A Photochromic Bioconjugate with Photoactivatable Fluorescence for

Article pubs.acs.org/JPCC

A Photochromic Bioconjugate with Photoactivatable Fluorescence for Superresolution Imaging Janet Cusido,† Sherif Shaban Ragab,†,‡ Ek Raj Thapaliya,† Subramani Swaminathan,† Jaume Garcia-Amorós,† M. Julia Roberti,§,# Beatriz Araoz,§ Mercedes M. A. Mazza,† Shiori Yamazaki,† Amy M. Scott,† Françisco M. Raymo,*,† and Mariano L. Bossi*,§ †

Laboratory for Molecular Photonics, Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146-0431, United States, ‡ Department of Photochemistry, Chemical Industries Research Division, National Research Centre (NRC), El behouth Street, Dokki, Giza, 12622, Egypt § INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428 − Buenos Aires, Argentina S Supporting Information *

ABSTRACT: A coumarin fluorophore and an oxazine photochrome can be integrated within the same molecular skeleton and connected covalently to a secondary antibody. Illumination of the antibody−dyad conjugate at an appropriate activation wavelength opens the oxazine ring reversibly and shifts bathochromically the ground-state absorption of the coumarin component. Selective excitation of the photochemical product then produces significant fluorescence and allows the detection of activated bioconjugates at the single-molecule level. Such fluorescence activation events can be exploited to resolve temporally individual emitters and reconstruct images of immunolabeled cells with subdiffraction resolution. Relying on a similar conjugation protocol, a model compound, incorporating the same chromophore of the photochemical product, can also be connected covalently to a secondary antibody. Stimulated emission can be exploited to deplete the excited state of the bioconjugated chromophores and switch their fluorescence off. These operating principles for fluorescence switching also permit the imaging of immunolabeled cells with subdiffraction resolution. Thus, these photoswitchable molecules, in combination with the labeling ability of antibodies, can evolve into valuable probes for bioimaging with superresolution.

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resolution of conventional fluorescence images.19−26 As a result, mechanisms for the optical control of fluorescence with photochromic compounds can ultimately evolve into invaluable operating principles for the visualization of biological specimens with unprecedented resolution. Indeed, representative examples of photochromic probes with optimal properties for subdiffraction imaging have been designed already, relying on diarylethenes,27−32 imidazole dimers,33 rhodamines,34−44 and spiropyrans.45−50 We developed a family of fast and stable photochromic compounds based on the photoinduced opening and thermal closing of an oxazine ring.51−63 We demonstrated that their reversible interconversion can be exploited to control the efficiency of electron- and energy-transfer processes and switch the emission of fluorescent partners.64,65 Additionally, the structural changes associated with the photoinduced opening of

INTRODUCTION Photochromic compounds1−10 switch reversibly between states with distinct absorption spectra under the influence of optical stimulations. Their reversible transformations can also cause significant emission changes, if one of the interconvertible states is fluorescent.11 Alternatively, nonemissive photochromes can be paired covalently or noncovalently to emissive chromophores in molecular or supramolecular constructs respectively to modulate fluorescence.12−18 In the resulting assemblies, the exchange of electrons or energy between the fluorescent component and one of the interconvertible states of the photochromic switch can be engineered to control the excitation dynamics of the former and regulate fluorescence. Similarly, the significant structural modifications that accompany the interconversion of the photochrome can be designed to alter the ability of the fluorophore to absorb exciting radiations and, as a consequence, its emission intensity. In turn, the possibility to switch fluorescence with optical inputs offers the opportunity to overcome the limitations that diffraction imposes on the spatial © 2016 American Chemical Society

Received: March 27, 2016 Revised: May 27, 2016 Published: May 30, 2016 12860

DOI: 10.1021/acs.jpcc.6b03135 J. Phys. Chem. C 2016, 120, 12860−12870

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The Journal of Physical Chemistry C

Figure 1. Photoinduced and reversible conversion of 1a into 1b together with model compound 2.

procedure.79 ESIMS was performed with a Bruker micrOTO-Q II spectrometer. NMR spectra were recorded with a Bruker Avance 400 spectrometer. Absorption spectra were recorded with a Varian Cary 100 Bio spectrometer, using quartz cells with a path length of 1.0 cm. Emission spectra were recorded with a Varian Cary Eclipse spectrometer in aerated solutions. Emission decays were measured with a HORIBA Jobin Yvon IBH 5000U fluorescence lifetime system in aerated solutions. Fluorescence quantum yields were determined with a coumarin 153 or a rhodamine 101 standard, following a literature protocol.80 Nanosecond transient absorption (TA) measurements were performed with an apparatus based on a commercial amplified Ti:sapphire laser system (Spectra-Physics Mai Tai Oscillator (80 MHz) and 5 W Spitfire Ace Amplifier pumped by a 527 nm Nd:YLF Empower laser) operating at 1 kHz and pumps an optical parametric amplifier (TOPAS, Spectra-Physics) to produce excitation pulse at 400 nm (IRF ∼ 70 fs, 8 μJ/pulse). The probe pulse (IRF ∼ 2 ns) was set to detect in the visible region (400−800 nm). Time-resolved absorption data were recorded with an EOS spectrometer (Ultrafast System), using a 2 mm quartz cell in which the solution was bubbled with argon before each measurement. 5. A solution of 3 (203 mg, 1 mmol) and 4 (188 mg, 1 mmol) in MeCN (30 mL) was heated under reflux for 24 h. After cooling to ambient temperature, the solvent was distilled off under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL). The resulting solution was washed with H2O (20 mL) and dried over Na2SO4, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography [SiO2: hexane/EtOAc (1:1, v/v)] to afford 5 (240 mg, 70%) as a yellow solid. ESIMS: m/z = 355.1308 [M + H]+ (m/z calcd for C19H19N2O5 = 355.1294); 1H NMR (CDCl3): δ = 1.22 (3H, s), 1.26 (3H, s), 1.58 (3H, s), 4.69 (2H, s), 6.61 (1H, d, 8 Hz), 6.77 (1H, d, 8 Hz), 7.82 (1H, s), 7.92 (1H, d, 8 Hz), 7.99 (1H, d, 8 Hz), 8.09 (1H, s); 13C NMR (CDCl3): δ = 17.7, 22.5, 29.6, 43.9, 51.5, 106.4, 111.6, 122.2, 122.6, 126.3, 127.2, 127.9, 128.0, 135.0, 142.0, 144.5, 155.0, 162.7, 172.9. 7a. A solution of 5 (50 mg, 0.1 mmol), 6 (34 mg, 0.1 mmol), and TFA (20 mg, 0.2 mmol) in EtOH (20 mL) was heated under reflux for 18 h. After cooling to ambient temperature, the solvent was distilled off under reduced pressure and the residue was dissolved in CH2Cl2 (20 mL). The resulting solution was washed with H2O (2 × 20 mL) and dried over Na2SO4, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography [SiO2, hexane/EtOAc (1:1, v/v)] to afford 7a (65 mg, 80%) as a blue solid. ESIMS: m/z = 582.2278 [M + H]+ (m/z calcd for C33H32N3O7 = 582.2235); 1H NMR [(CD3)2CO]: δ = 1.20 (6H, t, 8 Hz), 1.26 (6H, bs), 3.50 (4H, q, 8 Hz), 4.73 (1H, d, 16 Hz), 5.07 (1H, d, 16 Hz), 6.54 (1H, s), 6.72 (1H, d, 8 Hz), 6.82 (1H, d, 16 Hz), 6.69−7.03 (3H, m), 7.39 (1H, d, 8 Hz), 7.83−7.86 (2H, m), 7.92 (1H, s), 8.01 (1H, d, 8 Hz), 8.15 (1H, s); 13C NMR (CD3CN): δ = 13.5, 25.9, 44.3, 49.5, 54.1, 96.6, 104.0, 108.6, 109.8, 115.0, 117.7, 122.7, 123.6, 123.8,

the oxazine ring can be engineered to control reversibly the electronic structure of a covalently connected fluorophore.66−75 These transformations alter the ability of the latter to absorb exciting photons and emit as a result. In fact, our mechanism for fluorescence photoactivation can be replicated at the singlemolecule (SM) level to allow the stochastic reconstruction76−78 of images with spatial resolution at the nanoscale.70,71,74 Compound 1a (Figure 1) is a representative example of the fluorophore−photochrome dyads developed in our laboratories.66,70 The absorption spectrum of an acetonitrile solution of this molecule shows a band at 412 nm for the coumarin component. Illumination at 355 nm with a pulsed laser excites the 4-nitrophenoxy fragment, cleaves the adjacent [C−O] bond, and opens the oxazine ring to generate 1b within the laser pulse and with a quantum yield of 0.02. In the resulting structure, the coumarin fluorophore can extend its electronic conjugation over the photogenerated 3H-indolium cation with a concomitant bathochromic shift in absorption to 580 nm. Consistently, the resulting transient absorption band resembles the steady-state absorption of 2 (Figure 1). The photogenerated isomer (1b) has a lifetime of 0.2 μs and eventually reverts to the original state with first-order kinetics. Illumination of the sample within 0.2 μs at a wavelength positioned within the transient absorption can be exploited to excite selectively the zwitterionic isomer and produce fluorescence. Specifically, simultaneous illumination at 355 nm, to convert 1a into 1b, and at 532 nm, to excite the photochemical product, shows an emission band at 650 nm for 1b. This band resembles the emission of 2, confirming that it corresponds to the fluorescence of the cationic fragment of the zwitterionic isomer. Thus, these structural design and illumination conditions offer the opportunity to turn fluorescence on under optical control. Indeed, we reconstructed images of polymer nanoparticles, encapsulating 1a, with subdiffraction resolution relying on the photoactivatable fluorescence of 1b.70,71,74 This behavior suggests that the covalent attachment of such a coumarin−oxazine dyad to an antibody could, in principle, be exploited to implement immunolabeling strategies for the acquisition of subdiffraction images of biological preparations. On the basis of these considerations, we envisaged the possibility of modifying the structural designs of dyad 1a and model 2 in order to incorporate reactive functional groups, compatible with bioconjugation, within their covalent skeletons. In this article, we report the synthesis of these compounds and their covalent attachment to antibodies, together with steadystate and time-resolved spectroscopic characterizations of the resulting conjugates and their application to image cellular substructures with subdiffraction resolution.

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EXPERIMENTAL PROCEDURES Materials and Methods. Chemicals were purchased from commercial sources and used as received. CH2Cl2 and MeCN were distilled over CaH2. H2O (18.2 MΩ-cm) was purified with a Barnstead International NANOpure DIamond Analytical system. Compound 6 was prepared according to a literature 12861


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characteristic absorption of the 3H-indolium chromophore at 600 nm were used for further spectroscopic and imaging experiments. Fluorescence Imaging. Confocal images were acquired with a Leica TCS SP5 laser scanning confocal microscope, with excitation wavelengths of 405 or 561 nm, and custom detection range selected by means of an acusto-optical-beam splitter (AOBM). An oil immersion objective lens (Leica HCX PL APO CS 63× / NA1.40) was used. Stimulated emission depletion (STED) images were acquired in an Abberior Instruments 2C STED 775 QUAD Scan, with excitation at 640 nm and pulsed STED at 775 nm. SM localization superresolution microscopy was performed in a custom-built wide-field epifluorescence setup previously described.70,71 Briefly, single molecule images were acquired on a wide-field microscope equipped with an EMCCD (IXON-DU-897; Andor Technology) detection camera, a 532 nm DPSS laser (SDL-532−200T; Shanghai Dream Lasers Technology) for excitation (5−30 kW cm−2), and a 405 nm diode laser (SDL-405-LM-20-T; Shanghai Dream Lasers Technology) for photoswitching (0.6 kW cm−2). The latter was switched on and off using a homemade pulse generation unit to irradiate the sample in between frames. Laser beams were collinearly overlapped with a dichroic mirror (DCLP 425 DCXR), and focused (Achromat f200 lens) onto the back focal plane of a Leica PLAN 100× objective (1.25 NA, oil immersion), to achieve wide-field illumination. SM fluorescence emission was collected by the same objective and separated from the excitation beams by means of a dichroic mirror (ZT532rdc; Chroma Technology Corp.). A bandpass emission filter (FF01-640/40, Semrock Inc.) was placed in front of the detector. Excitation residual light was further cleaned with a notch filter (NF01532U-25; Semrock Inc.). The pixel size of 83 × 83 nm2 on the focal plane was calibrated with a resolution test pattern (USAF 1951 RES TARGET 2IN, Edmund Optics). Typically, sequences of images were recorded onto a 12 × 12 μm area. Image acquisition was performed with the software of the provider of the camera (Andor Solis; Andor Technology) at an exposure time of 30 ms. Mechanical stability of the system was assured to better than 5 nm over several minutes. Data analysis for image rendering was performed with homemade routines previously described.74 Cell Preparation and Immunostaining. HeLa and Vero cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin and kept in an incubator at 37 °C and 5% CO2. HeLa cells stably expressed H2B-EGFP in the nucleus, which facilitated finding them under the microscope. For immunostaining, cells were first fixed in methanol at −20 °C for 15 min and then washed three times with PBS. A blocking step with PBS−BSA 3% was carried out at room temperature for 30 min, and afterward a solution of anti-α-tubulin antibody (mouse monoclonal antibody, DM1A, Sigma) diluted 1:500 in PBS−BSA 3% was applied at room temperature for 1h. Cells were washed three times with PBS−BSA 3%, 5 min each washing step. Then a secondary antibody covalently coupled with the fluorescent compound of interest (10a or 13) was titrated, and the optimal dilution was found to be 1:50 (in PBS−BSA 3%) from the quality of confocal images. Incubation with this solution was carried out at room temperature for 1 h. Finally, the cells were washed three times for 5 min with PBS and immediately mounted.

124.2, 124.6, 125.0, 127.6, 129.6, 130.6, 131.3, 138.4, 141.7, 142.0, 151.3, 156.1, 159.0, 160.0, 166.7. 8. DCC (54 mg, 2.0 mmol) was slowly added to a solution of 7 (130 mg, 0.2 mmol), NHS (28 mg, 0.2 mmol), and DMAP (2 mg, 0.02 mmol) in CH2Cl2 (30 mL) maintained at 0 °C under Ar. The mixture was allowed to warm to ambient temperature, stirred under these conditions for 15 h, and filtered. The solvent of the filtrate was distilled off under reduced pressure, and the residue was purified by column chromatography [SiO2, hexane/ EtOAc (1:1, v/v)] to afford to afford 8 (75 mg, 50%) as a green solid. ESIMS: m/z = 679.2438 [M + H]+ (m/z calcd for C37H35N4O9 = 679.2404); 1H NMR [(CD3)2CO]: δ = 1.21 (6H, t, 8 Hz), 1.30 (6H, s), 2.92 (4H, s), 3.53 (4H, q, 8 Hz), 4.77 (1H, d, 16 Hz), 5.15 (1H, d, 16 Hz), 6.50 (1H, s), 6.73 (1H, d, 8 Hz), 6.82 (1H, d, 16 Hz), 6.97 (1H, d, 16 Hz), 7.04−7.09 (2H, t, 8 Hz), 7.40 (1H, d, 8 Hz), 7.89−7.96 (3H, m), 8.03 (1H, d, 8 Hz), 8.16 (1H, s); 13C NMR (CDCl3): δ = 12.8, 18.7, 25.8, 26.4, 26.6, 41.4, 45.4, 50.5, 97.4, 103.6, 109.4, 110.0, 110.3, 116.0, 117.6, 118.9, 121.6, 124.4, 124.9, 125.0, 125.2, 130.6, 132.6, 132.9, 140.2, 142.8, 152.3, 154.2, 157.2, 160.0, 161.0, 162.6, 170.9. 10a. The pH of an aqueous solution of 9 (Jackson ImmunoResearch Inc. 515-005-003, 1 mg, 450 μL) was adjusted to 8.8 with BCB (1 M, 50 μL). The resulting mixture was combined with a solution of 8 (7.4 mM) in DMF (20 μL), stirred for 1 h at ambient temperature, passed through a Sephadex G25 column, and eluted with PBS. The collected fractions (0.5 mL each) were analyzed by absorption spectroscopy, and those showing the characteristic absorption of the coumarin chromophore at 425 nm were used for further spectroscopic and imaging experiments. 11. A solution of 3 (500 mg, 2.5 mmol) and MeI (0.55 mL, 2 mmol) in MeCN (20 mL) was heated under reflux and Ar for 14 h. The mixture was allowed to cool to ambient temperature, and the resulting precipitate was filtered off and washed with EtOH (5 mL) and hexane (20 mL) to afford the iodide salt of 11 (700 mg, 80%) as a white solid. ESIMS: m/z = 218.1197 [M]+ (m/z calcd for C13H16NO2 = 218.2716); 1H NMR [(CD3)2CO]: δ = 1.79 (6H, s), 3.13 (3H, s), 4.32 (3H, s), 8.12 (1H, d, 8 Hz), 8.34 (1H, d, 8 Hz), 8.45 (1H, s); 13C NMR [(CD3)2CO]: δ = 21.0, 34.2, 54.6, 114.9, 123.9, 131.0, 132.2, 141.7, 145.2, 166.6. 12. A solution of the iodide salt of 11 (545 mg, 2.5 mmol) and 6 (612 mg, 2.5 mmol) in EtOH (20 mL) was heated under reflux for 12 h. After cooling to ambient temperature, the solvent was distilled off under reduced pressure and the residue was purified by column chromatography [SiO2, CH2Cl2/MeOH (9:1, v/v)] to afford the iodide salt of 12 (670 mg, 60%) as a blue solid. ESIMS: m/z = 445.2136 [M]+ (m/z calcd for C27H29N2O4 = 445.5302); 1H NMR (CD3OD): δ = 1.31 (6H, t, 8 Hz), 1.87 (6H, s), 3.63 (4H, q, 8 Hz), 4.06 (3H, s), 6.65 (1H, s), 6.95 (1H, d, 9 Hz), 7.65 (1H, d, 9 Hz), 7.96 (1H, d, 8 Hz), 7.99 (1H, d, 16 Hz), 8.26 (2H, d, 10 Hz), 8.36 (1H, d, 15 Hz), 8.48 (1H, s); 13C NMR (CD3OD): δ = 11.4, 25.5, 32.8, 45.2, 51.5, 96.4, 109.4, 110.3, 111.4, 112.4, 113.6, 123.4, 130.8, 132.7, 143.0, 150.6, 155.0, 158.3, 160.0, 183.2. 13. A solution of 12 (4.5 mM), NHS (12 mM) and EDC (18 μM) was prepared in DMF and stirred for 30 min at ambient temperature. Then 25 μL of the mixture was added to an aqueous solution of 9 (sheep antimouse IgG, Jackson ImmunoResearch Inc. 515-005-003, 1 mg, 450 μL) whose pH was previously adjusted to 8.8 with BCB (1 M, 50 μL). The mixture was stirred for 1 h at ambient temperature and passed through a Sephadex G25 column. The collected fractions (0.5 mL each) were analyzed by absorption spectroscopy, and those showing the 12862


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Figure 2. Synthesis of antibody−oxazine−coumarin conjugate 10a.

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RESULTS AND DISCUSSION Synthesis. Following our imaging experiments with 1a,70 we envisioned introducing an active ester in position 5 of the 2H,3Hindole fragment of the coumarin−oxazine dyad to allow the subsequent bioconjugation to an antibody. The resulting compound was synthesized in three steps from commercial reactants (Figure 2). Specifically, reaction of 3 with 4 gave 2H,4H-benzo[1,3]oxazine 5 in a yield of 70%. Condensation of 5 with 6, under the assistance of trifluoroacetic acid (TFA), resulted in the formation of coumarin−oxazine dyad 7a in a yield of 80%. Esterification of 7a with N-hydroxysuccinimide (NHS), under the influence of dicyclohexylcarbodiimide (DCC), gave 8 in a yield of 50%. Treatment of 8 with sheep antimouse IgG (9 in Figure 2) in bicarbonate/carbonate buffer (BCB, pH ∼ 8.5) resulted in the formation of 10a. Following a similar synthetic protocol (Figure 3), we introduced a reactive functional group also on position 5 of the

absorption spectroscopy. In particular, the corresponding spectra (A in Figures 4 and 5) show the characteristic absorption band of the coumarin component of 10a and 13 at 425 and 572 nm, respectively. Additionally, a very weak absorption is also observed in the red region for 10a and 7a (A and B in Figure 4). These bands are indicative of the spontaneous opening of the oxazine ring with the formation of the corresponding ring-open isomers 10b and 7b (cf. 1b in Figure 1). In fact, they are positioned in the same region of wavelengths (ca. 570−600 nm) where 13 and 12 absorb (A and B in Figure 5), which have essentially the same 3H-indolium chromophores of 10b and 7b. These observations are fully consistent with literature data,70 which indicate that an equilibrium between the ring-closed and -open isomers (e.g., 1a and 1b in Figure 1) of related oxazines is established in protic solvents. Nonetheless, this equilibrium is strongly displaced toward the ring-closed isomer in the bioconjugate 10a. The absorption spectrum of conjugate 10a closely resembles that of corresponding model 7a (A and B in Figure 4), while that of conjugate 13 is broader than that of its model 12 (A and B in Figure 5) and appears to have a shoulder red-shifted by ca. 20 nm. A possible explanation for these minor differences is the presence of dye molecules in different environments. In particular, the conjugation of the antibody to the 3H-indolium cation of 13 must be responsible for the red-shifted shoulder, presumably because the adjacent biomolecule imposes a relatively hydrophobic environment on the cationic chromophore and/or restricts the torsional freedom of the two [C−C] bonds along its conjugated platform and affects the equilibration of its conformers. The characteristic absorption band of the protein can be observed at ca. 280 nm for both conjugates (A in Figures 4 and 5). Comparison of the absorbance of this band to those of the conjugated chromophores suggests that the degree of labeling is 3.0 for 10a and 1.2 for 13. However, these values were estimated relying on the molar absorption coefficients of the chromophores measured in acetonitrile and do not take into account the possible influence of conjugation on the latter parameters. A further comparison of the spectra of 13 and 12 (A and B in Figure 5) shows that a band at 400 nm is present only in the spectrum of the conjugate. This absorption is presumably a consequence of the nucleophilic addition of a hydroxyl group to the 3H-indolium cation of 13 with the formation of the corresponding hemiaminal in the course of the antibody-labeling

Figure 3. Synthesis of antibody−indolium−coumarin conjugate 13.

3H-indolium fragment of 2. Specifically, methylation of 3 gave the iodide salt of 11 in a yield of 80%. Condensation of 11 with 6 afforded the iodide salt of 12 in a yield 60%. Treatment of 12 with 9 in BCB, under the influence of NHS and 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC), resulted in the formation of 13. Spectroscopy. The two antibody conjugates (10a and 13) were purified by size-exclusion chromatography, and the presence of the chromophoric labels was confirmed by 12863


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Figure 4. Absorption (A and B) and emission (C and D, λEx = 410 nm) spectra of PBS solutions (25 °C) of 10a (A and C, ∼0.2 mg mL−1) and 7a (B and D, 4 μM).

Figure 5. Absorption (A and B) and emission (C and D, λEx = 580 nm) spectra of PBS solutions (25 °C) of 13 (A and C, ∼0.04 mg mL−1) and 12 (B and D, 0.34 μM).

Figure 6. Transient absorption spectra (A) of a PBS solution (25 °C) of 10a (∼0.2 mg mL−1 μM) recorded after 400 nm photoexcitation with 8 μJ/ pulse (IRF ∼ 2 ns) together with the kinetic traces at 445 (B), 537 (C), and 625 nm (D).

5). The fluorescence quantum yield is 0.002 for 10a and 0.07 for 13. These values are 60% and 24% smaller than those measured for 7a and 12, respectively, indicating that the attachment of these fluorescent chromophores to the biomolecules depresses the radiative efficiency. This behavior is fully consistent with literature precedents82,83 and is ascribed to self-quenching

reaction, which is performed in aqueous medium at a pH of 8.5. In fact, the conversion of 3H-indolium cations to hemiaminals, under basic conditions, is well established.81 The emission spectra of 10a and 13 (C in Figures 4 and 5) show bands at 456 and 660 nm respectively that closely resemble those observed for the corresponding models (D in Figures 4 and 12864


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Fluorescence Imaging. The two conjugates were tested in fluorescence imaging experiments, using standard immunolabeling protocols (see Experimental Procedures). Briefly, HeLa or Vero cells were labeled with a primary antibody against tubulin and then with the secondary antibodies 10a and 13. The samples were mounted in Mowiol medium on standard slides and imaged with the different techniques described throughout this section. Confocal images (Figure 7) with both conjugates show good

resulting from crowding effects produced after attaching multiple chromophores to a protein molecule. Hydrophobic compounds are more prone to such effects in aqueous solutions. Indeed, a reduction in fluorescence quantum yield is generally observed, even in bioconjugates with low degree of labeling, for common fluorescent dyes upon antibody conjugation, and this effect can be mitigated with the incorporation of spacers (e.g., polyethylene glycol) or charged groups (e.g., sulfonates) in the emissive chromophores.84,85 The fluorescence of 7a and 12 decays monoexponentially with lifetimes of 2.8 and 0.2 ns, respectively. By contrast, the temporal evolutions of the emission intensities of 10a and 13 display biexponential dependence. The corresponding average lifetimes are 3.5 ns (4.0 ns −86%−/0.9 ns −14%−) for 10a and 1.0 ns (1.2 ns −80%−/0.13 ns −20%−) for 13. The different behavior of free dyes and conjugates suggests the coexistence of, at least, two distinct environments for the fluorescent chromophores covalently attached to the antibody. Transient absorption spectra (A in Figure 6), collected on a nanosecond time scale upon excitation at 400 nm in PBS, confirm the photoinduced conversion of the ring-closed species 10a into the ring-open form 10b. Specifically, they show (1) a negative band at 445 nm for the ground-state bleach of 10a, (2) a positive band at 537 nm for the ground-state absorption of the 3H-indolium chromophore of 10b, and (3) a negative band at 625 nm for the emission of the 3H-indolium chromophore of 10b. This spectroscopic response is fully consistent with that observed for 1a in acetonitrile66,70 and demonstrates that the covalent attachment of the photochromic dyad to the antibody does not suppress the photoinduced ring-opening process, allowing the formation of 10b upon photoexcitation. Interestingly, the bands detected for 10b at 537 and 625 nm in the transient spectra (A in Figure 6) are shifted hypsochromically by ca. 40 nm, relative to those observed for 13 in the steady-state spectra (A and C in Figure 5). Both species incorporate essentially the same 3H-indolium chromophore, which can adopt four distinct geometries differing in the conformations about the two [C−C] bonds connecting the two heterocycles. Presumably, the steady-state populations of the four conformers of 13 differ from those produced under photoexcitation with the photogeneration of 10b. Such a difference might be responsible for the hypsochromic shift and narrowing of the transient band relative to its steady-state counterpart. The photogenerated species 10b reverts to the original one 10a in microseconds. Consistently, the two negative transient bands at 445 and 625 nm recover and the positive one at 537 nm decays on this time scale (B−D in Figure 6). The corresponding kinetic traces, however, display multiexponential temporal dependence (Table S1, Supporting Information), suggesting the coexistence of dyads in different environments within the bioconjugates. Furthermore, all traces do not return to zero on the microsecond time scale. A fraction of the photogenerated and emissive isomers appears to persist for relatively long times (i.e., hundreds of microseconds to milliseconds). This population of long-lived states is presumably responsible for the fluorescence signal detected at the single-molecule level (vide infra). These species eventually revert to the original ring-closed form and, in fact, steady-state spectra recorded before and after up to 5.4 × 106 excitation pulses are virtually identical (Figure S1, Supporting Information). Thus, the photochromic dyads can be interconverted multiple times between their two states with outstanding fatigue resistance, even after their covalent attachment to the biomolecule.

Figure 7. (A and B) Confocal images of HeLa cells immunostained with 10a. (A) Image recorded under excitation at 405 nm and detection set at 420−480 nm (blue channel). Scale bar =10 μm. (B) Image acquired under excitation at 561 nm with detection at 620−740 nm (red channel). The lower cell shows also emission from GFP in the nucleus. Scale bar =10 μm. (C) Confocal image of Vero cells immunostained with 13. The image was recorded under excitation at 561 nm and detection set at 620−740 nm. Scale bar = 10 μm. The primary antibody as well as the fixing and labeling protocols was identical for both cell lines.

selectivity and low background. Because of the moderate brightness of 10a, images were recorded with a slightly increased power in the excitation laser (ca. 2-fold, compared to samples labeled with typical commercial dyes, e.g., rhodamines, fluoresceins), in order to avoid long recording times. Some bright spots were detected (Figure 7A) with 10a, probably resulting from antibody aggregation. This effect was not observed in samples labeled with 13 (Figure 7C), presumably because of the hydrophilic character of the cationic chromophore incorporated within this conjugate. While centrifugation of the solutions of 10a, before labeling, could also improve the images, chemical modifications of the chromophoric component with polar or charged groups (e.g., sulfonates) are desirable to improve its performance and avoid the potential loss of antibody activity for the conjugate over time.82,84 Interestingly, samples labeled with 10a can be imaged in two detection channels (Figure 7A and 7B) with resolved spectral regions (blue and red emission), indicating that the ring-closed (10a) and -open (10b, cf. 1b in Figure 1) forms of the dyad coexists in aqueous solutions of the conjugate. 12865


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The Journal of Physical Chemistry C Next, the performance of both conjugates for superresolution applications was investigated. Conjugate 10a can be photoactivated with a laser operating at 405 nm and, thus, is a good candidate for SM approaches [i.e., photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)].76−78 In order to explore this possibility, we first studied the SM behavior of 10a in a poly(vinyl alcohol) (PVA) film. Figure 8 shows a typical frame of a series containing

Figure 8. SM activation and detection of 10a in a PVA film. One of the frames is shown in the upper panel (left), along with a SM trace of a 10 000 frames series, corresponding to the highlighted area in the frame. On the lower panel, the accumulated amount of SM events localized is shown (left) and with the distribution of detected photons per SM (right). From the latter, an average amount of 146 photons per event was calculated.

several photoactivated molecules. A time trace in the highlighted area demonstrates that a high signal-to-noise ratio can be obtained. However, it can be observed that, even in PVA films, there are several spots with low intensity that are hard to identify and localize by standard routines. In a more realistic imaging environment (e.g., fixed cells with more dense labeling), these molecules can contribute to the background and complicate the localization process required in SM superresolution techniques. Nevertheless, the average of photons detected per SM is 146, allowing for a good theoretical resolution enhancement.86 We must stress that dim spots are not localized and, therefore, not considered in this distribution (see discussion below). The accumulated number of events also shows a mild spontaneous (thermal) activation at the beginning (only the excitation laser 532 nm was on in the first 2000 frames) and a strong increase after the activation laser (405 nm) is turned on to reach finally saturation, once most of the probes are photobleached. Encouraged by these results, we imaged cells in a homemade microscope for stochastic SM localization microscopy. Excitation and activation were performed under conditions similar to those used in the experiment presented in Figure 8. Activation laser (405 nm) was gradually increased through the series of frames, to obtain a mean number of detected events (SM) per frames of ∼15 (Figure S2, Supporting Information). However, a larger threshold was set to discard dim events (i.e., molecules that yield low number of photons per incursion to the bright open isomer) and, as a result, the average number of photons per SM for Figure 9 is 600 (Figure S2). Before the beginning of a measurement, labels in the open isomer had to be bleached. However, this

Figure 9. Superresolution imaging with 10a. Wide-field (WF, A) and stochastic SM localization (B) images of Vero cells immunostained with 10a. The WF image was recorded with excitation at 405 nm and detection was performed with a bandpass filter 460/60 nm. The SM image was recorded under excitation at 532 nm and activation at 405 nm, while detection was performed with a bandpass filter 630/75 nm. A line profile along the dotted line is shown in C. SM image reconstructed from 300 900 localized single molecules, from 83 000 frames; pixel size = 40 nm; scale bar = 1 μm and 350 nm in the zoomed area shown in the insets.

fraction of bleached probes that cannot be used for superresolution imaging is rather small, as stated above. The images obtained (Figure 9) show a good resolution enhancement. Single tubulin filaments are observed with sizes of ∼70−80 nm, close to the expected value from their actual size plus the size of the primary and the secondary antibodies. However, they are of relatively low quality because of the fact that under the imaging conditions, the samples showed low signal-to-noise ratio. This is a consequence of the low emission efficiency of the open isomer and the contribution from the fraction of molecules decaying in the microsecond range to the background signal (vide supra). The latter is evidenced by the fact that a better resolution was observed near the borders of cells (filaments are better defined in such regions, e.g., in Figure 9) where they are thinner. It is also evident that the “spottiness” of the images is higher than that of their confocal counterparts. Despite the fact that this behavior 12866


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The Journal of Physical Chemistry C has already been observed in superresolution imaging,87,88 mostly because of antibody-labeling discontinuities that are normally undetected in standard low-resolution techniques, the effect seems to arise from other factors in our case. Presumably, the activation and/or the emission efficiencies of 10a depend on the environment. As a result, we may be selectively imaging those probes that are less exposed to the polar medium. Alternatively, the average burst time (i.e., the time the system stays in the bright state, in this case isomer 10b) is also strongly dependent on the polarity of the environment, resulting in the same effect. Therefore, only molecules with long residence time in the bright state produce enough photons to be localized, while the rest only contribute to background (Figure 9). Finally, we cannot rule out that the hydrophobicity of the marker may hamper the selectivity of the antibody and result in a more disperse or inhomogeneous labeling of the microtubules (i.e., some discontinuities in the labeling of the filaments). While this effect has little or no observable influence in a diffraction-limited image (Figure 7), it becomes evident in a subdiffraction technique. This complication has recently stimulated the development of biomolecules smaller in size than full IgG antibodies for superresolution microscopies.89,90 Conjugate 13 has no isomerization mechanism that can be used for photoswitching in SM strategies, such as PALM and STORM.19−26 Relying on its photophysical states, however, the fluorophore can be used in STED microscopy,19−26 provided the emission from the first singlet excited state can be depleted by stimulated emission, the dye is sufficiently photostable at the excitation, and, more importantly, depletion high intensities required for such approaches. To this end, the samples already tested in confocal imaging were imaged in a commercial STED microscope. As Figure 10 shows, images with a significant resolution improvement can be obtained, despite the label not being optimized for the available laser lines (excitation at 640 nm and depletion at 775 nm). Again, tubulin filaments present transversal section of ∼70 nm, comparable to the value observed in the images obtained with bioconjugate 10a in the single molecule approach.

Figure 10. Superresolution imaging with 13. Confocal (A) and STED (B) images of Vero cells immunostained with 13. The images were recorder under excitation at 640 nm and STED at 775 nm. A line profile along the dotted line in A is shown in C. Pixel size = 30 nm; scale bar = 0.5 μm.

preparations with resolution at the nanometer level on the basis of stochastic schemes. In fact, the conjugated biomolecule retains its binding affinity for a complementary primary antibody and permits the immunostaining of subcellular structures with the photoswitchable labels. Their sequential photoactivation and SM localization within the stained cells then enables the reconstruction of images with approximately a 5-fold enhancement in resolution compared to their diffraction-limited counterparts. Furthermore, experiments with a model bioconjugate, incorporating the very same 3H-indolium chromophore of the photogenerated isomer of the dyads, demonstrate also that stimulated emission can be exploited to deplete the excited state of this particular fluorophore and switch its emission off. Specifically, tubulin filaments can be immunostained with this bioconjugate and imaged with also an approximately 5-fold enhancement in resolution, relative to their diffraction limited counterparts, on the basis of STED schemes. Thus, our photoswitchable fluorophores, in conjunction with conventional immunolabeling protocols, can indeed be exploited to image subcellular targets with nanoscale resolution and can evolve into an entire family of probes for bioimaging with superresolution.

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CONCLUSIONS A functional group suitable for bioconjugation, in the shape of an active ester, can be introduced on the photochromic component of a coumarin−oxazine dyad in three synthetic steps from commercial precursors. The resulting compound can be attached covalently to a secondary antibody in an additional step. Bioconjugation has minimal influence on the photochemical and photophysical properties of the dyad. Even after covalent attachment to the biomolecule, the oxazine ring of the photochromic component opens upon illumination and then closes thermally on a microsecond time scale. Furthermore, the photoswitchable dyad retains its characteristic fatigue resistance, even under these conditions, and can be switched multiple times between its interconvertible states with no sign of degradation. The photoinduced ring opening brings the coumarin fluorophore into electronic conjugation with the resulting 3Hindolium cation. This structural transformation shifts bathochromically the ground-state absorption of the fluorophore by ca. 90 nm and allows the selective excitation of the photochemical product with concomitant fluorescence. A significant fraction of the photogenerated and emissive isomers lives for a sufficiently long time to allow the collection of enough photons for SM localization. In turn, this behavior offers the opportunity to overcome diffraction and to reconstruct images of biological

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.6b03135. Fitting parameters, absorption spectra, and events and photons histograms (PDF) 12867


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; tel: +1 305 284 2639. *E-mail: [email protected]; tel: +54 11 4576 3380. Present Address #

EMBL, Heidelberg, Germany.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS F.M.R. acknowledges support from the National Science Foundation (CHE-1049860). M.L.B. acknowledges support from CONICET, the University of Buenos Aires, and ANCyPT.

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