A Strategy to Lengthen the On-time of Photochromic Rhodamine

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A Strategy to Lengthen the On-time of Photochromic Rhodamine Spirolactam for Super-Resolution Photoactivated Localization Microscopy Zhiwei Ye, Haibo Yu, Wei Yang, Ying Zheng, Ning Li, Hui Bian, Zechen Wang, Qiang Liu, Youtao Song, Mingyan Zhang, and Yi Xiao J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11369 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Journal of the American Chemical Society

A Strategy to Lengthen the On-time of Photochromic Rhodamine Spirolactam for Super-Resolution Photoactivated Localization Microscopy Zhiwei Ye,a,b‡ Haibo Yu,a,*‡ Wei Yang,b‡ Ying Zheng,b‡ Ning Li,b Hui Bian,b Zechen Wang,a Qiang Liu,a Youtao Song,a Mingyan Zhangc and Yi Xiaob* a. College of Environmental Sciences, Liaoning University, Shenyang 110036, P.R.China. b. State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, P.R.China. c. Liaoning Center of Disease Prevention and Control, Shenyang 110001, P.R. China. ‡.

These authors contributed equally to this work.

ABSTRACT: Rhodamine derivatives and analogues have been widely used for different super-resolution imaging

techniques, including photoactivated localization microscopy (PALM). Among them, rhodamine spirolactams exhibit great superiority for PALM imaging due to a desirable bright-dark contrast during the photochromic switching process. Although considerable attention has been paid to the chemical modifications on rhodamine spirolactams, the on-time of photochromic switching, one of the key characteristics for PALM imaging, has never been optimized in previous developments. In this study, we proposed that simply installing a carboxyl group close to the lactam site could impose an intramolecular acidic environment, stabilize the photoactivated zwitterionic structure and thus effectively increase the on-time. Based on this idea, we have synthesized a new rhodamine spirolactam, Rh-Gly, that demonstrated considerably longer on-time than the other tested analogues, as well as an enhancement of single molecule brightness, an improvement on signal-to-noise ratio and an enlargement of total collected photons of single molecule before photobleach. Finally, super-resolution images of live cell mitochondria stained with Rh-Gly have been obtained with a good temporal resolution of 10 s, as well as a satisfactory localization precision of ~25 nm. Through self-labeling protein tags, Rh-Gly modified with HaloTag ligand enabled super-resolution imaging of histone H2B proteins in live HeLa cells; through immunostaining antibodies labeled with an isothiocyanate-substituted Rh-Gly, super-resolution imaging of microtubules was achieved in fixed cells. Therefore, our simple and effective strategy provides novel insight for developing further enhanced rhodamine spirolactams recommendable for PALM imaging.

Introduction Single molecule localization microscopy (SMLM) techniques, including photoactivated localization microscopy (PALM)1–3 and stochastic optical reconstruction microscopy (STORM)4–6, call for high performance fluorescent dyes.7–12 Recently, rhodamine derivatives and analogues have been widely applied for PALM imaging. Generally, there are two kinds of rhodamines for PALM imaging, i.e., caged rhodamines13–16 and rhodamine spirolactams.17,18 The first subclass requires a caging group (usually an o-nitrobenzyl moiety) that quenches the fluorescence and undergoes irreversible photocleavage to recover fluorescence upon UV irradiation. Unfortunately, the applications of caged rhodamines in live cell imaging are limited because the potential toxicity of the photoreleased fragments remains unclarified. The second subclass, i.e., rhodamine spirolactam, undergoes a reversible structural transformation from nonfluorescent leuco form to highly fluorescent zwitterion under UV light irradiation. Such reversible off-on switching avoids the additional release of

poisonous species and, thus, represents an important advantage of the rhodamine spirolactam over the caged rhodamine. In 2007, for the first time, a photoactivatable rhodamine spirolactam (Rh-Pht, in Scheme 1a) was introduced into localization-based super-resolution imaging by Bossi, Hell and their coworkers.18 Since then, a variety of rhodamine spirolactams have been applied in subcellular super-resolution imaging to study the architecture of tubulin,18,19 clathrin20 and keratin,19 dynamics of actin filaments,9 the 3D structure of the bacterial cell membrane.17 By rational design of an amide substituent for rhodamine spirolactam, Moerner et al.17 shifted the activation wavelength to the visible region, which greatly enhanced the biocompatibility of PALM imaging. Recently, Belov and Bossi et al.19 extended the spirolactam design with enhanced hydrophilicity, feasible for super-resolution imaging in live and fixed cells. However, in PALM imaging, there always exists a tradeoff between spatial and temporal resolutions.1,21 To investigate fine architectures of organelles in fixed cells, earlier PALM imaging required an acquisition time of

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several minutes to hours. To monitor the dynamic changes inside live cells, such a time requirement is impractical. Therefore, various mathematical or instrumental methods, e.g., algorithms for solving the multi-emitter problems22–24 and faster speed cameras,25 have been developed to improve the temporal resolution of PALM imaging. Chemists also seek to solve this dilemma by rational design of fluorophores with enhanced brightness. Remarkably, Lavis et al.26,27 doubled the quantum yield of rhodamine by replacing N,N-dialkyl units with four-membered azetidine rings, and have generalized this strategy to other fluorophores. Later, Liu, Xu et al.28 further introduced a three-membered azetidine replacement to achieve enhanced brightness in highly polar fluorophores. However, as every acquired single molecule signal consists of hundreds to thousands of photon emission events, the sustainability of emission, reflected by the duration of on-time, constitutes another major impact on the brightness besides ensemble fluorescence performance. Thus, a lengthened on-time and a stabilization of bright state can enhance the single molecule brightness. This enhancement further generates blink events with high signal-to-noise ratio (SNR), and ultimately improves the spatial resolution of obtained PALM images during a shortened time of image acquisition. Hence, the design of photoswitchable rhodamines with prolonged on-time leads to possibilities of achieving high quality PALM imaging.

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spirolactam (Scheme 1a), the key to prolong on-time, in fact, is to stabilize the transitional bright zwitterionic structure.30,31 In the fields of chemosensors where rhodamine derivatives are studied intensively, it is widely accepted that an acidic environment (e.g., lower pH solution or coordination with transition metal ions, Scheme 1b) is a stabilization factor for the zwitterionic structures.32,33 Based on this principle, a massive number of rhodamine-spirolactam-based fluorescent sensors has been reported.29,34,43,44,35–42 This knowledge inspires us that if we place the rhodamine spirolactam molecules into such an acidic environment, then the on-time -- i.e., the lifetime of the photogenerated zwitterionic structures -will be extended. However, for super-resolution imaging of live cells, it might be impractical to lower pH or to add highly toxic metal ions safely, precisely and selectively at the intracellular sites where the rhodamine spirolactam molecules are located. Here, we report a simple and effective strategy to design rhodamine spirolactams with prolonged on-time of photogenerated zwitterions. Our idea is the installation of a carboxyl group close to the lactam site, imposing an intramolecular acidic environment upon rhodamine spirolactams. The new dye, Rh-Gly, outperforms its esterified counterpart Rh-MGly as well as a pioneering model Rh-Pht in terms of on-time and single molecule brightness. Finally, through PALM imaging of live cells stained with Rh-Gly, we obtain high-quality superresolution images of live cell mitochondria with relatively good spatial and temporal resolutions. Through HaloTag technique and immunostaining method, we further achieved super-resolution images of histone H2B proteins and microtubules with derivatives of Rh-Gly in HeLa cells.

Results and Discussion Molecular design and synthesis

Scheme 1. (a) Photochromic mechanism of common rhodamine spirolactam, e.g., Rh-Pht and Rh-MGly. (b) Photochromic mechanism of a rhodamine spirolactam stabilized by zinc ion.29 (c) Proposed hypothesis on photochromic reaction of Rh-Gly with light-induced activation (hν) of fluorescence (zwitterion) and thermal relaxation (∆) to the deactivated state (spirolactam).

The optimization of photochromic on-time has never been attempted in previous designs of PALM-applicable rhodamine spirolactams. Considering the rapid relaxation from photogenerated rhodamine zwitterion back to dark

Our idea in the design of a photoswitchable rhodamine spirolactam with prolonged on-time is to add an acidic carboxyl group in close proximity to the lactam (Scheme 1c). Photoactivation of Rh-Gly will first generate an unstable and short-lived amide anion. Simultaneously, the carboxylic acid could immediately protonate the amide nitrogen in an intramolecular fashion. Since the nucleophilicity of carboxylic anion is much lower than amide anion, the final zwitterionic structure of Rh-Gly should be more stable and long-lived. Therefore, we expect Rh-Gly to possess a longer on-time of the bright zwitterionic state and superior potential for high-quality PALM imaging in live cells. Rh-Gly and Rh-MGly were readily prepared from commercial starting materials, and detailed procedures were provided in the supporting information. The esterified intermediate Rh-MGly, lacking acidity, is a good control compound for Rh-Gly to estimate the intramolecular acidic effect on the on-time of the zwitterion.

Ensemble photochromic and pKa study


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Journal of the American Chemical Society Figure 2. (a) Spontaneous spirocyclization equilibrium of rhodamine spirolactam. (b) Integrated emission intensity vs pH of both Rh-MGly and Rh-Gly in C2H5OH/H2O (v:v=3:7). (c) Emission spectral changes of Rh-Gly vs pH in C2H5OH/H2O (v:v=3:7).

Figure 1. (a) Emission spectral changes of Rh-Gly and RhMGly (5×10-7 M) in glycerol (containing 30% CH3OH, mixed well) before and after UV irradiation (Ac stands for after activation, λuv= 365 nm). Inset: (left) magnified spectrum of the boxed region in (a) and (right) photographs of Rh-Gly solutions (5×10-4 M) in glycerol (containing 30% CH3OH, mixed well) before and after UV irradiation. (b) Emission spectral changes of Rh-Gly and Rh-MGly in thin PVA film before and after UV irradiation.

Rh-Gly exhibits excellent photoactivation performance upon irradiation with UV light both in solution and in film (Figure 1). The absorption band of Rh-Gly and RhMGly tails beyond 350 nm (Figure S1), which is sufficient for sparse activation required for PALM.20 A majority of Rh-Gly molecules remain nonfluorescent in glycerol (containing 30% methanol); however, the color of solution changes to pink (Figure 1a inset) upon exposure to UV light, with an appearance of the characteristic emission peak of rhodamine fluorophore at 585 nm (Figure 1a). In addition, both absorption and emission spectra of Rh-Gly in glycerol upon UV irradiation match those measured in acidic aqueous solution (Figure S2), suggesting the ring-opening transformation from spirolactam leuco to zwitterionic chromophore upon photoactivation. In contrast to the 6.1-fold photoactivated fluorescence enhancement of Rh-Gly, its esterified counterpart Rh-MGly exhibits essentially no change in glycerol solution. The photoactivation phenomenon was further studied in thin PVA film. As recorded in Figure 1b, fluorescence of Rh-Gly in PVA film shows a 7-fold increment (λfl = 570 nm) after 375 nm irradiation for 5 s, while the enhancement of Rh-MGly is just 3.5-fold under identical conditions. The above results demonstrate that Rh-Gly possesses an enhanced photoactivation capability versus Rh-MGly.

To better understand the acidity effect on the existence in terms of spirolactams or zwitterions, we further measured the pKa values of both dyes under identical conditions. The spirocyclization equilibrium of rhodamine spirolactam45–48 corresponding to the pKa is shown in Figure 2a. As shown in Figure 2b, the pKa value of Rh-Gly is 5.86, which is ca. 1 pH unit higher than RhMGly. The result reveals that transformation of Rh-Gly into the corresponding zwitterion does not require a strong acidity as in the case of Rh-MGly. This decreased demand for acidity also proves that zwitterion transformed from Rh-Gly can be stabilized to some extent through a “self-stabilized” structure (Scheme 1c), while there is no such stabilization effect for Rh-MGly. This stabilization extends the lifetime of the zwitterion and contributes to the improved photoactivation efficiency of Rh-Gly (Figure 1). In addition, the corresponding fluorescence spectra of Rh-Gly during pH titration are shown in Figure 2c. The dye shows a significantly enhanced fluorescence (186-fold) at 585 nm, which results from lowering pH values from 10.45 to 3.87. At neutral pH, a small peak in the fluorescence spectrum (red curve in Figure 2c) is detected, indicating that a small subset of Rh-Gly molecules exist in zwitterion forms. The existence of open-ring zwitterions is also evidenced by the small visible band of the absorption spectrum in PBS in Figure S1b. As rhodamine spirolactams continuously undergo spontaneous ring-opening in neutral medium,47 the slightly acidic pKa of Rh-Gly guarantees that the majority of its fluorophores exist in the nonfluorescent spirolactam form under the physiological condition, thus avoiding signal overlap during live cell PALM imaging.

Photoactivation and single molecule fluorescence on-time measurements of Rh-Gly To investigate the repeatable activation performance of Rh-Gly, we performed a photoactivation experiment in PVA film. Rh-Gly molecules dispersed were continuously excited with a strong 532 nm laser (~2 kW/cm2) and periodically activated by UV irradiation of 375 nm. Upon exposure to UV irradiation, newly formed zwitterions sharply improve the total detected number of activation events to approximately 6.0 × 102, as shown in Figure 3a. The activation cycle is repeated 46 times, and the process lasts for over 10 min until the majority of Rh-Gly fluorophores are bleached. The photoactivation images of the investigated region are further shown in Figure S3b. The fraction of fluorescent signals observed before UV photoactivation is due to the proton-controlled ringopening equilibrium (Figure 2a). Thus, Rh-Gly demonstrates a repeatable photoactivation performance. This light-controlled characteristic between dark



spirolactam and bright zwitterion of Rh-Gly satisfies the photoswitching nature of PALM imaging. The photoswitching behaviors were further identically investigated at the single molecule level upon continuous UV photoactivation for Rh-Gly, Rh-Pht and Rh-MGly. All studied PVA films show sparsely distributed single molecule signals (Figure 3b inset) with distances significantly greater than the full width of the single molecular spot of approximately 330 nm, thus avoiding the overlap between close signals. The blink behavior is described by a kinetic model following Bustamante et al.49 A fluorophore could be activated from dark state to active (bright) state. Upon continuous excitation, the active state either transforms to dark state or bleach state. Based on the model, photoswitching rates between different states are estimated through the fittings of the probability distribution of four characteristic parameters: the on-time of bright state (ton), the dark time of nonfluorescent state (tdark), the number of blinks after fluorophore activation (nblinks) and the photobleach time (tbleach). Figure 3b exhibits an example of an integrated intensity trace from a single Rh-Gly fluorophore along with its measurement of blink parameters. The theoretical kinetic model provided good fittings and predictions on the probability distributions of the experimental blink parameters (R2 > 0.90, Figure S4, detailed discussion in supporting

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information section 4.4). These results further allowed us to propose a structural transformation model to explain the blink behaviors of three dyes. As shown in Figure 3c, the activation of spirolactam (dark state) is a doublerecovery (kr1 and kr2) process under UV irradiation. The slow recovery rate kr1 possibly corresponds to the UV photoactivated transfer to non-protonated zwitterion (A1), containing an unstable amide ion, while the fast recovery rate kr2 probably corresponds to the transformation into protonated zwitterion (A2) through either photoactivation or spontaneous ring-opening during spirocyclization equilibrium. However, once the fluorophore is in active state, the two states (A1 and A2) are undistinguishable as they represent the same bright state of ring-opened zwitterion. After a short residence time in the active (bright) state, the zwitterion either transforms to the ring-closed dark state with rate kd or bleaches with rate kb. To compare the rates of photoswitching transformations of spirolactams, we performed the single molecule experiment for 5 times independently (details of obtained results are shown in Table S1 and discussed in supporting information section 4.4); since the results exhibit a similar tendency, one result close to median is listed in Figure 3d (from n > 1000 single molecule intensity traces for each fluorophore).

Figure 3. Single-molecule characterization of Rh-Gly, Rh-MGly and Rh-Pht embedded in PVA film in conventional (wide-field imaging) mode. (a) Time trace of numbers of activation events of Rh-Gly upon periodically controlled 375 nm laser and continuous irradiation of 532 nm laser. (b) One single molecule intensity trace of Rh-Gly and the measurement of its blink parameters. Inset presents two fluorescence images of single molecule signals recorded in control and Rh-Gly-dispersed PVA film, respectively. Control shows almost no noise signals. Scale bar: 10 μm. (c) Proposed mechanism explains the blink behavior of rhodamine spirolactam. Upon self-ring-opening or photoactivation, the dark spirolactam transforms to its non-protonated (A1, kr1) or protonated (A2, kr2) zwitterionic structure (active state). The zwitterion is stabilized by an intramolecular proton transform (Rh-Gly) or an attraction on an environmental proton (Rh-Pht and Rh-MGly). After some dwelling time at active state, the ring-opened structure either transforms to dark state (kd) or bleaches (kb). (d) Summary of kinetic rates and photon statistics. α is the fraction of kr1 recovery; ‘SNR’ is the signal-to-noise ratio. (e) Probability density distribution of on-times from Rh-Gly fits well to a single exponential decay. Ton was calculated as the inverse of summed rates (kd + kb). (f) Molecular structures of Rh-Asp, Rh-dMAsp, Rh-Ser and Rh-MSer.


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To avoid bias introduced by large values, Ton, i.e., the average on-time of bright state, was properly calculated from the probability distribution fit of on-times during photoswitching quantification analysis (as shown in Figure 3e; other fits are provided in Figure S4), providing an accurate estimation of the mean lifetime of active states. Among three dyes, Rh-Gly exhibits a prolonged on-time of 68.5 ms, which is apparently longer than the other two analogs (37.7 ms for Rh-MGly and 29.9 ms for Rh-Pht, respectively). The prolonged on-time of Rh-Gly demonstrates the existence of a stabilization effect on the zwitterion, consistent with the one described in the ensemble study. As the average on-time (Ton) was calculated as the inverse of the sum of rates (kd + kb), its extension can be further explained by the analysis of photoswitching rates (kd and kb) for three compared spirolactams. For Rh-Gly, the short-lived negative nitrogen atom of the active state is stabilized by a following intramolecular proton transfer, which induces a lower kd of 12.7 s-1, whereas the others rely on the small possibility of catching nearby protons from hydroxyl groups of the PVA molecule, resulting in faster transformations to dark states (Rh-MGly kd = 23.8 s-1 and Rh-Pht kd = 29.9 s-1). In other words, the ring-close reaction for Rh-Gly is slowed by the intramolecular proton transfer. In addition, Rh-Gly exhibits the lowest bleach rate (kb = 1.9 s-1), supporting the enhanced stabilization effect in an intramolecular fashion. As all compared analogs show much lower rates of kb (< 4 s-1) than the rates of kd (> 12 s-1), the transformation to dark spirolactam constitutes a more significant effect than the photobleach process on on-time (discussions on other obtained rates are presented in supporting information section 4.4). Therefore, it is the intramolecular proton transfer that stabilizes the zwitterionic structure of bright state and further extends the fluorescence on-time of RhGly. Enhanced single molecule fluorescence signals are also observed following the on-time increase. As listed in Figure 3d, Rh-Gly exhibits robust single molecule brightness (average photon emission rate: 3.57 × 104 photons/s), significantly higher than the compared analogs (2.44 × 104 photons/s for Rh-Pht and 2.85 × 104 photons/s for Rh-MGly). The enhancement of the brightness for Rh-Gly is consistent with its prolonged ontime, owing to the rigidification of the zwitterion through intramolecular hydrogen bonding. Following the enhancement of single molecule brightness and the lengthening of on-time, Rh-Gly also exhibits the highest SNR (5.48). On the other side, the prolonged on-time and the enhanced emission rate increases the times of excitation to unstable excited states during one switching cycle. The integration of excitations further increases the possibilities of photobleach during each cycle, leading to a decreased number of switching cycles of Rh-Gly (7.7, Table S2), compared to the other analogs (Rh-Pht: 9.3; Rh-MGly: 9.8). Nevertheless, these numbers are still comparable to previous reported values of fluorophores, which require excessive imaging enhancing additives.11,50 Although the number of switching cycles of Rh-Gly is

fewer, its overall photostability is improved as indicated by the significant enlargement of total collected photons of single molecule before photobleach (1.90 × 105), compared to the others (Rh-Pht: 0.79 × 105; Rh-MGly: 1.34 × 105). The improvement is attributed to the same rigidification of zwitterion through hydrogen bonding, consistent to the on-time extension. Moreover, the enhanced single molecule signals further benefit the localization reconstruction. The average localization uncertainty of Rh-Gly is 22.1 (Table S2), which is lower than its analogs (Rh-Pht: 26.1; Rh-Pht: 24.6). Therefore, owing to the carboxylic group, the single molecule fluorescence of Rh-Gly is synergistically strengthened by both the intramolecular proton transfer and the hydrogen bonding, making the fluorophore recommendable for single molecule localization-based super-resolution imaging. To further understand the relevance between the ontime extension and the acid substituents, four additional analogues, i.e., Rh-Asp, Rh-Ser, Rh-dMAsp and RhMSer, were prepared (synthesis procedures are provided in supporting information) and investigated at single molecule level under identical conditions. According to their substituents, the added four fluorophores (Figure 3f) are classified into two groups i.e., acid installed spirolactams and esterified analogues. As listed in Figure 3d, the acid installed spirolactams (Rh-Asp, Ton = 67.1 ms; Rh-Ser, Ton = 63.3 ms) exhibit extended on-time in a uniform fashion, compared to their esterified analogues (Rh-dMAsp, Ton = 41.3 ms; Rh-MSer, Ton = 36.5 ms). This result confirms the stabilization effect of intramolecular proton transfer on the zwitterionic structure of rhodamine spirolactams. In addition, the on-times of RhAsp and Rh-Ser, are in proximity to the on-time of RhGly (as listed in Figure 3d and Table S1), suggesting that extra hydrophilicity of the substituents does not prolong the lifetime of zwitterionic bright state. On the other side, as listed in Figure 3d, the single molecule brightness of these acid installed spirolactams (Rh-Asp, 3.34 × 104 photons/s; Rh-Ser, 3.55 × 104 photons/s) is enhanced than their esterified analogues (Rh-dMAsp, 2.82 × 104 photons/s; Rh-MSer, 2.62 × 104 photons/s), further proving the stabilization effect through intramolecular hydrogen binding. Furthermore, the stabilization leads to strengthened SNR (Rh-Asp, 5.14; Rh-Ser, 5.41), enlarged total collected photons before photobleach (Rh-Asp, 1.76 × 105; Rh-Ser, 2.27 × 105) and improved localization uncertainties (Rh-Asp, 22.6; Rh-Ser, 22.0) of acid installed spirolactams. Therefore, the rigidification of zwitterion, caused by the proximate acid group, significantly extends the on-time and enhances the single molecule fluorescence of rhodamine spirolactams.

Rh-Gly for PALM imaging of mitochondria in live cells



Figure 4. Two-color colocalization analysis of Rh-Gly in HeLa (panel a) and MCF-7 (panel b) cell lines. In each panel, from left to right: confocal fluorescence image of Rh-Gly after UV irradiation, colored in red; confocal fluorescence image of Mitotracker Deep Red (Mitotracker), colored in blue; merged image of two fluorescent images; intensity profiles from two fluorophores along the yellow line marked in the merged image. Scale bar: 25 μm.

Before applying Rh-Gly to live cell PALM imaging, we analyzed the distribution of Rh-Gly in different cell lines. After UV photoactivation, fluorescence of Rh-Gly exhibits mitochondrial networks in both HeLa cells and MCF-7

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cells. The majority of fluorescent signals of Rh-Gly overlap well with the signals of Mitotracker Deep Red (commercial mitochondrial tracker) in both cell lines as revealed by merged images in Figure 4a and b. Plots from Figure 4a and 4b right column show synchronous intensity distributions between two fluorophores along the yellow line marked in the merged images, proving the co-occurrence of fluorescence of the two dyes. In addition, more fields of view from colocalization experiments are presented in the supporting information (Figure S5). The correlations between two stainings are further quantified, yielding high average Pearson’s coefficients at 0.86±0.07 (MCF-7 cells, n = 52) and 0.83±0.06 (HeLa cells, n = 67). All results demonstrate that Rh-Gly specifically stains mitochondria in live cells with few nonspecific signals. The accumulation of the dye inside mitochondria is probably caused by the protonation equilibrium on spirolactam (Figure 2a). Under neutral cytosolic conditions (pH = 7.2) ,51 Rh-Gly molecules partially protonate and exist in forms of positively charged zwitterions; therefore, they are attracted into mitochondria by the negative potential of inner membranes.52 Once they enter mitochondria, the hydrophobic microenvironment will force them to transform back into the colorless spirolactam form of uncharged and hydrophobic nature, which also leads to their continual sequester within mitochondria. Therefore, Rh-Gly accumulates in mitochondria mostly in its ringclosed form and could be photoactivated to bright zwitterion form by UV irradiation.

Figure 5. Super-resolution imaging of mitochondria-enriched regions in live HeLa (panel a) and MCF-7 (panel b) cells stained with Rh-Gly. Inside each panel, the top left image is a wide-field fluorescence image of the region of interest and the right image is a PALM image of the same region reconstructed from ~2000 consecutive frames (10 s total duration) under inclined illumination53,54; yellow arrows mark the regions with high localization density, whereas green arrows indicate domains with low localization density; bottom row, from left to right: histogram of single molecule brightness, histogram of localization precisions and time trace analysis of Nyquist resolutions (orange color band shows the standard deviations of the resolutions estimated from >16 domains during the PALM imaging). Scale bars: 2 μm.


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Finally, we performed PALM imaging of mitochondria in live HeLa and MCF-7 cells stained with Rh-Gly under a total internal reflection fluorescence (TIRF) microscopy in inclined illumination mode.53,54 The fluorophore exhibits good photoswitching performance (shown in Movie S1 and S2) in live cells upon simultaneous irradiation by green and UV light. The blink events are generated in normal culture media free from additional oxygen depletion regents and toxic reductants,11,55,56 which inhibit ATP generation in live cells.57 Though PALM imaging certainly requires UV irradiation, the intensity of the laser is relatively low (~2 W/cm2) and causes no apparent cell damage during the short imaging time.58,59 In sharp contrast to the blurred wide-field images of mitochondria, the PALM images reveal detailed morphologies in Figure 5a and b (top row). Moreover, the super-resolution images indicate mitochondrial activity. Because of the prolonged on-time of protonated Rh-Gly and the acidinduced ring-opening tendency of a rhodamine spirolactam, the number of obtained localizations increases in regions where active proton pumping happens. Thus, the corresponding localization density also reflects the mitochondrial activity. The yellow arrows indicate typical dense regions with intense mitochondrial respiration, whereas the green arrows point to low density domains with lower activity. To further demonstrate the capability of mitochondrial super-resolution imaging with Rh-Gly, six additional results are shown in Figure S6 for each cell line, respectively. Therefore, Rh-Gly offers great potential for super-resolution imaging of mitochondria in live cells. Two core parameters to quantify the quality of PALM imaging are the localization precision and the localization density (ρ) of rendered molecules. The precision describes the uncertainty of each localization.60,61 As shown in Figure 5a, b (second plot in the bottom row of each panel) and Figure S7, the average localization precision of RhGly in each mitochondrial imaging is approximately 25 nm, corresponding to average photon numbers of 400800 per single molecule per frame (first bottom plot in Figure 5a, 5b; Figure S7). On the other hand, the localization density defines the sampling interval of reconstructed images. According to the Nyquist-Shannon criterion,1,62 intervals of discrete sampling (localization spaces) for target structures must be less than half of the desired resolution of the image. Hence, the Nyquist criterion-based resolution (Nyquist resolution) of a 2D localization image is calculated as 𝑅𝑁𝑦𝑞𝑢𝑖𝑠𝑡 = 2/ 𝜌 and is utilized for the evaluation of the overall spatial resolution of reconstructed images. Through the equation above, Nyquist resolutions (bottom right column of Figure 5a, b and Figure S7) were consequently determined at each second during PALM imaging. Following the elongation of imaging time, more localizations are gathered until Nyquist resolutions improve to ~50 nm at 10 s. The corresponding Fourier ring correlation (FRC) analysis63,64 of these images at 10 s also shows a relatively high spatial resolution of 128 nm (for HeLa cells in Figure 5a) and 122 nm (for MCF-7 cell in Figure 5b) in Figure S8. Therefore, through staining of Rh-Gly, balanced ~25 nm localization

precisions and ~50 nm Nyquist resolutions are obtained for super-resolution imaging of mitochondria in live cells within 10 s. Despite the tradeoff between spatial and temporal resolutions during super-resolution imaging,1 the PALM imaging of mitochondria stained with Rh-Gly does not sacrifice the imaging speed to obtain high quality. The temporal resolution of mitochondrial super-resolution imaging is shortened to 10 s, which is comparable to the best reported single molecule localization superresolution imaging results.1,5,25,46,59,65,66(Table S3) Although previous imaging of mitochondria has obtained a temporal resolution of 2 s,58 the imaging demand toxic blinking enhancing additives or heavily intensified laser power (Detail comparison is discussed in supporting information section 5.4). The high temporal resolution is probably a consequence of the on-time improvement of Rh-Gly. The combination of prolonged on-time and the stabilization effect on the zwitterion of Rh-Gly increases photon emission rate and enhances the single molecule brightness (Figure 3d). The brightness improvement further enlarges the number of localizations obtained during imaging, leading to improved spatial resolution within shortened time. Therefore, the inherent nature of on-time extension of the zwitterion makes Rh-Gly a good dye for PALM imaging of mitochondria in live cells. The dye exhibits a balanced tradeoff between preserving the localization precision and improving the temporal resolution of PALM imaging. In addition, the prolonged on-time also provides applicable potential for obtaining extended single molecule trajectories in live cells.



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Right: model of HaloTag protein covalently bounded to RhGly-Halo. Image of Rh-Gly-Halo coupled haloalkane dehalogenase (1BN6)67 is designed with chimera.68 (b) PALM image of H2B-Halo fusion proteins labeled with Rh-GlyHalo in live HeLa cell under inclined illumination. Inset shows the wide-field image of the same H2B-Halo fusion proteins labeled with Rh-Gly-Halo. (c) Histogram of single molecule brightness. (d) Histogram of localization uncertainties. Scale bars: 3 μm.

Rh-Gly labelled HaloTag for PALM imaging of fusion proteins in live cells

Figure 6. Super-resolution imaging of histones in nucleus of live HeLa cell. (a) Left: molecular structure of Rh-Gly-Halo;

Through established technique of HaloTag fusion proteins,69 we further obtained the super-resolution imaging of histones with Rh-Gly in live HeLa cells. To achieve the labeling of target enzymes (HaloTag protein), we prepared a chloroalkane ligand conjugated derivative of Rh-Gly, i.e. Rh-Gly-Halo, as shown in Figure 6a (details of the synthetic procedures are presented in supporting information). This probe is cell-permeable, and covalently binds to H2B-Halo fusion proteins in live HeLa cell as presented in Figure 6b. The reconstructed image of histone H2B proteins exhibits a significant enhancement of resolution compared to the wide-field image (Figure 6b inset), with an average photon number of 1040 per single molecule per frame (Figure 6c) and an average localization precision of 21 nm (Figure 6d). Moreover, additional four cases of super-resolution imaging of H2B proteins are exhibited in Figure S9, validating the efficiency of Rh-Gly-Halo in imaging HaloH2B fusion proteins. Therefore, Halo ligand conjugated derivative of Rh-Gly demonstrates an extended functionality for specific labeling other cellular structures besides mitochondria, and retains the capacity for PALM imaging in live cells.


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Figure 7. Super-resolution imaging of microtubules in fixed HeLa cell. (a) Molecular structure of Rh-Gly-NCS. Super-resolution image (b) and corresponding wide-field image (c) of microtubules immunolabeled with primary antibody against α-tubulin and secondary antibody labeled with Rh-Gly-NCS under inclined illumination. Super-resolution image was reconstructed from ~15000 frames. The imaging media is phosphate buffer saline (PBS). (d) Histogram of the localization precisions. (e) The intensity profiles of microtubule filament in super-resolution image (b) and wide-field image (c) indicated with blue lines. A single gaussian function was fitted to the data, estimating that the full width at half maximum (FWHM) of microtubule filament was 62 nm in the super-resolution image and 396 nm in the wide-field image. Scale bar: 3 μm.

Rh-Gly labelled antibody for PALM imaging of microtubules To further extend the functionality of Rh-Gly, a derivative with an isothiocyanate group, i.e., Rh-Gly-NCS, was prepared for super-resolution imaging of microtubules in HeLa cells (details of the synthesis are provided in the supporting information). Through immunolabeled fixed HeLa cells with primary antibodies against α-tubulin and secondary antibodies labeled with Rh-Gly-NCS, the super-resolution image of microtubules was successfully obtained in simple PBS solution, without the aid of special imaging enhancing additives (Figure 7b). The reconstruction image demonstrates a notable clarity improvement versus the wide-field image (Figure 7c), which is blurred by diffraction. Quantitative measurement of the PALM imaging provides an average single molecule brightness of 981 photons per frame of Rh-Gly-NCS (Figure S11) and an average precision of 15 nm of the corresponding localizations (Figure 7d). In addition, the super-resolution image of tubulin reveals a 62 nm width of antibody coated microtubule, close to previously reported value,70 and the result demonstrates a robust improvement on resolution versus the crude estimation of 396 nm width of microtubule in the widefield image (Figure 7e). Two appended cases of super-

resolution imaging of microtubules are shown in Figure S12. In short, owing to its capability of PALM imaging, bioconjugates labeled with Rh-Gly enable superresolution imaging of microtubules in fixed cells.

Conclusion In conclusion, we have proposed a simple and effective strategy to lengthen the on-time of photochromic rhodamine spirolactam, which proves to benefit PALM imaging. Our idea is to introduce a carboxyl group close to the lactam site in order to exert an inner acidity effect to stabilize photogenerated zwitterions. Rh-Gly shows repeatable and UV-controllable activation ability with a prolonged on-time of the zwitterion at the single molecule level, through comparing with both its esterified product (Rh-MGly) and another inspiring analog (a pioneering PALM dye, Rh-Pht) reported by Hell et al.18 The stabilization effect of the carboxyl group on zwitterion not only increases total photon collection but also enhances single molecule brightness. Furthermore, Rh-Gly also exhibits a specific mitochondrial staining in different cell lines (HeLa and MCF-7). As a consequence of the lengthened on-time, the temporal resolution of PALM imaging of mitochondria in live cells is shortened to 10 s, preserving localization precisions of approximately 25 nm. Because of the balanced tradeoff between spatial and temporal resolutions, a series of robust super-



resolution images of mitochondria in live cells are further obtained by Rh-Gly. The functionality of Rh-Gly for specific cellular labeling is extended with a HaloTag ligand derivative and an isothiocyanate derivative; through self-labeling protein tags and immunostaining method, these derivatives enable super-resolution imaging of histones and microtubules in HeLa cells. Therefore, our simple design paves a new path towards prolonging on-time for the development of rhodamine spirolactams to improve PALM imaging in the future.

ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on ACS Publications website at DOI: XX.XXXX/XXX. Experimental methods, synthesis procedures and characterization spectra, Table S1-S3 and Figure S1-13. Movie S1. Blink behaviors of Rh-Gly in Figure 5a. Movie S2. Blink behaviors of Rh-Gly in Figure 5b.

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AUTHOR INFORMATION (11)

Corresponding Author * [email protected] (Y. Xiao) * [email protected] (H. Yu)

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Author Contributions ‡ These

authors contributed equally to this work.

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ACKNOWLEDGMENT This work is supported by National Natural Science Foundation of China (Nos. 21421005, 21576040, 21776037 and 21804016), the Fundamental Research Funds for the Central Universities (No. DUT17LK43), Natural Science Foundation of Liaoning Province (20180510044), Program Funded by Liaoning Province Education Administration (No. L2014010) and National Water Pollution Control and Treatment Science and Technology Major Project (2015ZX07202012).

Special thanks to Prof. Xinfu Zhang, at State Key Laboratory of Fine Chemicals, Dalian University of Technology, for providing the comparison analog Rh-Pht; to Prof. Wei Ji, at Institute of Biophysics, Chinese Academy of Sciences, for help in building up the single molecule localization microscopy; and to Prof. Youjun Yang at East China University of Science and Technology, for inspiring discussions on the mechanism of prolonged on-time and for his help in improving the English of this manuscript.

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A Strategy to Lengthen the On-time of Photochromic Rhodamine

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