Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels

Abstract The use of photoactivatable dyes in STED microscopy has so far been limited by two‐photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Herein, we report ONB‐2SiR, a fluorophore that can be both photoactivated in the UV and specifically de‐excited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water‐soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general.

Antibody Conjugation and Purification: All necessary chemicals for antibody conjugation were purchased from Sigma-Aldrich. For NHS-ester conjugated antibodies 400 µL (~1 mg) secondary goat anti rabbit antibody (111-005-003, Dianova) was mixed with 40 µL 1 M NaHCO3 and 200 µL DMF premixed with 100 µg NHS-ester dye. After one hour reaction under rigorous stirring, the sample was purified using either a standard size exclusion purification with a PD-10 (GE Healthcare) column or by phase separation. For the phase separation purification 1.2 mL distilled water, 182 µL saturated (NH4)2SO4 and 1.82 mL tert-butanol was added, quickly vortexed and separated into two phases after a short centrifugation pulse. The aqueous phase (~400 µL) was diluted to two times its volume with PBS. The conjugated antibodies were aliquoted and stored at -20 °C. The antibodies did not show any degradation even after months of storage at 5 °C.
To modify antibodies with azide-groups, 1 mg (~400 µL) secondary goat anti rabbit antibody was mixed with 40 µL 1 M NaHCO3 and 1.32 µL DMSO premixed with 13.2 µg N-hydroxysuccinimidyl azidoacetate. The mixture was stirred for 1 h and purified using a PD-10 (GE Healthcare) size exclusion column. TBS was used as elution buffer. The glycan labelled antibodies (250 µg) were produced using the commercial GlyClick (Genovis) enzyme system and eluted in TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.6). To the final volume of 200 µL (1 mg N-hydroxysuccinimidyl azidoacetate modified antibodies or 250 µg glycan azide antibodies), 50 µL DMF and 50 µg of azide reactive DBCO-dye was added. The mixture was stirred over night at room temperature. Then it was diluted with 600 µL distilled water, 90 µL saturated (NH4)2SO4 and 900 µL tert-butanol, quickly vortexed and separated into two phases after a short centrifugation pulse. The aqueous phase (~ 200 µL) was diluted to two times its volume with TBS. Optionally, the buffer can be exchanged using a regenerated cellulose MWCO spin filter (Microcon YM-50 50 kDa MWCO). The conjugated antibodies were aliquoted and stored at -20 °C.
Cell fixation and staining: HeLa and U2OS cells were grown on coverslips and then fixed with 4% (w/v) paraformaldehyde for 15 min or with cold methanol (-20°C) for 4 min. The cells were permeabilizied with 0.1% Triton X-100 in PBS for 5 min. After blocking with 2% (w/v) BSA in PBS, the cells were treated with antibodies diluted in the same buffer for 1 h. Between multiple antibody treatments, the cells were washed with 2% (w/v) BSA in PBS. The phalloidin conjugates were dissolved in DMSO and applied on the fixed cells for 1 h at a final concentration of ~100 µg/mL in PBS. The samples were mounted in PBS (wide-field, confocal and STED imaging) or 20 mM Hepes buffer (pH 7 + 150 mM NaCl) and sealed using silicon resin (TwinSeal Picodent). Used antibody: ATP-Synthase B (ab5432, Abcam), TOM 20 (sc-11415, SantaCruz), DNA (61014, Progen), alpha-Tubulin (ab18251, Abcam).

Imaging:
The wide-field images were taken on a Leica DM6000 microscope (Leica HCX Pl APO 100×/1.4-0.7 NA oil) using an A4 filter cube (Leica, excitation: 360/40 nm, detection: 470/40 nm) for activation and APC HC filter cube (AHF F36-540, excitation: 600/37 nm, detection: 675/67 nm) for imaging. For the STED images a commercial Abberior Instruments Expert Line microscope equipped with a UPLANSAPO 100×/1.4 NA oilimmersion objective and a 775 nm 40 MHz STED laser of ~ 1.2 ns pulse duration was used. The fluorophores were excited with a 640 nm and a 561 nm 40 MHz pulsed laser and detected in two color channels 615/20 nm and 685/70 nm. Between the acquisitions of the multiplexed color channel, the sample was bleached using a 595 nm laser. The caged dyes were activated using a wide-field 405 nm led illumination (CoolLED) with a broad spectrum extending far below 400 nm. The single molecule and PALM images were performed on a home-built microscope and analyzed as previously described [2] . In brief, the sample was illuminated through an oil immersion objective (Olympus UPLANSAPO 100×/1.4 NA oil) using a 642 nm laser in total internal reflection (TIRF) mode and imaged onto an EMCCD camera using a bandpass emission filter (700/75 nm). The molecules were sparsely activated using a 405 nm laser.  [3] A mixture of 2-bromoterephthalic acid (3.0 g, 12.2 mmol), thionyl chloride (10.0 mL), and DMF (2 drops) was stirred at reflux for 3 h. The mixture was concentrated in vacuum and co-evaporated twice with toluene. The crude acid chloride was taken up in CH2Cl2 (18 mL) and was added to a solution of 3-methyl-3oxetanemethanol (3.12 g, 30.6 mmol, 2.5 eq) and pyridine (2.2 mL, 61 mmol, 5 eq) in CH2Cl2 (12 mL) at 0 °C, and stirred for 10 min. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with water and extracted with CH2Cl2 (3 ×). The combined organic solutions were washed with brine, dried (Na2SO4), filtered, and evaporated. Flash chromatography (0-75% EtOAc/hexanes, linear gradient) afforded the diester (S2) as a colourless gum (4.90 g, 90%). The NMR spectra corresponded to the published data [

x 2-NBnOC-QSiR-6-Carboxylic acid (2-H-H):
A vial was charged with 8 (23.4 mg, 18.9 µmol), sealed, and evacuated/backfilled with nitrogen (3 ×). Freshly degassed THF (1.5 mL) and 1 M aq LiOH solution (0.40 mL, 0.38 mmol, 20 eq) were added, and the mixture was stirred at 55 °C for 48 h (protected from light). The reaction mixture was cooled to room temperature, acidified with 1 M HCl (1 mL), diluted with water, and extracted with CH2Cl2 (3 ×). The combined organic solutions were dried (Na2SO4), filtered, and evaporated. The residue was purified by flash chromatography on silica gel (0-10% methanol in CH2Cl2) to get the desired leucodiacid 2-H-H (6.0 mg, 37%) as an off-white solid which was used in the next step.The di-acid (5.0 mg, 5.8 µmol, 1 eq) was dissolved in 10:1 CH2Cl2/H2O (1.1 mL); DDQ (6.6 mg, 29 µmol, 5 eq) was added, and the reaction mixture stirred at room temperature for 18 h (protected from light). The mixture was concentrated in vacuo and was purified by flash chromatography on silica gel (10 g; 0-5% methanol/CH2Cl2, linear gradient) to afford diacid 2-H-H as an off-white solid ("closed" form, 4.5 mg, 90%). Analytical HPLC: >98% HPLC area (4.  Compound 11-H-H: To a solution of acid 2-H-H (4.1 mg, 4.8 µmol) and TSTU (2.9 mg, 9.6 µmol, 2 eq) in anhydrous DMSO (300 μL) was added N,N-diisopropylethylamine (21.9 µL, 125 µmol, 5 eq). The reaction mixture was protected from light and stirred at room temperature for 1 h (monitored by HPLC). To NHS ester formed in the reaction mixture, hydrophilizer (9) [6] (4.0 mg. 14.4 μmol) was added and the reaction mixture was sonicated in the ultrasonic bath at room temperature (shaken from time to time) for 15 min. Water (20 µL) was added and the reaction mixture was further stirred at room temperature for 18 h (monitored by HPLC). After completion of the reaction (HPLC comtrol), 9 μL of acetic acid were added. The reaction mixture was frozen and dried by lyophilization.     The total integrated emission vs. the absorption at the excitation wavelength (620 nm) is shown for compounds 1-OMe-H (triangles), 2-H-H (circles) and 3 (squares), in the same solvent. For the "caged" (masked) compounds, starting solutions were stepwise irradiated, while the concentration for compound 3 was increased by additions of a stock solution. An emission quantum yield of 0,60 was calculated, using Nile Blue in ethanol as a reference dye (Fluo = 0.27) [7] .