-linking motif between cellulose microfibrils and lignin. 5 To achieve the maximum economic potential of an integrated biorefinery, challenges associated with the separation, dissolution and upgrading of these three biopolymers must be overcome, exploiting differences in solubility and reactivity under process conditions. 6,7 An estimated 56 × 10 9 tonnes of biomass is generated globally each year (as of 2005), 1 of which cellulose accounts for the largest mass fraction (typically ≥40 wt% of dry biomass). 2 In addition to its extraordinary abundance, the high carbon content and homopolymeric structure of cellulose render it an attractive candidate for valorisation to paper products, textile fibres, biofuels and composite materials (Fig. 1). However, despite the high concentration of hydrophilic hydroxyl substituents along the polymer backbone, cellulose exhibits vanishingly low solubility in water and the majority of conventional solvents because of the extensive network of hydrogen bonds (and van der Waals interactions).

Matthew T. Clough
Despite significant historical advances in the development of cellulose dissolution technologies, leading to established and successful large-scale industrial procedures (e.g., the Lenzing process, Austria), frequently-encountered problems associated with solvent toxicity, instability and limited recyclability point out the need for fundamental and applied research toward more sustainable alternatives. A solvent for cellulose should ideally meet several important criteria: (i) high solubility; (ii) rapid dissolution; (iii) facile solvent recyclability; (iv) minimal toxicity and flammability; and (v) insusceptibility to side reactions between solvent and solute. In this context, so-called 'organic electrolyte solutions' (OESs), mixtures of a (room-temperature) ionic liquid with an organic co-solvent, are promising candidates and have received growing attention since their first reports in the literature. 8 This article is divided into six sections. Initially, a succinct overview of the key structural and bonding properties of cellulose is provided. Secondly, the evolution of cellulose dissolution strategies through the 19 th and 20 th centuries is described. Thirdly, the properties and behaviour of OEScellulose systems are reviewed in detail. Next, examples of the successful application of OESs to cellulose derivatisation and valorisation are evaluated. The fifth section addresses the important question as to whether OESs can be considered 'green solvents' and, finally, key conclusions and future prospects of OESs are discussed.
The primary purpose of this review is to clarify the thermodynamic, kinetic and mechanistic properties that underpin cellulose dissolution in OES solvents, and to describe OES-based methodologies for the dissolution and valorisation of isolated cellulose.
OES solvents have been successfully applied to the fractionation of lignocellulose, for example purification of cellulose pulp by selective extraction of hemicelluloses in [C 2 C 1 im][OAc]-EtOH/acetone (IONOCELL-P 9 ). Moreover, inclusion of an organic co-solvent (e.g., ethanolamine 10 ) during pretreatment has been found to influence the efficiency of enzymatic hydrolysis of cellulose. Nevertheless for brevity, these topics are excluded from this review, instead focusing on procedures for the dissolution and valorisation of pre-isolated cellulosic materials. The interested reader is directed to a recent review article on the fractionation of biomass in ionic liquids and OESs. 11

Cellulose structure and bonding
As the largest single component of hardwoods (43-47 wt%), softwoods (40-44 wt%) and bagasse (ca. 40 wt%), 2 cellulose is the world's most abundant bio-renewable resource. It is a strong candidate for targeted upgrading towards textile fibres, 12-18 biofuels, [19][20][21][22][23] (nano)composites [24][25][26][27][28] or hydrogels, 29,30 not only because of its high natural abundance but also because of its well-defined structure of repeating glucopyranose residues, linked via β-1,4-glycosidic bonds. The β-configuration at the anomeric carbon gives rise to an extended linear geometry (differentiating cellulose from starch, with α-1,4 configuration and helical structure). The number of glucopyranose residues in a single cellulose chain (denoted the 'degree of polymerisation', DP) may reach 10 000 (e.g., in cotton or bacterial cellulose). Agglomeration of approximately 40-70 individual cellulose strands gives rise to a single 'microfibril', whereby strands are held together through an ordered arrangement of intramolecular and intermolecular hydrogen bonds and van der Waals interactions. 31 Effective dissolution of cellulose hinges on the solvent's ability to dismantle this network of interactions and peel away individual strands from the microfibril.
Various crystalline allomorphs of cellulose exist either in native organisms (cellulose I α and I β , collectively referred to as cellulose I), or are attainable through annealing or treatment (cellulose II, III I , III II , IV I and IV II ). 2 In native cellulose I, each glucopyranose residue participates in two intramolecular hydrogen bonds with neighbouring monomers (specifically O3H⋯O5 and O2H⋯O6), and one intermolecular hydrogen bond (linking O3 and O6 groups) with an adjacent strand in the same sheet, via the all-equatorial hydroxyl substituents. The arrangement of the heavy atom skeleton is identical in cellulose I α and I β although the hydrogen-bonding patterns are subtly different. In contrast to cellulose II, neither cellulose I α nor I β contain inter-sheet hydrogen bonds (sheets instead interact via van der Waals interactions). 32 In nature, cellulose I α is always accompanied to some degree by cellulose I β . The proportion of cellulose I α may be as high as ca. 70% in primitive organisms (e.g., bacteria) and as low as 20% in higher Fig. 1 Selected target materials from cellulose: regeneration (e.g., electrospinning) into textile fibres; depolymerisation and fermentation to biofuels; or derivatisation, mixing and fabrication into composite materials (or hydrogels).
plants. Diagrams showing representations of structures, hydrogen-bonding patterns and interconversion pathways of different cellulose allomorphs are displayed in Fig. 2.
Although naturally-occurring cellulose I is metastable, it typically undergoes a transformation to cellulose II during regeneration or mercarisation, suggesting that biosynthesis of cellulose fibrils does not proceed via the crystallisation of preformed chains. Instead, it has been proposed that chain growth occurs by sequential addition of glucopyranose units concurrent with crystallisation into fibrils. In higher plants, biosynthesis occurs with the participation of cellulose synthase complexes tethered to the cell plasma membrane (the interested reader is directed to recent review literature 33,34 ).
When characterizing the mechanism of cellulose dissolution or derivatisation in a given solvent system, an understanding of the changes in (hydrogen-)bonding properties and specific solvent-solute interactions is essential. A brief over-view of the history of cellulose dissolution strategies is provided in the next section, highlighting differences between early derivatizing solvent systems and more recent (formally non-derivatizing) technologies.

Evolution of cellulose dissolution strategies
Dissolution is likely to be a crucial stage of any cellulose valorisation technology. However, as discussed in the previous section, dissolving cellulose is challenging on account of the need to break the ordered and extensive network of strong intramolecular and intermolecular hydrogen-bonding 35 and van der Waals 32 interactions. Consequently, cellulose does not dissolve in the great majority of conventional solvents. Fig. 3 shows the evolution of cellulose dissolution technologies through the 19 th and 20 th centuries. Early technologies typically involved derivatisation to enhance its solubility (usually in water). In this manner, cellulose was shown to be soluble as cellulose nitrate (Schönbein, 1846), in the form of organo-soluble cellulose acetate (Schützenberger, 1865), or as cellulose xanthogenate, attainable by treatment with NaOH and CS 2 (Cross, 1892). 36 The latter procedure is the so-called 'Viscose' process and is still conducted today at industrial scale. Cellulose was shown to be solubilised by a cuprammonium  hydroxide solution (Schweizer, 1857), 37 whereby in the presence of 'Schweizer's reagent' ([Cu(NH 3 ) 4 ](OH) 2 ), cellulose reacts to form a complex with copper involving bidentate interaction of the O2 and O3 hydroxyl residues (shown in Fig. 3). In each of the above processes, the added functional group enhances the solubility of the resultant polymer in the solvent media.
Contemporary ionic liquids (ILs), salts that are molten at (or close to) ambient temperatures, were first successfully applied to the dissolution of cellulose by Swatloski et al. in 2002. 54 The authors noted that up to 25 wt% of high-molecular-weight cellulose (DP ≈ 1000) dissolved in the archetypal IL 1-butyl-3-methylimidazolium chloride, [C 4 C 1 im]Cl, assisted by 3-5-second microwave heating pulses, leading to a clear viscous solution. Moreover, this article first demonstrated the strong sensitivity of IL-cellulose mixtures to the anti-solvent effect of water, a recurrent theme in subsequent literature. [55][56][57][58][59] It is now established that pairing strongly hydrogen bondbasic anions (e.g. [60][61][62] with moderately hydrogen bond-acidic cations (e.g., 1-alkyl-3methylimidazolium, [C n C 1 im] + ; 1,8-diazabicyclo [5.4.0]undecenium, [HDBU] + ; tetraalkyl-phosphonium) generates ILs that are effective cellulose-dissolving agents (Fig. 4). 27,54,[61][62][63][64][65][66][67][68][69][70][71][72] Hauru et al. proposed that the difference between hydrogen bond basicity and acidity is a useful parameter for predicting the ability of a solvent to dissolve cellulose, whereby effective solvents exhibit β − α values in the range ca. 0.35-0.9. 73 Although the associated cost 74,75 and susceptibility to undergoing side reactions with cellulose [76][77][78][79][80][81][82] have to be considered carefully, ILs could nevertheless offer profound benefits over traditional cellulose dissolution systems on account of their typically low toxicities (low vapour pressures) and their potential for continuous recycling. [83][84][85] Questions on the mechanism of cellulose dissolution in ILs, and the specific roles of the cation and anion in dismantling the network of hydrogen-bonded strands, have formed the basis of various experimental 72,86-89 and theoretical 55,59,[90][91][92][93][94][95][96][97] studies. There is a broad concensus that bonding interactions between the IL anion and cellulose are the predominant driving force for dissolution of the biopolymer. 59,87,90,92 It has been suggested that in [C 4 C 1 im]Clcellulose mixtures, the anion interacts with the equatorial hydroxyl substituents in a specific and directional manner (with 1 : 1 stoichiometry between Cl − anions and hydroxyl residues), 87 although a Molecular Dynamics (MD) investigation of cellulose dissolved in the same IL demonstrated interactions of Cl − from both axial and equatorial directions. 91 In addition, MD simulations have provided evidence that the cation occupies positions at the upper and lower faces of cellulose, interacting in a relatively non-specific/non-directional manner via hydrophobic or dispersion interactions. 59,90,94,96,97 The cation may intercalate between individual cellulose strands, 96 aiding in separation of the chains. Dissolution of cellulose in ILs is likely to be accompanied by increased polymer conformational flexibility 55,86,95 and disruption of the O3H⋯O5 intramolecular hydrogen bonds between neighbouring glucopyranose monomers. 55 Therefore, the chemical and electronic properties of both the anion and cation are of critical importance for effective cellulose dissolution in an IL.

Organic electrolyte solutions and cellulose: thermodynamic and mechanistic considerations
Although electrolytes of inorganic salts have been known as solvents for cellulose for many years (e.g., LiCl in DMA), [39][40][41][42][43][44] analogous 'organic electrolyte solutions' (OESs)mixtures incorporating a room-temperature ionic liquid (IL) and a polar organic co-solventwere characterized in detail only recently (Rinaldi, 2011). 8 Remarkably, it was shown by Rinaldi that complete dissolution of 10 wt% Avicel cellulose was possible even at very low mole fractions of the IL, e.g., χ IL = 0.08 8 Moreover, it was observed that complete dissolution was achieved far more rapidly in the OES than in the corresponding neat IL (e.g., <3 min for 10 wt% cellulose in 50 : 50 wt/wt [C 4 C 1 im]Cl-DMI, cf. >10 h for 5 wt% cellulose in [C 4 C 1 im]Cl 65 ). The inclusion of a co-solvent has, in one instance, been shown to enhance the solubility of cellulose 98 and may even enable dissolution where the neat IL is incapable of doing so. 99 Example ions and cosolvents that contribute to effective cellulose dissolution are shown in Fig. 4. A flow diagram to aid in the appropriate choice of cation, anion and co-solvent combinations for cellulose dissolution is shown in Fig. 5. A variety of different experimental methods for dissolving cellulose in OESs have been described. In many instances cellulose was addedeither in small aliquots or as a single additionto a pre-mixed solution containing the desired stoichiometry of IL and co-solvent. [99][100][101][102] For instance, Huang et al. described addition of various cellulose materials to mixtures of tetra(nbutyl)ammonium acetate and DMSO at 25°C. 101 The authors observed that the time required to dissolve 4 wt% of microcrystalline cellulose (MCC) was influenced by the mass fraction of IL in the solvent, such that complete dissolution was most rapid (T < 2 min) at W IL = 0.15. Understandably, dissolution time increased incrementally with the DP of the cellulosic material. Comparative studies of this type are of great value in elucidating the little-known effects of e.g. IL mole fraction and DP on the practicalities associated with preparing OES-cellulose mixtures.
Another method for preparation of OES-cellulose mixtures is the initial dispersion of cellulose in the co-solvent, followed by addition of the IL. In one early example, cellulose was suspended in DMSO prior to the addition of tetra(n-butyl) ammonium fluoride (TBAF), after which complete dissolution was attained within 15 min. 103 Similarly, by first preparing a suspension of Avicel in DMI at 100°C, agglomeration of biopolymer fibres was prevented; upon subsequent addition of the IL, complete dissolution was attained in a remarkably short time frame (<3 min with [C 4 C 1 im]Cl or instantaneous with [C 2 C 1 im][OAc]). 8 In preparing dope solutions for electrospinning of cellulose fibers (refer to section 5.1, below), Härdelin et al. describe treating small pre-dried square pieces of pulp material first with co-solvent, followed after 1 minute by addition of the IL. 104 The authors observed that complete dissolution was less time-consuming following this procedure, and they rationalised that absorption of the co-solvent enhanced the rate of ion diffusion into the pulp. Future investigations into the exact influence of co-solvent pretreatment on the uptake of IL into the microfibrils would be of significant interest.
An important, recurring observation in studies of OESs and cellulose is that the specific electronic (and steric) properties of the co-solvent are of critical importance in determining cellulose solubility. For example, from the outset it was noted that OESs derived from DMI and 1,1,3,3-tetramethylurea (TMU), close structural analogues, required vastly different quantities of [C 2 C 1 im][OAc] for complete dissolution of 10 wt% cellulose (χ IL = 0.18 and 0.59, respectively). 8 Similarly Holding and coworkers demonstrated that, although OES solvent systems composed of tri(n-octyl)methylphosphonium acetate and DMSO were capable of dissolving up to 19 wt% cellulose, the analogous mixtures with DMF exhibited negligibly low cellulose solubility. 99 Pinkert put forward a qualitative argument for the difference in co-solvent capability of DMI and TMU, suggesting that the increased structural rigidity of DMI and static nature of the N π-orbitals (which can readily hybridize to sp 2 ) enables DMI to better assist with correct positioning of the ions at the hydrophilic and hydrophobic sites of cellulose. 105 However, gas-phase Natural Bonding Orbital (NBO) calculations suggested that the energies of DMI and TMU frontier molecular orbitals are approximately equal. 106 More likely, differences in dipole moment and subtle steric properties explain the contrasting abilities of co-solvent species to aid in cellulose dissolution (Fig. 6). 106 The capabilities of IL solvents to dissolve cellulose have been shown to correlate 62 with the KT empirical solvation parameters α (hydrogen bond acidity), 107 β (hydrogen bond basicity) 60 and π* ( polarity/polarisability). 108 Interestingly, for OES solvents derived from [C 2 C 1 im][OAc] and DMI, the β and π* values do not change significantly over the range χ IL = 0.1-1.0. The hydrogen bond basicity (β, largely influenced by the nature of the anion) and polarisability properties are therefore not significantly perturbed up to χ DMI ≈ 0.90. By contrast, the hydrogen bond acidity (α, largely influenced by the nature of the cation) appears to partially decrease in the direction χ IL = 1.0 → 0.1. 8 Below χ IL ≈ 0.1 the β, α (and to a lesser extent π*) values fall sharply, associated with the observed loss in cellulose solubility (Fig. 7). 8 Gericke et al. studied the influence of adding aliquots of a co-solvent (or co-solvent binary mixture) to 10 wt% cellulose solutions in the ILs 1-allyl-3- 109 It was concluded that, in order to be miscible with the initial mixtures of IL and cellulose, a co-solvent must typically have solvent parameter values within certain thresholds (β > 0.4, π* > 0.8, normalized empirical polarity (E T N ) > 0.3). Strongly dipolar and hydrogen-bond-basic cosolvents (e.g. DMSO, DMF) could be added in ≥1 : 1 wt co-solvent / wt IL-cellulose quantities before cellulose precipitation occurred, for all three investigated ILs. By contrast, protic co-solvents, characterized by high α values, caused immediate and permanent cellulose precipitation at 0.2 : 1 wt co-solvent /wt IL-cellulose , attributed to the tendency of the co-solvent to compete with cellulose for interactions with the IL anions.
Although OESs have emerged as an independent class of solvent for cellulose, the mechanisms that underpin dis-solution remain only partially understood. As for pure ILs, 59,87,90,92 there is almost unanimous agreement that strong, directional hydrogen-bonding interactions that form between the anions and the hydroxyl residues of cellulose are the driving force behind dissolution in an OES. 101,[110][111][112][113] Nevertheless, the precise role of the co-solvent in enabling rapid or instantaneous cellulose dissolution is the subject of some debate.
Xu et al. demonstrated that the addition of aliquots of DMSO to [C 4 C 1 im][OAc] brought about a considerable increase in ion conductivity (maximum conductivity occurred at χ DMSO /χ IL ≈ 5). 110 They hypothesized that preferential solvation of cations by DMSO helped to increase the availability of dissociated or 'free' [OAc] − anions, which were capable of accepting hydrogen bonds with the hydroxyl units of cellulose more readily. Similarly, from results of MD simulations on [C 4 C 1 im][OAc]-DMSO and [C 4 C 1 im][OAc]-DMF systems, Zhao and colleagues suggested a partial separation of the cationanion association by the co-solvent. Once again, a partial breakdown in associative interactions between cations and anions was thought to indirectly account for rapid dissolution of cellulose, though in this instance no preferential solvation of cations or anions by DMSO or DMF was evidenced by the simulations. 111 Subsequent studies have challenged the concept of ion pair loosening. Andanson 114 Conductivity was certainly enhanced with the inclusion of DMSO, in agreement with Xu et al. 110 However, this was attributed to improved mass transport of ions (lower viscosity) and not due to a significant change to either ion-ion or ion-cellulose interactions, on the basis that the Walden plot indicated no increase in the number of charge carriers with increased DMSO concentration (Fig. 8). 114 This was further supported by an analysis of the  The extent of ion pair loosening may vary considerably between different IL-co-solvent combinations; trends that have been observed for [C 4 C 1 im][OAc]-DMSO OESs therefore may not be replicated in all instances. As an example, Huang et al. recently proposed that mixtures of tetra(n-butyl)ammonium acetate and DMSO exhibit distinct 'ion-split' and 'ion-paired' stages (in the absence of cellulose), at different mass fractions of the IL (0.05 ≤ W IL ≤ 0.15 and 0.15 ≤ W IL ≤ 0.25, respectively). 101 The authors recognised that balancing the ion concentration and ion 'mobility' was the key to ensuring maximum solubility of cellulose, occuring at W IL = ca. 0.15 for this specific OES. A recent NMR relaxometry investigation highlighted a similar duality for 1-allyl-3-methylimidazolium chloride-DMSO mixtures: at low χ DMSO , evidence suggested that the co-solvent molecules insert into the IL phase and loosen the cation-anion interactions, whereas high χ DMSO values (>0.5) promote clustering of ions, restricting the ability of ions to form favourable hydrogen-bonding interactions with cellulose. 115 These data confirm a phenomenon noted in the earlier literaturethat subtle differences in electronic and steric properties of the IL ions and cosolvent may strongly influence the cellulose-solvating behaviour of the resultant OES. 8,105,106 The above-described improvement in mass transport of ions in an OES, relative to a simple IL, probably explains the high rate at which cellulose dissolves, 8 whereby cations and anions can rapidly orientate themselves along the cellulose backbone and participate in favourable hydrogen-bonding interactions. Although this kinetic effect has been noted in multiple literature reports, thermodynamic data relating to dissolution of cellulose in OESs is relatively scarce. Polymer dissolution in a molecular solvent is usually an endothermic event (favoured by operating at higher temperatures). 116 However, cellulose dissolution in ILs is typically exothermic (ΔH < 0 kJ mol −1 , whereby formation of ion-cellulose interactions more than compensates for breaking of cellulose-cellulose and ion-ion interactions). Dissolution of any polymer is usually accompanied by a small entropic gain (ΔS > 0 J mol −1 ). 117 Therefore, to maintain spontaneous cellulose solubility in an OES (ΔG < 0 kJ mol −1 ), the enthalpy term must remain approximately neutral or exothermic after dilution by the cosolvent (ΔG = ΔH − TΔS).
Andanson et al. used solution calorimetry to compare the heat of cellulose dissolution in [C 2 C 1 im][OAc] against dissolution in equimolar [C 2 C 1 im][OAc]-DMSO. 89 The inclusion of 50 mol% DMSO brought about a surprisingly minimal reduction in exothermicity (at 80°C), from −132 ± 8 J g −1 to −106 J g −1 , despite a significant dilution of ions. In a subsequent study, solution calorimetry was also used to measure and compare the enthalpies of mixing for cellobiose (as a model for cellulose) in IL and OES solvents, Δ mix H (CB) . 118 The mole fractions of IL ([C 4 C 1 im]Cl or [C 4 C 1 im][OAc]) and cosolvent (DMSO, DMI or DMF) were systematically varied, and the Δ mix H (CB) values were determined (at 25°C). The Δ mix H (CB) profiles as a function of χ IL for mixtures with DMSO, DMI and DMF were strikingly different (Fig. 9). For the [C 4 C 1 im]Cl-DMI and [C 4 C 1 im][OAc]-DMSO series, Δ mix H (CB) rose sharply  towards endothermic values from χ IL = 0.1 → 0. However, in agreement with the observation of Andanson et al., 89 changes in Δ mix H (CB) in the broad range χ IL = 0.1-1 were comparatively small for all mixture series. Therefore despite a small loss in exothermicity by inclusion of a co-solvent, the dissolution of cellulose in an OES is likely to be rapid on account of both thermodynamic and kinetic favourability. When χ IL falls below a particular threshold (dependent on the specific choice of IL and co-solvent), cellulose solubilisation is no longer thermodynamically preferable, and spontaneous dissolution of cellulose does not occur.

Cellulose derivatisation and valorisation
Building on the success of ILs for the dissolution, derivatisation and regeneration of cellulosic materials, 27,62,71 OESs have received growing attention on account of the additional 'handle' for controlling the physicochemical properties of the solvent. Foremost, the ability to modify viscoelastic and rheological properties by inclusion of a co-solvent, whilst still retaining high cellulose solubility, is an attractive aspect. Key examples of the implementation of OES systems toward cellulose derivatisation and valorisation are summarized in the following subsections.

Fibre electrospinning
Continuous and regulated production of biopolymer fibres can be achieved with the use of an 'electrospinning' apparatus. Electrospinning functions by applying a high voltage difference between a solvent-polymer solution, contained within a syringe, and a collection vessel. A syringe pump regulates the rate at which the solvent-polymer solution is dispensed from the syringe, through a narrow 'spinneret' (often a needle). A pendant droplet forms at the spinneret and, once the voltage difference becomes great enough, the droplet deforms into a conical tip denoted a 'Taylor cone'. 119 A thin and heavilycharged stream of solvent-polymer solution is then drawn towards the collection vessel. When a volatile organic solvent is used, solvent evaporation occurs at this stage. Conversely with (non-volatile) IL or OES solvents, 13,14,18,120,121 the solution is discharged into a collection bath (e.g. water 14 or EtOH 122 ), causing precipitation of the polymer fibres. Therefore, use of an IL (or OES incorporating a relatively non-volatile co-solvent) mitigates environmental concerns associated with the release of volatile and hazardous organic components, which are typically not recycled. 123 A simplified diagram representing an IL or OES electrospinning apparatus is shown in Fig. 10. Electrospun fibers typically exhibit the beneficial properties of high porosity, 124 large specific surface area 125 and well-regulated fibre diameter. Such properties are readily controllable by modification of process parameters (e.g. the field strength, feed rate, distance between spinneret and collection vessel), solution parameters (e.g. concentration, polymer molecular weight, surface tension, viscosity), [126][127][128][129] or ambient con-ditions. 130,131 Considering solution parameters, it is established that low viscosity is a key property that broadly differentiates OESs from their corresponding ILs. For example, Lv et al. studied solutions of a cotton-derived cellulose (DP = 650) in [C 4 C 1 im]Cl-DMSO and [C 1 vC 2 C 1 im]Cl-DMSO solvents, at 25°C. 132 For mixtures incorporating 1.5 wt% cellulose, exponential reduction in viscosity was observed in the range χ DMSO 0 → 0.7, mirroring the behaviour of OES solvents without cellulose. In a later investigation of [C 4 C 1 im]Cl-DMSO-cellulose mixtures by Saba et al. (5 wt% cellulose, DP = 500), non-linear viscosity behaviour was observed in the range 5-8 wt% DMSO, at temperatures of 50-80°C (Fig. 11). 133 The non-linearity was attributed to microscopic aggregation or phase separation in this region. Although in one instance a higher viscosity was observed for an OES-cellulose mixture ([C 2 C 1 im][OAc]/DMSO 90 : 10 wt/wt) than the equivalent neat IL-cellulose solution, 104 the general trend of decreasing viscosity upon addition of the molecular co-solvent still holds.
Prior to OESs emerging as an independent class of solvents for cellulose, 8 the inclusion of a co-solvent had been used as a strategy for controlling the rheological properties of IL-cellulose mixtures for electrospinning processes. For example, Xu and co-workers explored the influence of DMSO concentration on the electrospinning properties of cellulose (from cotton linters, DP = 1600) dissolved in [C 1 vC 2 C 1 im]Cl. 12 DMSO reduced the viscosity, 114,132-134 surface tension and entanglement density, thereby contributing toward a continuous electrospinning jet. The authors identified that a sufficient cellulose loading was necessary to ensure effective entanglement. At a fixed cellulose loading of 5 wt%, excessive quantities of DMSO (e.g., 8 : 1 DMSO/[C 1 vC 2 C 1 im]Cl wt/wt) led to electrospraying of the mixture. Insufficient DMSO (e.g. 1 : 1 DMSO/[C 1 vC 2 C 1 im]Cl wt/wt) afforded a mixture with poor electrospinning capability, on account of the high viscosity. Therefore, it is worth noting that the solution para- meters cannot be considered in isolation when developing OES-cellulose electrospinning processes. 12 Härdelin et al. conducted a detailed investigation of the influence of DMA, DMF and DMSO co-solvents on the ability of [C 2 C 1 im][OAc] to electrospin cellulose fibres (from dissolving pulp, DP = 750). 104 Interestingly, although each of the three co-solvents brought about a reduction in the surface tension of the mixture, DMA and DMF had a significantly more pronounced effect than DMSO. The OES-cellulose mixtures incorporating DMSO exhibited the greatest degree of 'shear thinning', leading to optimal fibre formation ( Fig. 12).
Therefore, in analogy to the differing abilities of co-solvent species to aid and promote cellulose dissolution (see section 4, above), the specific choice of co-solvent plays an important role in determining the precise solution parameters for cellulose electrospinning procedures. Whilst the inclusion of a cosolvent may offer an additional handle for controlling viscoelastic properties of the mixture, the solution parameters must be considered collectively in order for successful electrospinning of cellulose fibres to be achieved.

Solution-state NMR spectroscopy of cellulose
The low viscosities of OES-cellulose mixtures offer potential benefits beyond tailored electrospinning. Typically, cellulose solutions are of prohibitively high viscosity for high-resolution NMR spectroscopy measurements, with broad and poorlyresolved 1 H signals. Because of this, whilst solid-state (e.g., 13 C 'Cross-Polarisation-Magic-Angle Spinning') NMR spectroscopy has been successfully applied to cellulose, 135,136 solution-state measurements are comparatively uncommon.
Nevertheless, Holding et al. were able to collect high-resolution 1D and 2D NMR data of high-M w cellulose, at ≥8 wt% concentration, dissolved in tri(n-octyl)methylphosphonium acetate-DMSO-d 6 (50 : 50 wt/wt). 137 The sharpening of cellulose 1 H signals in this low-viscosity medium enabled glucopyranose residues at the reducing and non-reducing ends to be distinguished from those embedded within the chain. Cellulose DP values could therefore be semi-quantified via integration of the respective signals. Whilst the DP values of low-M w celluloses (DP ≤ 100), calculated by NMR, aligned closely with the results of Gel Permeation Chromatography (GPC) analysis, the increased difficulty in spectral resolution for long-chain cellulose polymers (up to DP > 600) led to poor correlation between NMR and GPC assays. In spite of this, such methodologies employing OES solvents hold promise for future in situ NMR analyses of cellulose depolymerisation and derivatisation reactions.

Cellulose derivatisation
The addition of a co-solvent offers a dual benefit for homogeneous cellulose derivatisation reactions performed in IL-type solvents: reduced viscosity, and improved miscibility with hydrophobic reagents. 109 The co-solvent can aid in the misci-  bility of common derivatisation agents for cellulose, such as acetic anhydride or acetyl chloride. As early as 1999, Heinze and co-workers demonstrated the use of an OES-type solvent, tetra(n-butyl)ammonium fluoride trihydrate with DMSO, to dissolve high-M w cellulose (DP > 600). 138 This solvent system was successfully used to carboxymethylate 138 and acylate 103 cellulose samples. Nevertheless, the use of such systems was hampered by hydrolysis of anhydrides and acid chlorides as a result of residual water in the solvent, 103 and the high energy input required for solvent recycling. 139 More recently, homogeneous acetylation of microcrystalline cellulose (MCC, 5 wt%) was performed in solvent systems comprised of triethyl(n-octyl)ammonium chloride with acetone or DMA (9 : 10 wt/wt). Results were compared against reactions carried out in conventional LiCl/DMA and [C 4 C 1 im]Cl solvents. 140 Strikingly, solvents containing tetraalkylammonium salts were advantageous because they gave rapid dissolution of cellulose, without the need for prior activation or prolonged heating. Using acetyl chloride as the derivatising agent, a similar degree of substitution was observed when comparing reactions performed in the tetraalkylammonium systems against those using LiCl/DMA and [C 4 C 1 im]Cl.
Although examples of procedures for the derivatisation of cellulose using OESs are currently scarce, the potential benefits associated with improved IL-reagent miscibility are significant, serving as another example of how inclusion of a co-solvent could tailor the properties of an IL solvent toward improved outcomes.

Phase separation
Although OES solvents are capable of dissolving high quantities of cellulose at remarkably low values of χ IL , excessive dilution of the ions (e.g., χ IL ≤ 0.08 for [C 4 C 1 im]Cl-DMSO, 10 wt% Avicel cellulose 8 ) leads to systems where cellulose dissolution is no longer thermodynamically favourable. Recently, OES-cellulose mixtures that exhibit thermally triggered and reversible phase separation were reported. 141 The mixtures, derived from [C 2 C 1 im][OAc], DMI and MCC, could be easily handled as a single, homogeneous and low-viscosity mixture at elevated temperatures. Upon cooling, the mixtures passed through a well-defined 'temperature of phase separation' (T PS ), and separated into two distinct liquid phases. Cellulose was found to be wholly retained within the lower IL-enriched phase, whereas the upper phase was dominated by the co-solvent, DMI. Interestingly, the T PS parameter could be tailored over a wide range of temperatures (ΔT PS ≈ 75°C) by small changes to the IL and DMI mole fractions (Δχ ± 0.05) (Fig. 13a). Although such phase-separating OES-cellulose mixtures have not yet been applied to cellulose dissolution or derivatisation, it can be envisaged that they may find use for derivatisation to high-valueadded products (e.g., chiral stationary phases for liquid chromatography), exploiting trivial separation of the cellulosic product from residual lipophilic reagents. 141 Holding and co-workers reported a somewhat different phase separation phenomenon, enabling recovery of the expensive IL component tri(n-octyl)methylphosphonium acetate, [P 8881 ][OAc], from OES mixtures with DMSO. 99 The addition of water or aqueous sodium acetate to blank OES solvents brought about division into two phases, namely, an upper phase rich in the IL and a lower aqueous-rich phase (Fig. 13b). This strategy was then applied to 5 wt% MCC solutions in [P 8881 ][OAc]-DMSO (55 : 45 wt/wt), whereby addition of 10 wt% aqueous sodium acetate caused simultaneous phase separation and precipitation of the MCC. The costly phosphonium IL could be recovered in close to quantitative yield and high-purity, and the cellulose could be obtained as a white powder having undergone significant conversion from cellulose I to cellulose II (cf. Fig. 2). Therefore, a variety of phase separation protocols could be envisaged for versatile and sustainable recovery of high-purity IL and cellulosic materials from complex mixtures. Added to those above, various promising strategies have been put forward, incorporating (e.g.) kosmotropic salt solutions, [142][143][144][145] hydrophobic or fluorinated anions, [146][147][148] or distillable ILs. 67 These separation methods are likely to be of increasing interest within the field of organic electrolyte solutions.

Organic electrolyte solutionsgreen solvents?
If OESs are to find suitability as solvents for cellulose beyond the laboratory setting, they must not only fulfil various economic criteria, but also satisfy the condition of being 'green solvents'. To state this another way, the OESs themselvesor the processes in which they are involvedought to meet requirements of (among other factors) waste reduction, energy efficiency and hazard minimization. Questions as to whether ILs are (or could be) green solvents, for example as solvents for catalysis, 149 have been previously addressed.
With reference to the 12 established 'principles of green chemistry', 150,151 OESs could be considered as advantageous or disadvantageous relative to the parent IL, depending on the principle in question or the process/technology in which the solvent is to be used. In the paragraphs below, selected relevant principles of green chemistry are addressed individually, and the extent to which OESs could prove beneficial or problematic for the dissolution and regeneration of cellulose (compared to exisiting alternative solvents) is critically examined.

Principle 1 -Prevent waste
If a volatile co-solvent (e.g., acetone or DMI) is used, an OES solvent is likely to generate a greater quantity of non-recoverable waste (in the form of solvent vapours) relative to a pure IL with negligibly low vapour pressure under typical conditions of cellulose dissolution or electrospinning. 152,153 However, the co-solvent could in principle be recovered and purified by distillation, whereas the IL is susceptible to gradual (irreversible) decomposition during repeat cellulose dissolution-regeneration cycles. [76][77][78][79][80][81][82] Therefore, so long as vaporisation of cosolvent is minimized via carrying out a procedure in a closed system, diluting an IL with co-solvent could bring about a significant net reduction in waste by minimizing the fraction of the solvent that undergoes unintended degradation. IL and OES solvents could both offer a potential reduction in waste in comparison to existing industrial technologies (e.g., the Viscose process).

Principle 3 -Design less hazardous chemical syntheses
Relatively few examples of cellulose derivatization procedures using OES solvents have been shown in the literature. 103,[138][139][140] The flammability, volatility and toxicity of any co-solvent species will present a significant hazard over and above the analogous procedure performed in a pure IL. Nevertheless, the use of an OES-cellulose mixture that exhibits phase separation could offer simplified separation of cellulosic products from unspent toxic organo-soluble reagents.

Principle 4 -Use renewable feedstocks
Interesting preliminary studies have pointed towards the potential for renewable co-solvents to be used in OES-cellulose mixtures. For example, Gale et al. recently identified γ-valerolactone (GVL, Fig. 4c) as a promising co-solvent when paired with [C 2 C 1 im][OAc]. 102 GVL may itself be prepared from biomass, 7 and its credentials as a green solvent have already been established. 154 6.4 Principles 8 and 12 -Use safer solvents and reaction conditions and minimize the potential for accidents A severe disadvantage associated with OES solvents is the inclusion of often volatile, flammable and toxic organic species. This presents a significant hazard at large scale. OESs could be regarded as green solvents (according to principles 8 and 12) only where relatively benign co-solvents are used. Accordingly, future research must centre on co-solvents that share properties with DMSO, in contrast to solvents such as DMF or N-methylpyrrolidinone (which, e.g., present a risk to reproductive health). In most instances an OES solvent would pose less of a safety risk than N-methylmorpholine N-oxide or CS 2 , used for the established Lyocell and Viscose processes, respectively. However, great care would need to be taken to ensure that toxic co-solvent residues did not find their way into the cellulosic product.

Principle 9 -Increase energy efficiency
The low viscosities of OES-cellulose blendsin comparison to those based on pure ILscould bring about a substantial reduction in the energy costs associated with e.g. handling or stirring. Moreover, the high rate of cellulose dissolution (at close to ambient temperatures) in OES solvents may also improve energy efficiency, minimizing the extent of heating and stirring required in order to obtain homogenous mixtures. However, the need to prepare mixtures of IL and co-solvent in careful stoichiometric quantities prior to the dissolution of cellulose will likely add at least one extra process stage.

Principle 11 -Analyze in real time to prevent pollution
As has been demonstrated in recent literature, the low viscosities of OES-cellulose mixtures make possible certain analyses (e.g. 2D NMR spectroscopy 137,155 ) that are not feasible in pure IL-cellulose blends. Therefore, OESs could represent a more green alternative to pure ILs with respect to in-line analysis of cellulose dissolution and processing, thereby minimizing sampling and affiliated pollution.
In summary, questions as to whether OES-based technologies could be sustainable or 'green' processes must be answered on a case-by-case basis, taking into consideration the specific ions and co-solvent that are incorporated into the mixture. Certainly, there is scope for OESs to improve upon existing technologies in the field of cellulose dissolution, derivatisa-tion and regeneration. In the scientific literature to date, attempts to evaluate the green credentials of OES solvents are scarce, 102 yet such studies will become increasingly relevant in future.

Conclusions
The development of effective, stable and recyclable solvent systems for cellulose has posed a challenge to chemists for many decades. In order to peel away and solvate individual cellulose strands from the solid state, without chemical modification of cellulose itself, the solvent must be capable of participating in strong hydrogen-bonding interactions with the polymer strands. Throughout the 20 th and 21 st centuries various non-derivatising cellulose solvents have emerged, among which 'organic electrolyte solutions' (OESs) can be considered as a class of solvent in their own right, though sharing certain properties with the ionic liquids (ILs) from which they are derived.
The rapidity with which high quantities of cellulose dissolve in an OES is best explained as a delicate balance between thermodynamic and kinetic properties. So long as the condition of a negative Gibbs free energy is satisfied, enabling the spontaneous dissolution of cellulose, dilution with a cosolvent and the accompanying reduction in viscosity may enhance the mass transport of the ions. Consequently, diffusion of ions into the microfibril and formation of hydrogen bonds between ions and cellulose will occur more rapidly with the aid of the co-solvent. Though the co-solvent appears to 'loosen' the ion pairs, closer inspection of mixtures of [C 4 C 1 im][OAc] with DMSO 114 suggests that ion-ion interactions are (at least in this instance) not significantly perturbed in the presence of the co-solvent. However, the microscopic and macroscopic properties of the OES hinge upon the specific electronic (and structural) properties of the chosen cosolvent. Therefore, an alternative choice of co-solvent could lead to an OES in which the associative interactions between cations and anions are more heavily disrupted. 155 In this regard, OESs may be considered to share the reputation of ILs as 'designer solvents', whereby physicochemical properties of the solvent can be fine-tuned by choice of cation, anion and co-solvent.
OES solvents have been successfully applied to the electrospinning of high-quality cellulose fibres, to accurate solutionstate NMR measurements of cellulose, and also to cellulose functionalisation. Most of these methods benefit from the low viscosity (compared to the parent IL) of the OES solvent. Furthermore, phase-separable OES-cellulose mixtures extend the range of possibilities for cellulose derivatisation, purification, and solvent recycling over and above conventional ILs. Future research is likely to be similarly directed towards improved process sustainability and the development of green OES solvents. As has been demonstrated for ILs, 156 ab initio computational calculations could be able to aid the identification of sensible IL-co-solvent combinations, mitigating the need for time-consuming experimental trial and error.
In summary, OESs represent attractive and versatile alternatives to simple ILs for the dissolution, derivatisation and regeneration of cellulose. In light of the additional 'handle' for modifying physicochemical properties by inclusion of a cosolvent, and the flexibility that this affords, they may be expected to attract increasingly widespread interest in the coming years.

Conflicts of interest
There are no conflicts to declare.