Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant (K L-Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF526)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Introduction

Small-molecule fluorophores are fundamental tools for biological research.[1,2] The century-old[3,4] rhodamine dyes remain the most useful class of small-molecule fluorophores and serve as scaffolds for a variety of useful imaging probes including biomolecule labels, cellular stains, and environmental indicators.[5,6] This broad utility can be attributed to three key aspects of rhodamine dyes:[7−20] (i) exceptional brightness and photostability; (ii) a broad palette of spectral properties accessed through straightforward structural modifications; and (iii) the equilibrium between the colorless, nonfluorescent lactone (L) and the colored, fluorescent zwitterion (Z; equilibrium constant: KL–Z). Tuning KL–Z lower—toward the lipophilic nonfluorescent lactone form—can improve cell-permeability[20] and yield “fluorogenic” ligands,[14,16,20−24] molecules that show substantial increases in absorption and fluorescence upon binding their cognate biomolecular targets.

The inherent fluorescence increase of fluorogenic ligands is particularly useful for biological imaging as such compounds remain quiescent until bound to their target, making them universal platforms for imaging and sensing.[25] This property can avoid the need to wash out excess ligand[20] and allow exchange of labels to circumvent photobleaching.[26] Many useful fluorogenic ligands exploit solvatochromism,[27,28] pH sensitivity,[29,30] photoinduced electron transfer (PeT),[31] or quencher ejection[32] to translate the binding event into a change in fluorescence intensity. Recently, the tetramethyl-Si-rhodamine (SiR, 1; Figure a) has emerged as a remarkably versatile fluorogenic dye, demonstrated first by Johnsson and co-workers.[14] Compound 1 exhibits far-red wavelengths with an absorption maximum (λabs) of 643 nm, a fluorescence emission maximum (λem) of 662 nm, a modest fluorescence quantum yield (Φ = 0.41, Table ), and excellent photostability. An important feature of SiR-based ligands is the relatively low KL–Z value (0.0034), which means the dye preferentially adopts the nonfluorescent lactone in aqueous solution (Figure a). This results in a lower extinction coefficient in aqueous solution (εw = 28 200 M–1 cm–1) but makes SiR-based ligands highly cell-permeable due to the increased fraction of the lipophilic lactone. The lower KL–Z also makes SiR compounds fluorogenic as binding to biomolecular targets often shifts the equilibrium toward the fluorescent zwitterionic form (Figure b).

Figure 1

Fluorogenicity of rhodamines. (a) Lactone–zwitterion equilibrium of SiR (1). (b) Mechanism of improved cell-permeability and fluorogenicity of 1. (c) Structures of Janelia Fluor dyes 2–7. (d) Absorption at λabs vs SDS concentration for 1 and 2 in 20 mM Na2HPO4, pH 7.0; error bars show ± s.e.m.; shading indicates [SDS] above the critical micelle concentration (c.m.c.).[36] (e) Change in fluorescence over the basal fluorescence (ΔF/F0) of HaloTag ligands 1–10 upon labeling purified HaloTag protein vs the KL–Z of the corresponding free dyes 1–10; solid line indicates a linear fit (R2 = 0.90); shading indicates ΔF/F0 = 5–10 and KL–Z = 10–2–10–3.

Table 1

Properties of Rhodamine Dyes

The initial development of SiR-based labels focused on fluorogenic ligands for genetically encoded self-labeling tags like the HaloTag and the SNAP-tag[14] but soon expanded to stains for endogenous structures like microtubules, F-actin, and DNA,[22−24] as well as sensors for disparate analytes.[12,33,34] The cell-permeability, brightness, photostability, and far-red wavelengths of SiR ligands have enabled advanced imaging experiments using structured illumination microscopy (SIM) and stimulated emission depletion (STED) imaging.[14] The low KL–Z of SiR also spurred the development of hydroxymethyl (HM) derivatives of SiR that spontaneously blink under physiological conditions and are useful for single-molecule localization microscopy (SMLM).[35] In another extension, our laboratory discovered that replacing the N,N-dimethylamino groups in fluorophores with four-membered azetidine rings was a general strategy to improve properties. Applying this strategy to SiR yielded a brighter and more fluorogenic dye: “Janelia Fluor” 646 (JF646, 2; λabs/λem = 646 nm/664 nm, εw = 5000 M–1 cm–1, Φ = 0.56, KL–Z = 0.0012).[16] We could fine-tune this Si-rhodamine by incorporating 3-fluoroazetidine into the structure, yielding JF635 (3, λabs/λem = 635 nm/652 nm, εw ≈ 400 M–1 cm–1, Φ = 0.54), which exhibited a low KL–Z value (<0.0001) and high fluorogenicity. This universal approach could be applied to carborhodamine and standard oxygen-containing rhodamine scaffolds to yield bright fluorescent and fluorogenic dyes across the visible spectrum (4–7, Figure c, Table ).[16,20]

We set out to expand the palette of fluorogenic molecules with the goal of creating a green-emitting version of SiR. We first investigated the relationship between KL–Z of dyes 1–7 and the fluorogenicity of their respective HaloTag ligands to determine a quantitative framework for the rational design of new fluorogenic rhodamine dyes. We discovered that the KL–Z is sufficient to predict fluorogenicity, and we determined that KL–Z < 10–2 was an appropriate threshold for the design of highly fluorogenic ligands, validating this with the known Si-rhodamine 110 (SiR110, 8). We next turned to the oxygen-containing rhodamine scaffold, first preparing derivatives of a tuned dye, JF552 (9), and then the rationally designed fluorophore JF526 (10). This dye shows similar fluorogenicity to SiR, allows the creation of fluorogenic labels and stains, and can be further modified to a spontaneously blinking derivative that enables facile localization microscopy in cells.

Results and Discussion

Mechanism of SiR/JF646 Fluorogenicity

In previous studies, the fluorogenicity of ligands based on SiR and other rhodamines was attributed, in part, to the formation of weakly fluorescent aggregates.[14,37] The key evidence for this mechanism was the large increase in absorption that occurred when the detergent sodium dodecyl sulfate (SDS) was added to aqueous solutions of SiR-based compounds, presumably disaggregating the dye. We investigated this phenomenon and found it was detergent-specific; incubation of SiR (1) and JF646 (2) with commonly used laboratory detergents showed absorption increases only in the presence of SDS (Figure S1a). We further characterized the interaction between the Si-rhodamine dyes 1 and 2 and SDS by measuring the absorption of 1 or 2 versus [SDS] and observed the absorption increase only above the critical micelle concentration (c.m.c.) of SDS (Figure d). These results suggest another mechanism for this observed increase in absorption, where dyes 1 and 2 interact with the negatively charged SDS-micelle surface, thereby stabilizing the zwitterionic form and giving the observed increase in absorption. This is further supported by the bathochromic shift in λabs observed for 1 and 2 in the presence of SDS (Figure S1b,c), which is characteristic for rhodamine–SDS-micelle complexes.[38] These data reveal a problem with the use of SDS solutions to determine the spectral properties of fluorogenic dyes as the measurement is strongly concentration- and dye-dependent (Figure d). We instead use a solution of strong acid in alcohol solvents to shift the equilibrium to the open form by protonation of the o-carboxyl group. This decades-old procedure[39,40] allows estimation of the maximal extinction coefficient (εmax) and determination of KL–Z (Supporting Information).

On the basis of these results, we hypothesized that the low KL–Z value was the primary driver behind the fluorogenicity of SiR and similar fluorogenic rhodamines. In this mechanism, the fluorophores preferentially adopt the colorless lactone form in solution, largely independent of concentration, dye structure, or ligand moiety. Binding to the biomolecule shifts the equilibrium to the fluorescent zwitterionic form through steric interactions and other changes to the local chemical environment. Aggregation of such compounds can still occur, particularly for high concentrations of dyes that prefer the lipophilic lactone form, but this is a consequence of the low KL–Z and not the causal element behind the observed fluorogenicity. To test this premise, we examined the relationship between KL–Z for a series of fluorescent dyes (1–7, Figure c, Table ) and the increase in fluorescence of the corresponding HaloTag ligands upon conjugation to purified HaloTag protein (1–7, Figure e, Figure S1d). The KL–Z was determined using ε values measured in 1:1 dioxane:water (εdw) to ensure a broad distribution of values.[20] We found an inverse relationship between KL–Z and fluorogenicity, showing that KL–Z is sufficient to predict the increase in fluorescence of different rhodamine dyes upon binding the same target. This increase in fluorescence is primarily driven by a rise in absorption; chromogenicity is correlated with both KL–Z and fluorogenicity (Figure S1e,f). This trend holds across different rhodamine scaffolds including Si-rhodamines (1–3), carborhodamines (4 and 5), and classic, oxygen-containing rhodamines (6 and 7, Figure c). This inverse relationship yielded a simple rubric: a dye with KL–Z = 10–2–10–3 should yield a HaloTag ligand with 5–10-fold fluorogenicity (Figure e).

Si-Rhodamine 110

In previous work, we developed strategies to fine-tune the properties of rhodamines by incorporating substituted azetidines into the dye structure.[20] In particular, we developed the bright, fluorogenic carborhodamine JF585 (5, Figure c; λabs/λem = 585 nm/609 nm, Φ = 0.78) by replacing the azetidine rings in JF608 (4; λabs/λem = 608 nm/631 nm, Φ = 0.67) with 3,3-difluoroazetidine; the KL–Z–fluorogenicity trend holds for these dyes (Figure e). As an alternative strategy to create orange-emitting fluorogenic dyes, we investigated the Si-containing analogue of rhodamine 110 (8), which has been described as a scaffold for fluorogenic enzyme substrates in the patent literature[41] but has not been used as a fluorescent label in cellular experiments. We synthesized this compound using the Pd-catalyzed cross-coupling of the Si-fluorescein ditriflate (11) with t-butyl carbamate followed by deprotection with TFA (Figure a).[16,42] Compound 8 exhibited λmax/λem = 587 nm/609 nm, representing a ∼50 nm hypsochromic shift relative to SiR (1; λabs/λem = 643 nm/662 nm, Table ); we named this dye Si-rhodamine 110 (SiR110). It has an increased fluorescence quantum yield (Φ = 0.53) compared to SiR (Φ = 0.41, Table ), but a comparable KL–Z = 0.0043.

Figure 2

Synthesis and testing of SiR110. (a) Synthesis of SiR110 (8). (b) Synthesis of SiR110–HaloTag ligand (8). (c) Absorption spectra of 8 (5 μM) in the absence (black) or presence (orange) of excess HaloTag protein. (d) Confocal image of U2OS cells expressing histone H2B–HaloTag fusion protein and labeled with 8. Scale bar: 20 μm. (e) Relative photostability of 8 and JF585–HaloTag ligand (5) in live cells.

On the basis of our KL–Z versus fluorogenicity relationship (Figure e), we predicted that ligands based on 8 would be fluorogenic. This was a noteworthy test of our hypothesis since SiR110 has similar KL–Z values to SiR but a different structure—lacking the four hydrophobic CH3 groups in 1. We prepared the SiR110–HaloTag ligand (8, Figure b) starting from the 6-carboxy-Si-fluorescein ditriflate methyl ester (13).[43] Cross-coupling afforded the Boc-protected SiR11014, which was saponified to yield free acid 15. Formation of the N-hydroxysuccinimidyl ester in situ, amidation with the HaloTag ligand (16), and deprotection with TFA yielded 8. We observed a 7-fold increase in absorption and ΔF/F0 = 9 after conjugation of 8 to HaloTag protein, slightly higher than SiR–HaloTag ligand (1; ΔF/F0 = 5; Figure c, Figure S1d) and in line with the KL–Z trend (Figure e). This dye was an excellent label for live-cell imaging experiments (Figure d) and showed higher photostability than our previously described orange-emitting fluorogenic JF585–HaloTag ligand (5, Figure e). These results further support our hypothesis that a low KL–Z value is a primary factor for rhodamine fluorogenicity.

Janelia Fluor 552

We then turned to the standard, oxygen-containing rhodamine scaffold exemplified by JF549 (6, Figure c; λabs/λem = 549 nm/571 nm, Φ = 0.88). Creating a fluorogenic rhodamine is challenging since this dye type strongly prefers the fluorescent zwitterionic form; JF549 exhibits a high KL–Z = 3.5, which is >102-fold higher than the apparent KL–Z threshold for a fluorogenic ligand (Figure e). Incorporation of 3,3-difluoroazetidine motifs into the JF549 structure yields JF525 (7), which shows a lower KL–Z = 0.068 and elicits ∼25 nm hypsochromic shift with similar brightness (λmax/λem = 525 nm/549 nm, Φ = 0.91; Table ). This KL–Z tuning is insufficient to achieve substantial fluorogenicity although the JF525–HaloTag ligand (7) exhibits higher cell-permeability than the JF549–HaloTag ligand (6)[20] and can cross the blood–brain barrier.[44] Since we exhausted the available substitutions on the azetidine ring with JF525, we sought a complementary approach for further modulating KL–Z. We recently reported a JF549 analogue with fluorine atoms installed at the 2′ and 7′ positions on the xanthene ring. This modification reduced the KL–Z by 5-fold (KL–Z = 0.70) with only a minor shift in spectral properties (λabs/λem = 552 nm/575 nm, Φ = 0.83; Table ).[19] The resulting dye, Janelia Fluor 552 (JF552; 9), is of particular interest because it could show improved cell-permeability relative to JF549 and could be further modified to create a fluorogenic rhodamine.

We aimed to develop a general synthetic strategy for JF552 derivatives that would also allow late-stage incorporation of different azetidinyl functionality. Unfortunately, our standard Pd-catalyzed cross-coupling approach for rhodamines[42,45] gave low yield (<5%) when starting with 2′,7′-difluorofluorescein ditriflate due to the instability of the o-fluorophenyl triflate groups (data not shown). To circumvent this problem, we devised an alternative synthesis starting with 3-bromo-4-fluorophenol (17, Scheme a). Acid-mediated condensation of 17 and phthalic anhydride (18) yielded dibromofluoran 19. Pd-catalyzed cross-coupling with azetidine (20) provided JF552 (9). To introduce a carboxyl group for bioconjugation, we condensed phenol 17 and with trimellitic anhydride (21) to give an isomeric mixture; crystallization from 9:1 toluene:pyridine yielded the 6-carboxy isomer 22. This was protected as the t-butyl ester using N,N-dimethylformamide di-t-butyl acetal (23) to yield 24, followed by Pd-catalyzed cross-coupling with azetidine (20) to provide 6-carboxy-JF552t-butyl ester (25). Deprotection of 25 with TFA yielded carboxylic acid 26, and subsequent conjugation to the HaloTag ligand amine (16) gave JF552–HaloTag ligand 9 (Scheme b). On the basis of these results, we briefly attempted to transform 2′,7′-difluorofluorescein ditriflates to their respective dibromofluorans using Ru catalysts.[46] This reaction was successful for the synthesis of dibromide 19 but gave poor yields of t-butyl ester 24 (data not shown).

Scheme 1

Syntheses of JF552 and JF549 Derivatives: (a) Synthesis of 9, (b) Synthesis of 9, and (c) Synthesis of 6 and 9

We then evaluated JF552 ligands as cell-permeable fluorescent labels, comparing the JF552–HaloTag ligand (9) to JF549–HaloTag ligand (6, Figure S1d) in yeast expressing histone H2A.Z–HaloTag protein fusion. Although 6 showed relatively poor labeling (Figure a), the 9 molecule showed a high fluorescence signal from the yeast nuclei under the same imaging conditions (Figure b). This result is expected on the basis of the smaller KL–Z of JF552, which should improve cell-permeability. We also synthesized the trimethoprim (TMP) conjugates of JF549 and JF552 (6 and 9) by reacting the 6-carboxy derivatives of these dyes (26 and 27, respectively) with the amino-TMP 28 (Scheme c). TMP conjugates selectively bind to Escherichia coli dihydrofolate reductase (eDHFR), and this labeling strategy can be used for live-cell imaging.[47] We expressed histone H2B–eDHFR fusions in U2OS cells and labeled with 6 or 9. Nuclei labeled with JF552-based 9 were 8-fold brighter than cells labeled with 6, giving images with higher signal-to-background (Figure c,d). These results support our hypothesis that even modest decreases in KL–Z can improve cell-permeability across different cell-types and labeling strategies.

Figure 3

JF552 ligands show improved cell-permeability. Overlay of fluorescence and bright-field images of yeast cells expressing a histone H2A.Z–HaloTag fusion protein and labeled with 6 (a) or 9 (b). Overlay of fluorescence and bright-field images of U2OS cells expressing histone H2B–eDHFR fusion protein and labeled with 6 (c) or 9 (d). Scale bars for all images: 5 μm.

Janelia Fluor 526

We then combined the structural modifications of 7 (KL–Z = 0.068) and 9 (KL–Z = 0.70), positing that the two complementary alterations could additively shift the KL–Z < 10–2, thus yielding a fluorogenic rhodamine. To prepare the free dye we used Pd-catalyzed cross-coupling to attach 3,3-difluoroazetidine (29) to dibromide 19 yielding the hexafluorinated rhodamine 10 (Figure a). We termed the resulting compound Janelia Fluor 526 (JF526, λabs/λem = 526 nm/550 nm, Φ = 0.87), which exhibited the desired additive effect on the lactone–zwitterion equilibrium (KL–Z = 0.0050; Table ). We then synthesized the 6-carboxy derivative using a route akin to the JF552 ligands (Scheme b): cross-coupling of dibromide 24 with azetidine 29 to give t-butyl ester 30 followed by deprotection with TFA to give 31. This compound could be coupled to amine-containing ligand moieties to yield JF526–HaloTag ligand (10) and JF526–SNAP-tag ligand (10, Figure b, Scheme S1a). We compared this with the previously described JF525–HaloTag ligand[20] (7) in vitro and in live cells. Although 7 exhibited a modest <2-fold increase of absorption upon conjugation to purified HaloTag protein, 10 exhibited a substantial 9-fold increase (Figure S2a,b), again following the KL–Z–fluorogenicity trend (Figure e). JF526 showed superior signal-to-background compared to JF525 in “no-wash”, live-cell imaging experiments using either the HaloTag (Figure d,e) or SNAP-tag expressed as histone H2B fusion proteins (10 versus 7, Figure f,g).

Figure 4

Synthesis and no-wash imaging of JF526 ligands. Synthesis of JF526 (a) and JF526 (b) ligands. (c) Structures of JF525 and JF526–HaloTag and SNAP-tag ligands. Confocal images of COS7 cells expressing a histone H2B–HaloTag fusion protein and labeled with 500 nM JF525–HaloTag ligand (7, d) or JF526–HaloTag ligand (10, e). Confocal images of COS7 cells expressing histone H2B–SNAP-tag fusion protein and labeled with 1 μM JF525–SNAP-tag ligand (7, f) or JF526–SNAP-tag ligand (10, g). Scale bars for all images: 5 μm.

We then prepared other JF526 ligands (Figure b, Scheme S1b–d) to demonstrate the general utility of this dye for multicolor advanced microscopy experiments. On the basis of previous work with SiR (1), JF646 (2), and other dyes,[22−24,48,49] we synthesized the following conjugates: JF526–Hoechst (10) to stain DNA, JF526–Taxol (10) to image microtubules, and JF526–pepstatin A (10) to visualize lysosomes (Figures b and 5a). 10 showed a modest increase in absorption (<2-fold) and a large increase in fluorescence quantum yield (10-fold) upon binding purified AT-rich DNA (Figure S2c,d), showing that chromogenicity can be magnified by other photophysical effects. The JF526–Taxol (10) also showed increased fluorescence upon binding to polymerized tubulin in vitro; this fluorogenicity was comparable to that of “SiR-tubulin” (1)[22] and higher than JF525–Taxol (7; Figure S2e–h and Scheme S1e). Live-cell imaging with these compounds showed specific staining, enabling one-, two-, and three-color no-wash imaging experiments (Figure b–d). We then used JF526 ligands in advanced microscopy. We performed two-color 3D-SIM[50] in live cells using JF526–pepstatin A (10) and JF646–Hoechst[49] (2, Figure a). JF526 also enabled multicolor super-resolution STED microscopy[51] of microtubules using 10 depleted with 775 nm (Figure b, Figure S2i–k). Notably, the compatibility of JF526 with the standard 775 nm depletion line facilitated three-color live-cell STED imaging using JF526–Taxol (10, microtubules), JF646–SNAP-tag ligand (2) targeted to Sec61β (endoplasmic reticulum), and JF585–HaloTag ligand (5) targeted to TOMM20 (mitochondria, Figure c). Finally, the JF526–pepstatin A (10) could be used for live-cell, two-color lattice light-sheet microscopy[52] with 2 (Figure d).

Figure 5

Extending the repertoire of JF526 ligands. (a) Structures of JF526 ligands. (b) Confocal image of live U2OS cells stained with JF526–Hoechst (10). (c) Confocal image of mouse primary hippocampal neurons stained with JF526–Taxol (10) and JF646–Hoechst (2). (d) Confocal image of U2OS cells expressing histone-H2B–HaloTag fusion protein and labeled with JF526–pepstatin A (10), JF585–HaloTag ligand (5), and “SiR–tubulin” (1). All images were acquired without washing. Scale bars: 5 μm.

Figure 6

Advanced microscopy imaging using JF526. (a) Confocal and SIM images of mouse primary hippocampal neurons stained with 10 and JF646–Hoechst (2). (b) Confocal and STED microscopy images of U2OS cells stained with 10. (c) Three-color live-cell STED image of U2OS cells expressing Sec61β–SNAP-tag labeled with JF646–SNAP-tag ligand (2), TOMM20–HaloTag labeled with JF585–HaloTag ligand (5), and microtubules stained with 10. (d) Lattice light-sheet microscopy image of U2OS cells stained with 10 and 2. Scale bars for all images: 5 μm.

Hydroxymethyl JF526

The lactone–zwitterion equilibrium of rhodamine dyes can be further exploited for single-molecule localization microscopy (SMLM). Converting the o-carboxyl moiety in SiR to the more nucleophilic hydroxymethyl group elicits an additional shift to the closed form.[35,53] The resulting hydroxymethyl-SiR (HM-SiR, 32) exists primarily in a colorless, nonfluorescent spiroether form but spontaneously switches to a transient, fluorescent form upon protonation at physiological pH (Figure a). This blinking behavior enables facile SMLM imaging, bypassing the need for photoconvertible fluorescent proteins, photoactivatable dyes, or strongly reducing dSTORM buffers.[54]

Figure 7

Localization microscopy with HM-JF526. (a) Blinking behavior of HM-SiR (32). (b) Synthesis of HM-JF526 NHS (37). Immunofluorescence images of tubulin labeled with a 37–antibody conjugate: (c) SMLM image, (d) diffraction-limited image. (e) Transverse profiles of fluorescence intensity corresponding to boxed regions in parts c and d. Immunofluorescence images of TOMM20 labeled with a 37–antibody conjugate: (f) SMLM image; (g) diffraction-limited image. (h) Transverse profiles of fluorescence intensity corresponding to boxed regions in parts f and g. Solid lines in parts e and h indicate Gaussian fits; numbers indicate the full width at half-maximum (fwhm) determined by the Gaussian fits of the SMLM (green) and diffraction-limited imaging (black). Scale bars for all images: 5 μm.

Given the similarity of the KL–Z values for JF526 and SiR, we were curious if a hydroxymethyl derivative of JF526 would show comparable utility to 32 in SMLM. We devised an efficient, high-yielding approach to synthesize derivatives of hydroxymethyl JF526 (HM-JF526), leveraging our divergent synthesis of JF526 and the differential reactivity of carboxyl groups and lactones with borohydride reductants.[55] Treatment of dibromofluoran 22 with LiBH4 at ambient temperature selectively reduced the lactone to a cyclic ether leaving the 6-carboxy group intact, providing 33 in moderate yield (54%; Figure b). Formation of 6-t-butyl ester with acetal 23 gave 34, allowing Pd-catalyzed cross-coupling with 29. The resulting 6-tert-butoxycarbonyl-HM-JF526 (35) can be deprotected to yield carboxylic acid 36 and then converted to amine-reactive N-hydroxysuccinimidyl ester 37 (HM-JF526 NHS).

To evaluate the performance of HM-JF526 in SMLM experiments we used 37 to label a goat-antimouse secondary antibody, followed by immunostaining of an anti-β-tubulin primary antibody in fixed cells. SMLM imaging in standard phosphate-buffered saline (pH 7.4) revealed that the HM-JF526 label showed spontaneous blinking behavior throughout the imaging session and did not require short-wavelength activation light (Movie S1). Standard SMLM analysis transformed these movies into super-resolution images (Figure c,d); the HM-JF526 label yielded 571 photons on average with a localization accuracy (σ) of 25 nm. The SMLM images showed fine structures of microtubules with a full width at half-maximum (fwhm) of 86 nm; diffraction-limited images had an fwhm of 253 nm (Figure e). We also labeled mitochondria using an anti-TOMM20 primary antibody which gave SMLM images of mitochondria with improved resolution (fwhm = 143 nm) compared to diffraction-limited images (fwhm = 581 nm; Figure f–h). HM-JF526 constitutes a new label for SMLM that is spectrally distinct from HM-SiR and compatible with standard immunolabeling protocols.

Conclusion

Rhodamine dyes exist in equilibrium between a lipophilic, colorless lactone and a polar, fluorescent zwitterion. This property dictates many properties of rhodamines including cell-permeability and fluorogenicity. On the basis of the prototypical fluorogenic dye SiR (1) and the Janelia Fluor dyes (2–7), we showed that the equilibrium constant, KL–Z, is sufficient to predict fluorogenicity by comparing the KL–Z of rhodamines and the change in fluorescence of their respective HaloTag ligands upon binding their protein target. We found an inverse relationship between these two parameters and developed a quantitative framework for developing new fluorogenic molecules: tuning KL–Z between 10–2 and 10–3 gives ligands with fluorogenicity of 5–10-fold (Figure ). This rubric was validated with the orange-emitting dye, SiR110 (8), which is fluorogenic and shows improved photostability compared to our previously described JF585 (5, Figure ).

Our previous attempts to tune the KL–Z of standard rhodamine dyes using 3,3-difluoroazetidine substituents transformed JF549 (6, KL–Z = 3.5) to the highly bioavailable JF525 (7, KL–Z = 0.068). Nevertheless, this decrease in KL–Z was insufficient to meet the fluorogenic threshold of KL–Z < 10–2; ligands based on JF525 show low degrees of fluorogenicity. We used a complementary approach to further tune the KL–Z by fluorinating the xanthene system. This yielded the highly cell-permeant JF552 (9) as an intermediary product (Scheme , Figure ) and ultimately led to the fluorogenic rhodamine JF526 (10). Akin to SiR (1), JF526 is a versatile scaffold for fluorogenic ligands, including labels for genetically encoded self-labeling protein tags (Figure ) and stains for endogenous structures (Figure ). These green-emitting ligands can be used in concert with red- and orange-emitting fluorogenic dyes,[14,16,20] allowing multicolor SIM and STED imaging in live cells (Figure ). We further extended the utility of JF526 to SMLM by creating the spontaneously blinking derivative: HM-JF526 (Figure ).

Looking forward, our results demonstrate the importance of KL–Z in the rational design of fluorogenic and spontaneously blinking rhodamines, regardless of the specific dye structure. This general rubric we uncovered should enable rational design of fluorogenic and spontaneously blinking dyes using other rhodamine variants, especially the exciting near-infrared derivatives containing phosphinate, phosphine oxide, or sulfone groups,[15,17,18] pushing advanced microscopy to longer wavelengths. This quantitative approach could be applied to fluoresceins and rhodols, which bear o-carboxy groups and have environmentally sensitive equilibria between fluorescent and nonfluorescent lactone forms.[13,19,56−58] Finally, we posit that the environmental sensitivity of fluorogenic rhodamines can be exploited beyond preparing ligands for no-wash imaging experiments. Like other fluorogenic molecules,[59−61] transduction of protein conformational changes into fluorescence modulations will provide new hybrid small-molecule:protein sensors for functional imaging inside living cells and organisms.

Safety Statement

No unexpected or unusually high safety hazards were encountered.