Nitrogen-Doped Biocompatible Carbon Dot as a Fluorescent Probe for

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Nitrogen Doped Biocompatable Carbon Dot as a Fluorescent Probe for STORM Nanoscopy Navneet Chandra Verma, Chethana Rao, and Chayan Kanti Nandi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b12773 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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The Journal of Physical Chemistry

Nitrogen Doped Biocompatable Carbon Dot as a Fluorescent Probe for STORM Nanoscopy Navneet C. Verma, Chethana Rao and Chayan K. Nandi* School of Basic Sciences, Indian Institute of Technology Mandi, HP-175001, India. AUTHOR INFORMATION Corresponding Author * Dr. Chayan K. Nandi, School of Basic Sciences, Indian Institute of Technology Mandi HP-175001, India E-mail: [email protected]

Abstract: Fluorescent carbon dot (CD) has got tremendous applications in bioimaging, molecular sensors, light harvesting, photovoltaic, catalysis and drug delivery. This is mainly due to the superior photostability, high quantum yield, aqueous solubility and low toxicity. However super resolution nanoscopy using CD has rarely been reported. Here we report the super-resolution image down to ~64 nm in the actin filament and in self-assembled soft matter polymeric ring structure. The photon counts, on-off duty cycles and the number of switching cycles were good enough to get such high resolution. This opens up a new door to the realm of application of CD in live cell nanoscopy.

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Introduction: The breaking of diffraction limited spatial resolution in light microscopy has recently been revolutionized by stimulated emission depletion (STED)1, structured illumination microscopy (SIM)2, photoactivated localization microscopy (PALM)3, stochastic optical reconstruction microscopy (STORM), fPALM and dSTORM.4-6 Many natural and synthetic fluorescent probes such as organic dyes, fluorescent proteins and quantum dots are available for super resolution imaging.7 However, the main drawback of the organic dyes is that, sometimes they suffer from low photostability, fast photobleaching and poor brightness.8,9 On the other hand, although quantum dots are advantageous due to the high molar extinction coefficient, photostability and brightness, they suffer from the cytotoxicity.10, 11 In recent years, due to the simple and inexpensive one-step synthesis method, CD, a new class of carbogenic nanomaterial, have shown potential applications in sensors, catalysis, light harvesting solar cells and in drug delivery12, 13 In addition, the excitation-dependent multicolor emission makes CD even more versatile for bioimaging ranging from blue to red region. However, in spite of the superior photostability, high quantum yield, aqueous solubility and low toxicity super resolution nanoimaging using CD has rarely been reported.14,15 The main bottleneck of the CD is the lack of single molecule level information of its on-off blinking mechanism. Our group is continuously working on the understanding of the fluorescence origin of CD both in ensemble and single molecule level.16-19 While the reversible photoswitiching via creation of cationic dark states in an electron transfer mechanism was first reported in 2015,17 recently we are able to show the single molecule blinking mechanism in a single CD. The single particle fluorescence study shows that the mechanism of CD blinking has remarkable similarities with that of semiconductor quantum dots.18 In particular, the temporal behavior of CD blinking follows a power law both at room and at cryogenic temperatures. A Dexter type electron transfer between surface groups 2 ACS Paragon Plus Environment

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of a nanoparticle plays a major role in the transition of CD to off or grey states, whereas the transition back to on-states is governed by an electron tunneling from the particle’s core. We were further able to show that the CD can be localized with a localization precision of ~35 nm.19 Here we report localization based stochastic optical reconstruction nanoscopic imaging (STORM) with nitrogen-doped highly stable, biocompatable and nontoxic CD in real actin filament and in self-assembled soft matter polymeric ring structure. The important criterion of STORM nanoscopy is that the fluorophore should show sufficient fluorescence blinking in between on- (bright) and off- (dark) state, a large number of photon counts and a number of switching cycles.7 By optimizing the above parameters a full width half maximum (FWHM) of ~ 64 nm was achieved. This is approximately four times smaller than the diffraction limited resolution obtained in total internal reflection fluorescence (TIRF) microscopy image. Experimental Section: Materials: All Glassware’s were washed with aqua regia (3 HCl: 1 HNO 3 ), followed by rinsing several times with double distilled water. Chitosan (CHS), polyethylene glycol (PEG), imidazole, KCl, MgCl 2 , EGTA, DTT, CTAB, diamino octane (DAO), n-hexanol, unlabelled Phalloidin from aminta phalloisds, phalloidin labelled ATTO 647N and Cy3 dye were purchased from Sigma Aldrich. NaOH was purchased from Merck chemicals. Double distilled (18.3 MΩ) deionized (DI) water (Elga Purelab Ultra) was used throughout the entire process. Actin from rabbit mussel was purchased from Invitrogen.

Synthesis, purification and characterization of CD:



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CD was synthesized from the mixture of CHS and PEG, In the first step of the synthesis, CHS gel (2%) was prepared by dissolving 2 g CHS in 99 ml of water in the presence of 1 ml of acetic acid, under vigorous stirring condition. 25 ml of PEG was dissolved in 75 ml water (25 % PEG solution). 4.5 ml of the prepared CHS gel (2%) was mixed with 4.5 ml of the 25% PEG solution. In the resulting solution 1 ml of 5 M NaOH was added. The solution was heated at 600 W for 3 minutes in a microwave oven. The resulting material (chocolate brown) was diluted with 100 ml distilled water and the solution was filtered by Whatman filter paper. The resulting solution was ultra-centrifuged twice (sorvall Lynx 6000, thermo scientific) at 23000 rpm for 30 minutes. The pellets were removed each time and the final supernatant was collected. CD were dialyzed with a 3 kDa membrane for 3 days before use. Finally, the most purified homogeneous fractions of CD was achieved via column purification of the dialyzed sample. The detailed characterization of the synthesized materials was performed following standard methods. UV-VIS absorption and fluorescence emission were measured to study the optical properties. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were performed for size and shape analysis. For chemical characterization Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were carried out. The detailed analysis could be found in supporting Figure S1-S5. Poymerization of Actin Filament and CD labeling: Actin filaments were polymerized using a known protocol20. 5 M actin monomers in a buffer containing 25 mM imidazole, 25 mM KCl, 4 mM MgCl 2 , 1 mM EGTA, and 1 mM DTT were mixed together. 10 µg of phalloidin was also added in the reaction mixture to get stable filament. After 15 minutes, purified CD was added to stain the filament. After 20 minutes of incubation with CD, it was immobilized on a PolyL-lysin coated glass coverslip chamber slide. The unwanted free CD was removed from the glass slides by washing several times with the buffer. 50 mM of methyl viologen was added to get better blinking and 4 ACS Paragon Plus Environment

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super resolved results. To perform the experiment with Atto 647N, actin filaments were labelled by phalloidin conjugated Atto647 dye (sigma) following the same protocol mentioned above. Synthesis of CD labeled self-assembled polymer ring structure: Typically, 0.2 mg/ml CD solution and 0.05 mg/ml DAO were mixed and shaken well to get a clear solution. 0.036 g of CTAB was added and the solution was sonicated for 30 minutes to get uniformly disperse solution. Finally 80 µl of n-hexanol and 1 ml of phosphate buffer (concentration of 20 mM and at pH 6.0) were added with a final sonication for another 30 minutes. The obtained solution was centrifuged at 20000 rpm for 30 minutes to collect the supernatant and then diluted with water in the ratio of 1:10000 (conjugated solution : water). Finally, the solution were drop casted on cleaned coverslip-glass slides and kept in an incubator overnight at 350 C in the dark to get well shaped micron size ring structure.

Results and Discussion: The nitrogen doped CD was synthesized from a mixture of biocompatible chitosan and polyethylene glycol (PEG) via a simple microwave synthesis.21 Chitosan, which composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D- glucosamine (acetylated unit), has a number of biomedical, environmental and commercial uses. On the other hand, PEG is a hydrophilic linear polymer and is widely used as a surface passivation agent for various nanoparticles for biomedical applications. It facilitates effective radiative recombination and thus increases fluorescence intensity in materials.22 The detailed synthesis and characterization of the CD could be found in the experimental section later in the manuscript. The highly purified homogeneous size distribution of CD was obtained via sequential purification initially by dialyzing and then by column chromatography. It thus avoids the chemical heterogeneity, which is the most common problem with CD sample. The average size of the CD was found to be 4nm. This was confirmed by the height profile of the atomic force microscopy image and the statistical analysis of more than 60-70 CDs observed under the 5 ACS Paragon Plus Environment


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transmission electron microscope (Figure S1). The surface composition of the CD was also characterized by X-ray photoelectron spectroscopy (XPS) (Figure S2). The three peaks at 283.2, 401.0 and 529.8 eV in XPS spectrum (Figure S2a) can be attributed to C1s, N1s, and O1s, respectively. This suggests that the synthesized CD majorly contains C and O and small amount of N (% atom ratio of C: O: N is 67.04%: 30.50%: 2.46%). The XPS data further confirms the presence of nitrogen as a doping element in CD. The three peaks of the C1s spectrum (Figure S2b) at 283.5, 284.8 and 286.5 eV are assigned to C−C, C−N and C=O respectively. The two peaks at 530.7 and 531.3 eV in the O1s spectrum (Figure S2c) are assigned to C=O and C−OH/C−O−C groups, respectively, while the N1s spectrum (Figure S2d) shows two peaks at 397.8 and 398.2 eV which are assigned to the C−N−C and N−H groups, respectively. The FTIR spectrum (Figure S3) shows peaks at 1650 and 1574 cm-1 indicating the existence of -COOH and N-H bending respectively, while the broad peak with a maximum at 3331 cm-1 corresponds to the -OH and N-H stretching vibrations. The peak at 2904 cm-1 corresponds to the -CH stretching vibration, while the peak at 1071 cm-1 corresponds to C-O-C bond. The CD was highly stable in biological pH (pH 7.4) and almost nontoxic as confirmed by the cytotoxicity assay (Figure S4). The quantum yield of the CD was found to be 13.4%. The observed multicolor emission is due to either multiple emissive states or the presence of multichromophoric groups in a single CD as proposed recently (Figure S5).16, 23 Although the maximum fluorescence intensity of the CD was observed in the blue region, the multicolor emission helped us to use its emission at 570 nm (532 nm excitation) with sufficient photon counts, required to achieve high resolution STORM imaging. Reversible photoswitching, which is the fundamental criterion for a fluorescent probe to be STORM active has been efficiently observed in CD in the presence of electron acceptor such as methyl viologen (MV).17, 19 Figure 1 shows the single molecule time traces. Both the blinking and single step photobleaching were observed. Approximately 45% blinking and 55% single step photobleaching were observed from 200 such time traces. Interestingly, the blinking event was increased from 45% to 80% in presence of 50 mM MV. The image resolution in 6 ACS Paragon Plus Environment

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STORM nanoscopy depends on the photon counts, on-off duty cycle and the number of switching cycles.7 As the localization precision depends on the inverse-square-root of the number of photons, the high photon yield is desired for the accurate determination of a probe’s position.24, 25 On the other hand, the maximum number of fluorophores localized in a diffraction-limited area is inversely proportional to the duty cycle. Hence, a low duty cycle provides better resolution obeying that the maximum fluorophore density limits the image resolution according to the Nyquist sampling criterion. The stochasticity of the localization error is reduced by the detection of multiple switching cycles from the same fluorophore and hence the mean localization positions converge with the true positions of the fluorophores. As a result the more the number of switching cycles the better is the resolution.



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Figure 2. (a-c) and (d-f) are the measured photon counting histogram, number of switching events and on-off duty cycle with their exponential fitting for CD and Cy3 dye respectively. The number of detected photons for (a) CD is approximately three times less than that measured for (d) Cy3 dye. On the other hand, the number of switching cycles is approximately 2.5 times higher in (b) CD than (e) Cy3 dye. The inset of each figure shows the actual values of the entity vs. event number of the given measurement. 8 ACS Paragon Plus Environment

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We measured all the above parameters for the CD and compared the values with measured Cy3 dye in the same set up and also with the reported data as well (Figure 2). It should be mentioned that the measured photon counts, on-off duty cycle and number of switching cycles for Cy3 are very close to the reported value by Dempsey et.al (Table S1)7 Although the number of detected photons for CD is approximately three times less than that measured for Cy3 dye, the number of switching cycles is approximately 2.5 times higher in CD in comparison to Cy3 dye. Moreover, the number of detected photons was found to be better than several other reported dyes and fluorescent proteins. The usual photon counts of the dyes are around 103-104 and the best dye molecule emits a maximum of 106 photons,7 whereas the fluorescent proteins have the photon counts in the range of 700-1000 (supporting table S1).26 The on-off duty cycle is also comparable with the reported dyes or proteins. Further, the number of switching events in CD is also higher than for several reported dyes. The image quality can further vary significantly, if the on-off rate is not linearly dependent on the laser power. Fortunately, the present CD shows both the photon emission rate and off-switching rate linearly dependent on the excitation intensity (Figure S6). With these results, we believed that CD could be successfully employed to achieve localization based super resolved nanostructures. We first checked the effective super-resolution localization using CD directly coated on a coverslip. In each frame, the fluorescent spots were identified and then localized. From the each frame of recorded single molecule fluorescence burst, the point spread function of the molecule was fitted by a Gaussian function. Figure 3 represents the reconstructed spots from single molecule diffraction limited spots of CD using 532 nm excitation laser. Small regions marked with the square boxes are enlarged for detail observation. The comparative analysis of the two images demonstrates a drastic improvement in spatial resolution. Those spots that are unresolvable in the diffraction-limited image are clearly isolated in reconstructed image. Figure 3a is the normal TIRF image and Figure 3b shows the image of the localization spots on a cover slip. The average localization precision for CD obtained by the fitting of 3D 9 ACS Paragon Plus Environment


Gaussian distribution was ~35 nm (Figure 3c, bottom panel). We also carried out spot resolution of two very closely spaced spots on the cover slide. Figure 3c (top most panel) shows a representative histogram fitted in 2G Gaussian of two nearby spots. It is clearly evident that we are able to resolve the two spots separated by 98 nm.

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Figure 3: Single molecule localization of spin-coated CD on a clean coverslip. (a) The normal TIRF image of spin coated CD.(b) The image of the localized and reconstructed spots of CD on a cover slip. (c) The zoomed image of the localized spots from the rectangular box in (b). The top most panel of (c) shows a representative histogram fitted in 2G Gaussian of two nearby spots. It is clearly evident that, we are able to resolve the two spots by 98 nm. The bottom panel of (c) shows the average localization precision for a spot obtained by the fitting of 3D Gaussian distribution, which is ~35 nm. Analysis of 120 molecules show the standard deviation of ~5nm in localization precision. 10 ACS Paragon Plus Environment

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We performed the single molecule nanoscopic imaging in (1) actin filaments and in (2) self-assembled polymeric ring structure of soft matter. Actin filaments were polymerized using a known protocol20 and is described in the experimental detail section. Phalloidin is known to accelerate the polymerization reaction and finally to stabilize the filaments.27 As a result, 10µg of phalloidin was also added to the reaction mixture to get stable the actin filament. After staining with CD, 50 mM of methyl viologen was also added to get better blinking events and super resolution imaging. Figure 4a shows a typical diffraction limited wide field image (100x and 1.49 NA TIRF objective, Nikon) of the filament labeled with CD. To perform single molecule localization we, recorded a time series with 0.05s acquisition time per image. The 5min movie contained 6000 stacked images, which were analyzed with GDSC Single Molecule Localization

Microscopy (SMLM)

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at:https://github.com/aherbert/GDSC-SMLM) to screen out diffraction limited spots of individual particles and to calculate their centers from each localization event. Figure 4b shows reconstructed superresolved image of the filament. The super resolution image shows more detail and lesser background in comparison to the wide field images. The localization average pattern followed a Gaussian distribution (Figure 4c) with a minimum FWHM of ~76 nm. It should be noted that the analysis was done with the statistical average of more than 50 places along a single actin filament and the FWHM of the width of the filament were found to be ~80 nm. We also measured the super resolved structure of the same actin filament with phalloidin labeled ATTO647N and the minimum FWHM was obtained as 38 nm (Figure S7). This is two times higher than the reported value of 17 nm.7 It should be mentioned here that the large number of detected photons and number of switching cycles for ATTO 647N gave the high resolution structure.

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Figure 4. (a, b) TIRF and reconstructed image of actin filament respectively stained with CD. (c) Intensity profile along the dashed line (first from the left) and its Gaussian fitting gives the maximum filament FWHM of ~76 nm. (d) Intensity profile along the other (second from the left) line and its Gaussian fitting gives the filament FWHM of ~80 nm Next, we demonstrate the capability of the CD to visualize the ring structure of soft matter with a resolution much beyond the diffraction limit. Although, the in situ nano imaging of soft matter is of paramount importance, very little has been exploited using the super resolution nanoscopy. Recently using newly synthesized organic photo switchable probes, the super resolved structure of cylindrical micellar string structures that is made up from block copolymers has been reported.28, 29 In the present case we have used a very simple approach for the formation of CD labeled self-assembled ring structure by controlling the appropriate experimental condition as described in the experimental detail section. After formation of the rings, these were drop casted on cleaned coverslip-glass slides and kept in an incubator overnight at 350 C in dark to get well shaped micron size CD labeled ring structure. The larger diameter was produced to get a better resolved image. After sample preparation, using the drying mediated method, 12 Environment ACS Paragon Plus

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the nanoscopic imaging was performed following the same methods as mentioned earlier. The superresolved STORM image of the ring structure as represented in Figure 5a-c, showed the thickness of the ring with the FWHM of 64 nm with a diameter of 880 nm. The average measured thickness of the ring (measured from several ring structure) was ~85 nm, which is in very good agreement with the measured TEM data of the ring as shown in Figure 5d. It should be mentioned here that the diameter of the ring (880 nm) is little larger in the nanoscopic imaging than that measured in TEM (~650 nm). This could possibly be due to the different substrate mediated drying process used in microscopy and in TEM. In light microscopy, the air water drying mediated process was performed on the glass slide but in TEM, metallic copper grid was used. Also, the deformation can be observed in some of the actual ring structure compared to TEM image. This is most probably due to inhomogeneous labeling of the CD inside the ring structures. The measured thickness obtained in our case is exactly similar to the measured photo-activated localization microscopy (PALM) data (64 nm) and is little larger than measured via STORM (50 nm) in cylindrical micelles.28, 29 From the above data it is quite obvious to conclude that CD could be an easy alternative for the successful application in super resolution imaging of soft mater.

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Figure 5. (a) TIRF image of the self-assembled ring structure stained with CD and its (b) Superresolution reconstructed image (c) Transmission Electron Microscopy image of the ring structure (d) Intensity profile along the yellow line shown in figure (b) and its Gaussian fitting gives the FWHM of the periphery of the ring is ~64 nm.

Conclusion: In conclusion, we exploited CD for super-resolved localization based nanoscopic imaging in the actin filament and polymer ring structure. We rationalized its suitability for superresolved imaging by optimizing the photon counts, on-off duty cycle and number of switching cycles. By comparing the results with already reported known dyes, we show that the present CD is good enough to resolve the structure in actin filament and also self-assembled polymeric ring structure with a resolution down to ~64 nm. Our report suggests that the applicability of these CD is, however, not restricted to STORM, but can also be used for reversible saturable optical fluorescence transitions (RESOLFT) microscopy, super-resolution optical fluctuation imaging (SOFI) and photo-activable localization microscopy (PALM). We have improved the CD properties to achieve the optimal blinking for the super resolution imaging in artificial system. We believe that our results lay the foundation and opens up a new door to use CD for super resolution 14 Environment ACS Paragon Plus

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application in fixed and possibly in live cell in the near future. Further research is needed to functionalize the CD for the specific labeling of the cellular component and to get better super resolved live cell nanoscopic image.

AUTHOR INFORMATION Corresponding Author *Dr. Chayan K. Nandi, School of Basic Sciences, Indian Institute of Technology Mandi HP-175001, India E-mail: [email protected] ORCID Chayan K. Nandi: 0000-0002-4584-0738 Funding Sources Department of Biotechnology, Govt.of India.. Notes The authors declare no competing financial interests. Associated Content

Supporting Information. All used materials and detailed experimental procedures are available in supporting information. This material is available free of charge on the ACS Publications website. CND absorbance measurement,

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fluorescence measurement ,Transmission Electron Microscope (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM), X-ray photoelectron spectroscopy (XPS), Cytotoxicity assay (MTT), Single molecule time trace and photon counts analysis, localization based superresolution details.

Acknowledgment We acknowledge the AMRC facilities of IIT Mandi for our experiments. Department of Biotechnology India with Project number (Project No: BT/PR4067/BRB/10/1128/2012) is acknowledged for the financial support. Navneet Chandra Verma thanks Council of Scientific and Industrial Research (CSIR SRF: 9/1058(07)/17-EMR-1) Government of India for the financial support. References

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