Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.


Synthesis of DBOV-azide 12
To a Schlenk tube equipped with a stirring bar was added 11 (1.2 mg, 0.79 μmol) and NaN3 (20.0 mg, 307 μmol).The reaction tube was evacuated and backfilled with argon for three times before the addition of DMF (2 mL).The mixture was degassed by three freeze-pump-thaw cycles and heated at 80 ℃ for 12 h.After cooling down to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water, brine, dried over Na2SO4 and evaporated.The residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol = 20/1 v/v) to give the product (1.00 mg, 85% yield) as blue oil.

Sample preparation for single-molecule measurements
Coverslip cleaning and coating of polystyrene film were done according to the previous literature 4,5 .In brief, the coverslips were sonicated in 1% Micro 90 alkaline cleaning solution for 15 min.Then the coverslips were rinsed three times with Milli-Q water and finally dried with nitrogen flow.Afterward, those coverslips were cleaned by oxygenplasma cleaner (250 W, 10 min).
The purification of polystyrene was carried out using an anti-solvent crystallization method.The polystyrene solid was dissolved in anhydrous THF to obtain a clear solution.Then an equal volume of MeOH was added to the solution, and the resulting mixture was let stand until it became a clear solution again, and all the crystals were precipitated.The solvents were subsequently removed, and the precipitates were washed with MeOH, and then dried in a vacuum desiccator prior to the use.100 μL of the solution of polystyrene purified above (4 mg/mL in toluene) was spin-coated on the cleaned coverslip.The coverslip was first spun at 2,000 rpm for 20 s and then at 4,000 rpm for 40 s.The sample was dried on a hot plate by heating at 90 °C for 10 min.

Preparation of nanographene samples for single-molecule blinking property measurements in PBS and different pH values solutions
10 μL of a solution of nanographene (10 -12 M in toluene/ethanol=1/99) was spin-coated on the polystyrene-coated coverslip.The coverslip was firstly spun at 2,000 rpm for 20 s and then at 4,000 rpm for 40 s.The sample was dried on a hot plate by heating at 70 °C for 15 min.

Preparation of nanographene samples for single-molecule blinking property measurements in air
10 μL of a solution of nanographene (10 -12 M in toluene) was spin-coated on the cleaned coverslip.The coverslip was first spun at 2,000 rpm for 20 s and then at 4,000 rpm for 40 s.The sample was dried on a hot plate by heating at 70 °C for 15 min.

Preparation of Aβ fiber
Aβ (No. RP10017) was purchased from Genscript as Aβ1-42 "click peptide".These click peptides can be easily converted to native peptide at pH 7.4 or above.Aβ1-42 peptide received was diluted with DPBS to a final concentration of 100 µM and stored in a -80 o C freezer until use.This peptide solution is a mixture of monomers and oligomers without pre-monomerization.For fibrils formation, 80 μL of DBOV-OTEG (10 nM) in DMSO was added to 20 μL Aβ1-42 peptide solution, then was incubated for five days at 37 °C.After Aβ fibrils formation, ThT solution was added at a final concentration of 200 nM for 30 min.Subsequently, 10 μL of the above solution was dropped on a clean circular coverglass (#1.5, 170 μm thickness) and dried in a vacuum desiccator.The fiber-deposited coverglass was washed three times with water to remove excess salt and unfixed fibers and then dried again for further measurement.

Live cell SMLM imaging of lysosomes with DBOV-OTEG
U2OS cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), in a 5% CO2 humidified incubator at 37 °C.The cells were then plated into 35 mm diameter glass bottom Petri dishes (ibidi) and incubated overnight under the same condition.Live U2OS cells were stained for four hours with 1 μM DBOV-OTEG in DMEM (supplement 10% FBS) medium, rinsed three times in phenol red-free DMEM (each time for five minutes), and 75 nM LysoTracker Green in DMEM was added for 30 minutes, and then washed twice with DMEM, finally replaced the washing medium with DMEM (supplement 10% FBS) before imaging.

Preparation of neurons sample and imaging
DRG neurons were incubated with 10 μM o-propargyl-puromycin (OPP -custom synthesized) diluted in the cell culture medium for 15 min at 37 o C. Neurons were then washed three times with PBS and then fixed for 20 min with 4% PFA in PBS at room temperature.Neurons were permeabilized for 15 min with PBS + Triton X-100 (0.3% w/v) and washed three times with regular PBS.Neurons were then incubated for 2 h at room temperature with the click mix solution: PBS at pH 7.8, 2 mM TBTA (Tris((1benzyl-4-triazolyl)methyl)amine), 1 mM TCEP (tris(2-carboxyethyl)phosphine), 2 uM DBOV, 0.5 mM CuSO4.Neurons were washed three times for 10 min with the click wash buffer: 0.5 mM EDTA, 1% Tween 20, PBS pH 7.8, and washed with regular PBS twice.DAPI in PBS staining was done for 20 min followed by two washes with regular PBS, and then mounting coverslips in PBS sealed with nail polish.
Control experiment 1: As a control, neurons were pre-incubated with 40 μM anisomycin (Sigma, A9789) for 30 min followed by 10 μM o-propargyl-puromycin (OPP -custom synthesized) diluted in the cell culture medium for 15 min at 37 o C. Cells were then washed three times with PBS and then fixed for 20 min with 4% PFA in PBS at room temperature, and then subjected to the same click reaction using DBOV-azide.
Control experiment 2: Neurons were incubated in the cell culture medium for 15 min at 37 o C. Cells were washed three times with PBS and fixed for 20 min with 4% PFA in PBS at room temperature, and then subjected to the same click reaction using DBOVazide.

Measurement of single-molecule blinking characterization and super-resolution imaging
Both single-molecule blinking property measurement and super-resolution imaging were performed using the SR GSD microscope (Leica).642 nm (500 mW) and 488 nm (500 mW) laser were selected for fluorescence reactivation.For the 642 nm laser, the excitation filter (637-647 nm/400-410 nm), the dichroic beam splitter (637-647 nm/400-410 nm), and the emission filter (660-760 nm/449-451 nm) were used.For the 488 nm laser, the excitation filter (483-493 nm/400-410 nm), the dichroic beam splitter (483-493 nm/400-410 nm), and the emission filter (500-550 nm/449-451 nm) were used.The objective lens HCX PL APO 160×1.43NA Oil CORR-TIRF was selected for single-molecule measurements and super-resolution imaging.The microscope was equipped with an EMCCD camera (iXonDU-897, Andor).The camera settings were 10 MHz at 14 bit and a pre-amplification of 5.1.Please note here that the double bandwidth of the filters/beam splitter were chosen for 405 nm back pumping and in our experiments mentioned in this work, such back pumping was not used.For single-molecule blinking measurement, 20,000 frames were recorded with an exposure time of 30 ms, EM gain of 100, 642 nm laser power of 5 kW/cm 2 .For super-resolution imaging of amyloid fibrils labeled with DBOV-OTEG in air and various pH value solutions, 20,000 frames were recorded with an exposure time of 50 ms, EM gain of 100, 642 nm laser power of 5 kW/cm 2 .We note here that at 642 nm, the absorption cross-section of DBOV-OTEG in water is around 3.57 x 10 -3 nm 2 .For live-cell superresolution imaging, 6,500 frames were recorded with an exposure time of 23 ms, EM gain of 100, 642 nm laser power of 1 kW/cm 2 .For super-resolution imaging of global nascent proteins labeled with DBOV-azide in PBS, 40,000 frames were recorded with an exposure time of 30 ms, EM gain of 100, 642 nm laser power of 5 kW/cm 2 .

SMLM image data analysis
All SMLM movies were analyzed with ThunderSTORM Plugin in ImageJ 6 .The peak intensity threshold was set as 1.7.And sigma > 90 and sigma < 150 were used to collect the true signal-molecule signal with a wavelength of 660 to 760 nm.Drift correction was done with cross-correlation function (number of bins of 3) in ThunderSTORM Plugin in ImageJ.For signal-molecule blinking characterization and super-resolution imaging of neurons, the continuous fluorescence signal was merged as one molecule.The localization precision of single-molecule was calculated based on Maximum-Likelihood methods 7 with ThunderSTORM Plugin in ImageJ 6 .All super-resolution images were reconstructed in ThunderSTORM, for SMLM image of amyloid fibrilspixel size of 10 nm, for SMLM images of lysosomes and neurons -pixel size of 5 nm.The localizations of global nascent proteins were used for Voronoi analysis (software: SR-Tesseler 8 ).All parameters are default (Density factor: 1, Min area: 2, Min # of locs: 5, Max are: 10,000, Max # of locs: 100,000).

Figure S18 .
Figure S18.Characterization of the DBOV-OTEG solubility by fluorescence correlation spectroscopy (FCS).Normalized FCS autocorrelation curve for DBOV-OTEG (red circles) and a reference dye ATTO643 (blue squares) measured in DMSO (left panel) and in PBS (right panel).In DMSO, the hydrodynamic radius (Rh) of DBOV-OTEG is around 1 nm indicating very good solubility.In PBS, the Rh of DBOV-OTEG is around 10 nm indicating the formation of very small aggregates.

Figure S19 .
Figure S19.Blinking properties of single molecule DBOV-OTEG in air, Left: Histogram of detected photons per switching event and single-exponential fit.Right: On-off duty cycle (fraction of time a molecule resides in its fluorescent state).

Figure S24 .
Figure S24.Wide-field images of neurons treated with anisomycin (ANISO), opropargyl-puromycin (OPP) and DBOV-azide (DBOV) in PBS.The top panel displays neurons labeled OPP in absence of ANISO.Newly synthesized proteins tagged with OPP were clicked with DBOV-azide (in red).The panel in the middle displays untreated neurons, which were still subjected to click reaction with DBOV-azide.Finally, the bottom panel displays neurons treated with ANISO for 30 min first, then treated with OPP and clicked with DBOV-azide.The parameters (exposure time and laser intensity) of microscope setting are the same for the three samples.The DAPI was excited with 405 nm laser and the DBOV-azide was excited with 635 nm laser.The brightness scale bar of images of DBOV-azide channel is showing on the right of images.The length scale bar for all images is 20 μm.