Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Abstract Recent developments in fluorescence microscopy call for novel small‐molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small‐molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples.


Introduction
Small-molecule fluorophores are essential tools for biological imaging,which is akey method in modern life sciences. With the rapid development of novel fluorescence-based imaging techniques,t he design and chemical synthesis of fluorophores with improved photophysical properties has experienced an enormous revival. [1] In particular, superresolution imaging techniques,s uch as single-molecule localization microscopy (SMLM), like direct stochastic optical reconstruction microscopy (dSTORM) or photoactivated localization microscopy (PALM), are heavily dependent upon individual properties of the applied organic fluorophores. [2] Active control of emissive and dark states required for the localization of individual emitters in SMLM typically requires high intensity illumination, addition of redox reagents or UV exposure,r endering SMLM in al ive specimen challenging. [3] Spontaneously blinking fluorescent probes [4] based on the silicon rhodamine (SiR) scaffold [5] have recently been developed to enable live-cell SMLM under physiological conditions.Inthis approach, ahydroxymethyl nucleophile causes the reversible intramolecular formation of an onchromophoric spiroether.T his chemical equilibrium of hydroxymethyl SiR (HM-SiR) leads to stochastically occurring transitions between the excitable quinoid and the nonexcitable spiroether isomer. Hence,s trenuous manipulation of the fluorophore excited state to populate dark states is not required. This enables SMLM recordings without additional stimulation of on-off transitions.
Thequality of image reconstruction in SMLM is governed by blinking kinetics,l abeling density,a nd signal specificity. [6] In STORM and PA LM the first is controlled by adapting excitation power and redox buffer properties.I nc ontrast, blinking kinetics of spontaneously blinking fluorophores is an intrinsic property that can be influenced by ac hange in the local microenvironment;L ocalizing HM-SiR to lipid membranes,for instance,has resulted in improved performance in SMLM. [7] Regarding labeling density and signal specificity, fast, quantitative,and highly selective labeling reactions with aminimum of unspecific binding are crucial. Among bioconjugation methods,bioorthogonal reactions such as the inverse electron demand Diels Alder reaction (DA inv )b etween 1,2,4,5-tetrazines and ring-strained alkenes and alkynes have lately received significant attention. DA inv is particularly popular due to its fast kinetics,c hemoselectivity,a nd biocompatibility,making it well-suited for live-cell applications. [8] Therefore,t etrazine-modified small-molecule fluorophores have abroad applicability as they can be installed at virtually any dienophile-tagged biomolecule.They allow specific labeling in combination with protein tags, [9] peptide tags, [10] and unnatural amino acids, [11] pushing the label size to aminimum. Moreover,D A inv enables labeling various biomolecules other than proteins like nucleotides, [12] sugars, [13] and lipids [14] as well as to exploit small-molecule-mediated targeting. [15] Additionally,tetrazine-based labeling can be used to reduce unspecific signal;Carefully designed tetrazine probes have been shown to undergo an increase in fluorescence when the tetrazine is consumed in DA inv ,t hat is,w hen the dye label is covalently linked to its target structure. [15a,16] This is avaluable additional feature for live-cell fluorescence microscopy because it obviates the need for extensive excess dye wash-out and dramatically reduces background signal. [17] This is particularly advantageous for SMLM as the localization precision is affected by background signal. [18] Overall, we reason that ab roadly applicable probe for SMLM would comprise all of the above-mentioned features.I ts hould be as small as possible,self-blinking, fluorogenic,r eadily suited for bioconjugation, and equipped with generally favorable photophysical properties,such as high brightness and photostability.
Here,wereport the first merger of all these properties in the fluorogenic,f ar red-emitting, self-blinking silicon rhodamine f-HM-SiR.T his tetrazine-derivatized HM-SiR is initially strongly quenched and shows fluorescence enhancement upon bioconjugation in DA inv .W er eport the synthesis and photophysical properties of the novel fluorogenic dye and demonstrate its application in live-cell super-resolution microscopy.T he tetrazine functionalization was utilized to attach f-HM-SiR to intracellular targets by fast and selective click chemistry in living cells and to visualize intracellular dynamics by SMLM. Importantly,t he fluorogenicity of f-HM-SiR allows for minimal-or no-wash procedures in live-cell imaging while its self-blinking feature minimizes phototoxicity in SMLM experiments.

Results and Discussion
First, we examined synthetic strategies to access fluorophore probes that combine both tetrazine and the desired structural hydroxymethyl motif in the SiR scaffold. Synthesis with tetrazines requires mild reaction conditions as they are highly base sensitive and react with strong nucleophiles. Therefore,amild Lewis-acid-mediated Friedel-Crafts reaction [16e] was selected as the key step for the assembly of the HM-SiR derivative.W es et out to test the feasibility of this strategy for the synthesis of the simple unfunctionalized HM-SiR without tetrazine ( Figure 1) and found that this four step route using methoxymethyl (MOM) protection group chemistry enabled the synthesis of HM-SiR at 22 %o verall yield (Supporting Information, Scheme S2). This approach complements previous strategies relying on the addition of aryl lithium reagents to Si-anthrones [4a] and could offer ashort and mild synthesis route for similar fluorophores in the future. Our attention then turned to the synthesis of the tetrazinemodified HM-SiR derivative,inthe following termed fluorogenic HM-SiR (f-HM-SiR). Forthis purpose,wesynthesized the appropriate tetrazinyl benzaldehyde (5,S upporting Information, Scheme S1) as electrophile for the Friedel-Crafts reaction. Conversion with the diarylsilane (6,Supporting Information, Scheme S1) gave access to the desired f-HM-SiR,w hich carries both a3 -hydroxyl and a6 -tetrazine substituent ( Figure 1). Both substituents are crucial for the intramolecular modulation of the probesspectral properties: While fluorescence of f-HM-SiR is strongly quenched by the tetrazine moiety (Figure 1a), it is restored by conversion with dienophile-equipped biomolecular targets in DA inv (Figure 1a,b). Ther esulting Diels-Alder product of f-HM-SiR still exhibits the pH-dependent equilibrium between absorbing (open) and non-absorbing form (closed spiroether) and thereby spontaneously switches between fluorescent and non- fluorescent states.C onsequently,e xternal control of the emitter density,w hich is otherwise essential in SMLM, becomes obsolete.
In order to evaluate the fluorogenicity of f-HM-SiR in DA inv ,i tw as treated with bicyclo[6.1.0]non-4-yne (BCN) dienophile,which resulted in a10-fold fluorescence enhancement upon reaction ( Figure 1b). Furthermore,wedetermined as ubstantial increase in brightness of the respective isolated cycloadduct DA inv -Product (Supporting Information, Table S1). To assess the self-blinking properties of f-HM-SiR, we studied the reversible spirocyclization reaction. Both, the quinoid and the spiroether isomer of f-HM-SiR,c ould be observed in 1 HNMR (Supporting Information, Figure S3). ThepH-dependent equilibrium between the two isomers was investigated by absorbance (Supporting Information,Figure S2) and fluorescence (Figure 1c)s pectroscopy.H ere,w e determined an equilibrium constant of pK cycl = 4.0(AE 0.1), indicating that the non-fluorescent spiroether form of f-HM-SiR significantly prevails at physiological conditions (99.9 %). [4a] Thei solated DA inv -Product showed as ignificant shift to pK cycl = 5.2(AE 0.1), indicating that the proportion of the fluorescent quinoid form of the target-bound f-HM-SiR is increased but remains below 1% at physiological pH (99.4 % spiroether). Theh igher abundance of the quinoid isomer of target-bound f-HM-SiR infers that the emissive state will be more populated compared to unreacted dye leading to an overall reduction of unspecific signal. Consequently,t wo mechanisms of fluorogenicity could be observed:o ne corresponds to the loss of the quenching tetrazine,the other to the changed tendency of the product to form the spiroether. Following up on those results,w es et out to evaluate the general suitability of f-HM-SiR for bioconjugation. Apurified HaloTag-EGFP was modified with HaloTag ligand-BCN (HTL-BCN) and subsequently reacted with f-HM-SiR.I ngel fluorescence showed specific labeling,w hile the pHdependent emission of the EGFP-bound dye was retained (Supporting Information, Figure S7).
Based on these promising photophysical characteristics, we moved on to apply the probe in live-cell wide-field fluorescence imaging to further evaluate the fluorogenicity of f-HM-SiR in the context of cellular labeling. Given the flexibility of tetrazine-based click chemistry,w et ested av ariety of procedures ( Figure 2a). Protein labeling was demonstrated using the enzymatic self-labeling HaloTag as

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Research Articles 806 www.angewandte.org af usion with histone H2A or mitochondrial import receptor subunit TOM20 in combination with HaloTag ligand dienophiles.F urther,w eu sed triphenylphosphonium (TPP), asmall-molecule organelle marker,tolocalize the respective dienophile conjugate TPP-BCN to the mitochondrial matrix. [19] Staining and fluorescence imaging of nuclear and mitochondrial targets were carried out in HeLa cells.C ells were transiently transfected with H2A-HaloTag and treated with HTL-BCN (10 mm)for 30 min. Additional staining of unmodified HaloTag proteins with HTL-TMR subsequent to HTL-BCN incubation served as at ransfection and co-localization control.
After wash-out of excess HTL-BCN,c ells were incubated with f-HM-SiR and imaged without dye removal. Averaging 2000 frames of the recorded blinking signal resulted in an image with good co-localization to the HTL-TMR control (Figure 2b). In the same fashion, mitochondrial labeling with (E)-cyclooct-2-en-1-ol (TCO * )a sd ienophile was equally successful (Supporting Information, Figure S8). Similarly,H eLa cells treated with TPP-BCN and f-HM-SiR show expected mitochondrial structures (Figure 2c). Costaining with TOM20-HaloTag and HTL-TMR verifies the specificity of the mitochondrial staining at good contrast. This demonstrates the efficient background suppression when using f-HM-SiR in live-cell fluorescence microscopy.Incontrol experiments without HTL-BCN we noted slight unspecific signal upon addition of f-HM-SiR (Figure 2b). We attribute this to the presence of lysosomes with luminal pH % 4. In this context, we validated the previously observed fluorogenicity of f-HM-SiR in comparison to other silicon rhodamine dyes like HM-SiR (lacking at etrazine moiety), tetrazine-substituted SiR-Tz,a nd bare SiR-COOH (Supporting Information, Figure S6). To this end, we incubated wild type HeLa cells with the respective probes and quantified cellular fluorescence intensities (Figure 2d,e). Thea verage fluorescence from HeLa cells incubated with f-HM-SiR was comparable to untreated cells,while the other three SiR dyes caused significantly higher signal. Theo bserved signal intensities directly correlate to the residual background resulting from nonreacted dye excess in labeling experiments.T hese tendencies were even more pronounced under no-wash conditions (Supporting Information, Figure S9). This highlights the improved background suppression of f-HM-SiR compared to existing SiR derivatives owing to the close proximity of tetrazine to the chromophoric center.
Unspecific signal reduces contrast and structure representation quality in fluorescence imaging.I n SMLM it additionally leads to decreased localization precision. Therefore,e fficient background suppression is particularly important here.T hus,s mall-molecule probes bearing bioorthogonal attachment functionalities that come along with fluorogenic properties possess great potential for super-resolution microscopy.I nt he past, super-resolution microscopy with fluorogenic tetrazine dyes has been achieved in fixed cells. [16g,h] Encouraged by the advantageous features of f-HM-SiR in live-cell labeling,w es tudied its suitability for SMLM in living cells.HeLa cells transiently transfected with TOM20-HaloTag were incubated with HaloTag ligand-TCO * (HTL-TCO * ), washed and labeled with f-HM-SiR (2 mm). To minimize background signal for livecell SMLM excess fluorophore was washed out in this experiment. Imaging under highly inclined illumination con- Figure 3. Spontaneous blinking of f-HM-SiR enables SMLM with improved resolution and reduced background. a) COS-7 cells transiently expressing TOM20-mCherry-HaloTagw ere incubated with HTL-TCO * (10 mm), washed, and labeled with f-HM-SiR (2 mm)for SMLM imaging. See Movie S1 in the Supporting Information.b)Average projection of 500 raw data frames used for reconstruction in (a). c) Merged zoom-ins of boxed regions in (a) (red) and (b) (green). d) Line profile along boxed region in (c) comparing normalized intensities in averaged raw data (green) and SMLM reconstruction (red). e) Averaged cross-sectional profiles of labeled mitochondria AE 1 standard deviation, n = 16. Individual profiles were aligned to minimum between peaks (see methods and Figure S11 in the SupportingInformation for all profiles). f) Peak-to-peak distancesi ncross-sectional profiles shown in (e). g) Long-term stability of f-HM-SiR labeled TOM20-HaloTag in fixed COS-7 cells. Mean localizations per frame normalizedt onumbero flocalizations in first frame (black line) AE 1s tandard deviation, n = 8. h) Background localization rate for HM-SiR and f-HM-SiR in non-transfected HeLa cells, n = 20. Scale bars:a,b) 5 mm, c) 1 mm. ditions allowed the localization of individual spontaneously blinking emitters.T he image reconstructed from 500 frames shows the expected structure for outer mitochondrial membrane localized TOM20 (Figure 3a)and enabled us to follow dynamics at sub-diffraction resolution (Supporting Information, Movie S1). Comparison with an image averaged over 500 frames in Figure 3b highlights the achieved resolution improvement of f-HM-SiR in live-cell SMLM. From the reconstructed image,i ti sp ossible to differentiate the outer membrane of the mitochondrial network (Figure 3b,d) and we were able to determine amean mitochondrial diameter of about 375 nm from 16 cross-sectional profiles (Figure 3e,f and Supporting Information, Figure S11). SMLM image sequences were recorded at alaser intensity of 1kWcm À2 at 640 nm, at which we did not observe any considerable phototoxicity, even in long time experiments (Supporting Information, Movie S5). As these moderate laser intensities mitigate photobleaching,w ew ere interested in the capability of f-HM-SiR for long-term SMLM. In order to genuinely quantify the number of localizations over time unbiased by cellular movement, we chemically fixed the cells after addition of the probe.T he average number of localizations sampled over 200 ss howed no significant decrease (Figure 3g). At the same time,s uper-resolution images reconstructed from frame subsets do not indicate any noticeable loss in reconstruction quality.F inally,w et ested how the fluorogenicity of f-HM-SiR affects the localization of unspe-cifically bound label in comparison to the non-fluorogenic HM-SiR.Comparison of the background localizations within ac ell per area and time unit shows am ore than 2-fold reduction for f-HM-SiR (Figure 3h). Together,t hese observations suggest ah igh suitability of f-HM-SiR to probe cellular dynamics in live-cell SMLM.
To fully make use of excitation-independent on-off switching of f-HM-SiR,w ep erformed long-term SMLM experiments,t his time omitting unnecessary dye wash-out in order to fully utilize the probesf luorogenicity.F irst, we turned our attention to the protein H2A-HaloTag, localized in the nucleus.A fter incubating HeLa cells with HTL-BCN and subsequent linker wash-out, cells were labeled with f-HM-SiR.M ere buffer medium change and direct image acquisition was sufficient to afford high resolution in the reconstructed image (Figure 4a). In contrast to an average projection (Figure 4b,c), only the super-resolved image allows for an identification of individual H2A proteins and variations in local H2A density.Subsequently,tovisualize amore dynamic cellular target we localized the dienophile to the mitochondrial matrix with TPP.H eLa cells were incubated with TPP-BCN,washed and stained with f-HM-SiR.Medium was replaced once to minimize extracellular deposition of the probe before imaging the cells.W ec hoose am oderate excitation power of 1kWcm À2 to facilitate long time imaging. As TPP accumulates in the mitochondrial matrix, the reconstructed image in Figure 4d shows the expected pattern of homogeneously filled tubes.T he averaged intersection from eleven cross-sectional profiles (Figure 4e)has amedian FWHM of 346 nm (Figure 4f), which is in accordance with the median of the peak-to-peak distance of 375 nm determined for mitochondria labeled at the outer membrane (See Figure 3e,f and Figure S11 cinthe Supporting Information for adirect comparison).
We were also able to follow mitochondrial dynamics over 200 swith atemporal resolution of 10 sasshown in Figure 4g and Movie S2 in the Supporting Information. Considering the inherent trade-off between high excitation power (resulting in high photon output and thus localization precision) and cell viability,t he achieved spatial resolution is reasonable (for photon count and localization uncertainty see Figure S10 in the Supporting Information). Thet ime-resolved super-resolution imaging was not limited by fluorophore bleaching, emphasizing the performance of f-HM-SiR.Beyond visualizing motion dynamics,i tw as also possible to observe mitochondrial fusion and fission events (Figure 4g). Te mporal resolution in SMLM depends on the time during which emitter localizations are accumulated for reconstructing as uper-resolved image.I ncreased time resolution can be achieved by shortening the accumulation time at the cost of reconstruction quality,t hat is,t he captured localization density along the imaged structures.
To capture fast cellular dynamics,w ea dditionally made use of the exact time at which individual localizations were recorded. We visualized this additional information by color coding the appearance time of localizations within each reconstruction from red (beginning) over green to blue (end) of each reconstruction window (Figure 4h and Supporting Information, Movie S3). In this representation, structures which remain quasi stationary during data recording for one reconstruction will appear green while forming structures will be colored red and collapsing structure will be colored blue (Figure 4h,a rrows). Thef act that most mitochondria in the shown example are colored green further indicates that the achieved time resolution of 10 si ss ufficient to capture the observed mitochondrial dynamics.
Overall, these results show that the combination of fluorogenicity and self-blinking in f-HM-SiR greatly improves the potential of SMLM applications for live-cell experiments. While fluorogenicity significantly reduces artifacts from unspecific probe deposition, self-blinking enables live-cell SMLM over relatively long time scales due to the low excitation power required.

Conclusion
In summary,w ep resent the first combination of fluorogenic and self-blinking properties in as ingle fluorescent probe and demonstrate its advantage for live-cell localization microscopy.T he tetrazine moiety strongly quenches the fluorescence prior to reaction and enables fluorogenic bioconjugation to various dienophile-modified cellular targets. f-HM-SiR exhibits excellent cell permeability,h igh brightness,a nd photostability,a ll of which are important criteria for application in live-cell imaging. Exploiting the self-blinking properties,w ed emonstrate its application in live-cell SMLM without the necessity of stabilizing buffers or high excitation power. f-HM-SiR allows live-cell localization microscopy after simple media replacement. SMLM imaging of intracellular dynamics can be performed over long time periods and at ah igh spatiotemporal resolution. We expect that our molecular design will stimulate the future development of multifunctional fluorescent probes as tailor-made tools for bioimaging.