Carbon Dots for Single-Molecule Imaging of the Nucleolus - ACS

Jan 12, 2018 - These optical properties were used for performing blinking-assisted localization microscopy that shows organization of the nucleolar RN...
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Carbon Dots for Single-Molecule Imaging of the Nucleolus Syamantak Khan,* Navneet C. Verma, Chethana, and Chayan K. Nandi* School of Basic Sciences, Indian Institute of Technology (IIT) Mandi, Mandi, Himachal Pradesh 175001, India S Supporting Information *

ABSTRACT: Carbon dots are newly discovered bright fluorescent biolabeling probes that nonspecifically bind to multiple cellular structures. Here we report yellow-orange emissive carbon dots that spontaneously localize inside the nucleolus of HeLa cells, specifically binding to the RNA. Single-particle measurements of carbon dots show fluorescence-intensity fluctuations with superior brightness and photostability. These optical properties were used for performing blinking-assisted localization microscopy that shows organization of the nucleolar RNA with improved resolution. Our study opens up the opportunity for single-molecule imaging and super-resolution microscopy applications using fluorescent carbon dots. KEYWORDS: carbon dots, nucleolus, fluorescence, single-molecule imaging, super-resolution microscopy, HeLa cells, nucleolar RNA

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bioimaging probe,21,22 can spontaneously accumulate in the nucleolus of HeLa cells, enabling a rapid and easy process of nucleolus imaging. Although carbon dots have high cell permeability and low cytotoxicity, two common bottlenecks of these probes are their blue emission (excited by UV light) and nonspecific interaction with all cellular components.21,22 Therefore, studies toward wavelength tuning and specific biolabeling with carbon dots are highly valuable. Here we synthesized yellow-orange emissive carbon dots that spontaneously accumulate in the nucleolus of fixed HeLa cells. The carbon dots were synthesized from urea and citric acid using a solvothermal synthesis process23 and characterized by transmission electron microscopy, X-ray diffraction (XRD), and Raman and Fourier transform infrared spectroscopy. We chose citric acid as the precursor because carbon dots synthesized from citric acid are known to be nontoxic and more biocompatible than most of the commercial fluorescent probes. The carbon dots, with an average diameter less than 10 nm, show high crystallinity in selected-area electron diffraction (hexagonal spot pattern), XRD (002 plane), and Raman spectroscopy (D and G bands) results similar to those of graphitic carbon (Figure S1). It should be noted here that the formation mechanism of a large “graphitic” structure below 500 °C is debatable, and some of the characterization techniques might not be sufficient to confirm the formation of carbon particles.24−26 However, carbon dots are known to be bright and stable in physiological pH and temperature. In fact, they are generally stable through a long temperature range because they are formed in a high-temperature pyrolysis process.21,22 The UV−visible absorption spectrum shows a sharp maximum

he nucleolus is an essential organelle in the nucleus of eukaryotic cells. It is the site for the synthesis and assembly of ribosomal subunits. The function of the nucleolus is strongly linked to cell growth, cell proliferation, cell-cycle regulation, senescence, and stress responses.1−4 Disruption of the nucleolus function can induce a series of pathogenic conditions triggered by errors in ribosomal biogenesis.5 Tumors6 and viruses7 often induce alteration of the nucleolar size, organization, and function, which favors the propagation of pathogenesis. The nucleolus also regulates the response of tumor-suppressing genes.8,9 As a result, the nucleolus has begun to emerge as a potential target for therapeutic applications in various human diseases including cancer.8,10 Imaging of the nucleolus remained a challenging task because of the lack of specific targets. Nucleoli form around specific chromosomal regions called nucleolar-organizing regions and are made of proteins, DNA and RNA. While the nucleolus is known to host more than 700 proteins, most of them shuttle between the nucleolus and nucleoplasm, or even cytoplasm.11,12 As a consequence, labeling the nucleolus with high specificity is not easy. Electron microscopy reveals three main nucleolar componentsfibrillar center (FC), dense fibrillar component (DFC), and granular component (GC)that cannot be resolved by light microscopy.2,11,13 A few targets like RNA polymerase 1, upstream binding factor (target for FC), nucleolin, fibrillarin (target for DFC), nucleophosmin, nucleolar and coiled-body phosphoprotein 1 (target for GC or the whole nucleolus) have been identified14−18 as labeling targets using immunostaining-assisted techniques. In situ hybridization probes19 and arginine-rich peptides20 have also been used by targeting specific DNA and RNA sequences of the nucleolus. However, these techniques are complicated, timeconsuming, and costly. Here we show that fluorescent carbon dots, which are a newly discovered inexpensive bright © XXXX American Chemical Society

Received: November 9, 2017 Accepted: January 12, 2018 Published: January 12, 2018 A

DOI: 10.1021/acsanm.7b00175 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Nano Materials

Figure 1. (Top) Confocal micrograph of HeLa cells after fixation. Yellow: nucleolus, labeled with carbon dots. Blue: chromatins, labeled with DAPI. Red: actin filaments, labeled with phalloidin conjugated with Atto647. Inset: Fluorescence intensity decay of free and bound carbon dots. (Bottom) Images in the individual detector channels with three different laser excitations (401, 560, and 639 nm, respectively). Scale bar: 10 μm.

Figure 2. RNase digestion test confirming that carbon dots bind to RNA molecules present in the nucleolus. (a) Labeling of native HeLa cells showing that the carbon dots localize specifically in the nucleolus. (b) Labeling of HeLa cells after digestion with RNase enzyme showing that the carbon dots localize all over the nucleus with reduced concentration at the nucleolus. (Left) Transmission detector image. (Middle) Confocal image. (Right) Color map (red to yellow) of the confocal image. Scale bar: 10 μm.

at 542 nm corresponding to a π−π* transition, and the fluorescence spectrum shows an emission maximum at 612 nm (Figure S2). The fluorescence excitation spectrum nearly matches the absorbance spectrum. A notable property of these carbon dots is their spectral homogeneity. Unlike most of the reported carbon dots,21,22 the emission spectrum is

independent of the excitation wavelength (Figure S2), and therefore they can behave as conventional fluorophores. The Xray photoelectron spectroscopy data show the presence of carbon, nitrogen, oxygen, and sodium in the carbon dot. It also confirms the presence of carboxylic groups with a maximum at 289.10 eV in the C 1s spectra27 (Figure S3). The fluorescence B

DOI: 10.1021/acsanm.7b00175 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Nano Materials

Figure 3. Fluorescence time traces of a single carbon dot. (a and b) Two typical traces showing single-particle fluorescence blinking. (c) Steady signal with single-step photobleaching. (d) Photon count histogram showing an exponential distribution with an average of 1.6 × 104 photons per event. (e) Survival-time histogram showing superior photostability of carbon dots.

specificity, suggesting that carbon dots indeed bind to RNA. Figure 2 shows that carbon dots diffuse out of the nucleolus region and disperse throughout the nucleus after the degradation of RNA by an enzyme treatment. A color map (Figure 2, right panel) shows the intensity redistribution before and after RNase digestion. Thus, the RNA digestion test confirms that the carbon dot used in this study is specific to the nucleolar RNA. Because the abundance of RNA is commonly observed in most of the cell nucleolus, the carbon dots are expected to possess similar nucleolar affinities in other cells lines too. It should be mentioned here that, although a few recent studies have reported nucleolus-specific labeling with carbon dots with various cell lines,28−31 the exact origin of the RNA specificity is yet not understood. It is known whether the RNA is more reactive than DNA because of the presence of the 2′-hydroxyl group on the pentose ring. So, one can speculate that the carbon dot interacts with the 2′-hydroxyl group of RNA and may bind through chemical bonding. However, it should also be noted that RNA-specific fluorophores are very rare (only a few styryl dyes) in the literature and the binding mechanism is hardly understood. Therefore, with limited knowledge of the carbon dots’ chemical structures, it would be complicated to predict the binding mechanism at this stage. Nevertheless, because the carbon dots are highly specific to RNA present in the nucleolus, we extended their prospect for super-resolution imaging. The most popular approach to super-resolution imaging is to localize the positions of the individual molecules, commonly known as single-molecule imaging.32 However, they need a set of strict criteria of the optical properties, e.g., high photon count, photostability, and on−off switching at the singlemolecule level. Previous studies have proven that carbon dots have superior optical properties at the single-molecule level, which is appropriate for single-molecule imaging.33−35 They are known to show intensity fluctuation with ionic dark states that are controlled by charge-trapping dynamics similar to that of

quantum yield of the carbon dots was calculated to be 7.4% relative to Rhodamine B (Figure S4). The carbon dots were used for labeling HeLa cells (fixed with 10% paraformaldehyde) and studied using a confocal microscope (Nikon) with 560 nm laser excitation and a 60× oil immersion objective. Figure 1 (and Figure S5) shows carbon dot accumulation in the nucleolus (yellow) with chromatins counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The actin filaments of the cytoplasm were labeled (red) with Atto647-conjugated phalloidin. Individual panels show that the three regions were labeled very accurately without any significant overlap. The clear tricolor imaging reveals the high specificity of carbon dots for the nucleolus. It is worth noting that carbon dots do not get excited with other lasers and therefore are not visible in other detector channels. The superior imaging contrast can originate either from intensity enhancement in the nucleolus or by the physical accumulation of a large number of carbon dots. The fluorescent intensity decay (Figure 1, inset) shows that the fluorescence lifetime of free carbon dots slightly decreases with a more multiexponential nature when bound to the nucleolus. This eliminates the possibility of intensity enhancement upon labeling (like DAPI) where the fluorescence lifetime is expected to enhance. Therefore, it can be inferred that a large number of carbon dots accumulate selectively in the nucleolus. Next, we look into the possible origin of nucleolus specificity. While nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm, it is highly possible that a few of them have some natural affinity toward the carbon dot. However, the nucleolus is where rRNA synthesis and the assembly of ribosomes occur. Therefore, the specificity toward rRNA can also result in the accumulation of carbon dots inside the nucleolus. To check the possibility of RNA labeling, we digested the RNA of HeLa cells in situ using ribonuclease enzyme (RNase). The RNase-treated HeLa cells lost nucleolar C

DOI: 10.1021/acsanm.7b00175 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Nano Materials semiconductor quantum dots. 33,35 Chizhik et al. have performed stochastic optical fluctuation imaging, a type of super-resolution imaging with carbon dots.35,36 In this study, we observed that yellow-orange carbon dots also show singleparticle intensity fluctuation with superior photostability and photon count. Parts a and b in Figure 3 show two typical fluorescent time traces (∼80% of the molecules) with intensity fluctuations. A few (∼20%) of the time traces also show stable signal and single-step photobleaching (Figure 3c). A photon count histogram in Figure 3d shows an exponential distribution with an average of 1.6 × 104 photons per particle. This is comparable to other photoactivatable fluorescent probes like organic dyes, fluorescent proteins, and quantum dots, which typically yield on the order of 103 detected photons per switching event.37 Figure 3e shows a survival-time histogram, which illustrates the photostability of individual carbon dots. To elucidate the detailed localization of carbon dots, we performed single-molecule imaging inside the nucleolus (Videos S1 and S2 and Figure S6). HeLa cells were partially labeled (to avoid overstaining) with carbon dots without any counterstain for recording single-particle blinking. Figure 4a

the super-resolved (SR) image of the nucleoli. Instead of spherical symmetry, the SR image shows nucleolar ultrastructures with a reduced average diameter (∼500 nm). A magnified image of a single nucleolus is shown in Figure 4c. The SR images show more detail and lesser background in comparison to the WF images. To quantify the improvement, we plotted the intensity profiles along the dotted lines along two nucleoli (1 and 2) in both the WF and SR images. Figure 4d shows approximately 4 times improvement of the full-width half-maxima (fwhm) of the Gaussian profile after single-particle localization. Similarly, Figure 4e shows more detail of the intensity profile, indicating the resolved nucleolar ultrastructure in the SR image. The exact reason for RNA specificity of carbon dots inside the nucleolus remains unclear at this point and hence needs further investigation. Nevertheless, it is somewhat evident from the SR images that the carbon dots are localized toward the center of the nucleolus, which is known to host densely packed transcription units. Henceforth, it can be deduced that the carbon dots used in this study have a natural affinity toward some nucleolar RNA, which enable us to perform high-resolution imaging of the nucleolus. In conclusion, we present a type of fluorescent carbon dot for specific labeling of the nucleolus by targeting RNA molecules. The carbon dots emit in the yellow-orange region and show superior optical properties like excitation-independent emission and good quantum yield. Single-particle fluorescence displays intensity fluctuations with high photon budget enabling singlemolecule imaging. Blinking-assisted single-molecule imaging shows that carbon dots localize spontaneously in the central region of the nucleolus. This result opens up opportunities for future studies to rapidly monitor the dynamics of nucleolar RNA organization, especially in the propagation of various diseases. Because carbon dots are very easy to synthesize, they would be useful to some researchers working in this field. Further studies are required to elucidate the molecular interaction between carbon dots and different biomolecules to rationally design specific targets for biolabeling.



ASSOCIATED CONTENT

S Supporting Information *

Figure 4. Single-molecule imaging of carbon dots inside the nucleolus. (a) WF diffraction-limited image of three nucleoli of a HeLa cell. The yellow dotted circle shows the boundary of the nucleolus. (b) Reconstructed and SR image of three nucleoli. (c) Magnified view of a resolved nucleolus showing greater detail than the WF image. (d) Intensity profile along the dotted lines on nucleolus 1 showing 4 times improvement of the fwhm. (e) Intensity profile along the dotted line on nucleolus 2 showing the detail of some well-resolved ultrastructures inside the nucleolus.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.7b00175.



Experimental methods and supporting figures (PDF) Single-molecule blinking of a carbon dot (AVI) Carbon dots attached to the nucleolus (AVI)

AUTHOR INFORMATION

Corresponding Authors

shows a typical wide-field (WF) diffraction-limited image (100× TIRF objective, Nikon) of the HeLa cell nucleus with three nucleoli, which are labeled with carbon dots (excited with a 535 nm laser). The nucleoli show nearly spherical symmetry and an average diameter of ∼2 μm. To perform single-molecule localization, we recorded a time series with 0.05 s acquisition time per image. The 5 min movie contained 6000 stacked images, which were analyzed with the GDSC single-molecule localization microscopy (SMLM) algorithm (University of Sussex, Sussex, U.K.; code available at https://github.com/ aherbert/GDSC-SMLM), to screen out limited diffraction spots of individual particles and to calculate their centers from each localization event. Figure 4b shows a reconstructed image or

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Chayan K. Nandi: 0000-0002-4584-0738 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We acknowledge the AMRC facilities of IIT Mandi for our experiments and Department of Biotechnology India (Project BT/PR4067/BRB/10/1128/2012) for financial support. D

DOI: 10.1021/acsanm.7b00175 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Letter

ACS Applied Nano Materials



(26) Khan, S.; Sharma, A.; Ghoshal, S.; Jain, S.; Hazra, M. K.; Nandi, C. K. Small Molecular Organic Nanocrystals Resemble Carbon Nanodots in Terms of Their Properties. Chem. Sci. 2018, 9, 175−180. (27) Yang, Z.; Xu, M.; Liu, Y.; He, F.; Gao, F.; Su, Y.; Wei, H.; Zhang, Y. Nitrogen-Doped, Carbon-Rich, Highly Photoluminescent Carbon Dots from Ammonium Citrate. Nanoscale 2014, 6, 1890− 1895. (28) Kong, W.; Liu, R.; Li, H.; Liu, J.; Huang, H.; Liu, Y.; Kang, Z. High-Bright Fluorescent Carbon Dots and Their Application in Selective Nucleoli Staining. J. Mater. Chem. B 2014, 2, 5077. (29) Saini, A. K.; Sharma, V.; Mathur, P.; Shaikh, M. M. The Development of Fluorescence Turn-on Probe for Al(III) Sensing and Live Cell Nucleus-Nucleoli Staining. Sci. Rep. 2016, 6, 1−9. (30) Sun, S.; Zhang, L.; Jiang, K.; Wu, A.; Lin, H. Toward HighEfficient Red Emissive Carbon Dots: Facile Preparation, Unique Properties, and Applications as Multifunctional Theranostic Agents. Chem. Mater. 2016, 28, 8659−8668. (31) He, H.; Wang, Z.; Cheng, T.; Liu, X.; Wang, X.; Wang, J.; Ren, H.; Sun, Y.; Song, Y.; Yang, J.; Xia, Y.; Wang, S.; Zhang, X.; Huang, F. Visible and Near-Infrared Dual-Emission Carbogenic Small Molecular Complex with High RNA Selectivity and Renal Clearance for Nucleolus and Tumor Imaging. ACS Appl. Mater. Interfaces 2016, 8, 28529−28537. (32) Patterson, G.; Davidson, M.; Manley, S.; Lippincott-Schwartz, J. Superresolution Imaging Using Single-Molecule Localization. Annu. Rev. Phys. Chem. 2010, 61, 345−367. (33) Khan, S.; Verma, N. C.; Gupta, A.; Nandi, C. K. Reversible Photoswitching of Carbon Dots. Sci. Rep. 2015, 5, 11423. (34) Verma, N. C.; Khan, S.; Nandi, C. K. Single-Molecule Analysis of Fluorescent Carbon Dots towards Localization-Based SuperResolution Microscopy. Methods Appl. Fluoresc. 2016, 4, 44006. (35) Chizhik, A. M.; Stein, S.; Dekaliuk, M. O.; Battle, C.; Li, W.; Huss, A.; Platen, M.; Schaap, I. A. T.; Gregor, I.; Demchenko, A. P.; Schmidt, C. F.; Enderlein, J.; Chizhik, A. I. Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots. Nano Lett. 2016, 16, 237−242. (36) He, H.; Liu, X.; Li, S.; Wang, X.; Wang, Q.; Li, J.; Wang, J.; Ren, H.; Ge, B.; Wang, S.; Zhang, X.-D.; Huang, F. High-Density SuperResolution Localization Imaging with Blinking Carbon Dots. Anal. Chem. 2017, 89, 11831−11838. (37) Klar, T. a; Hell, S. W. Subdiffraction Resolution in Far-Field Fluorescence Microscopy. Opt. Lett. 1999, 24, 954−956.

REFERENCES

(1) Andersen, J. S.; Lam, Y. W.; Leung, A. K.; Ong, S.-E.; Lyon, C. E.; Lamond, A. I.; Mann, M. Nucleolar Proteome Dynamics. Nature 2005, 433, 77−83. (2) Leung, A. K.; Lamond, A. I. The Dynamics of the Nucleolus. Crit. Rev. Eukaryotic Gene Expression 2003, 13, 39−54. (3) Guarente, L. Link between Aging and the Nucleolus. Genes Dev. 1997, 11, 2449−2455. (4) Visintin, R.; Amon, A. The Nucleolus: The Magician’s Hat for Cell Cycle Tricks. Curr. Opin. Cell Biol. 2000, 12, 372−377. (5) Hetman, M. Role of the Nucleolus in Human Diseases. Biochim. Biophys. Acta, Mol. Basis Dis. 2014, 1842, 757. (6) Busch, H.; Byvoe, P.; Smetana, K. The Nucleolus of the Cancer Cell: A Reviews. Cancer Res. 1963, 23, 313−339. (7) Hiscox, J. A. RNA Viruses: Hijacking the Dynamic Nucleolus. Nat. Rev. Microbiol. 2007, 5, 119−127. (8) Woods, S. J.; Hannan, K. M.; Pearson, R. B.; Hannan, R. D. The Nucleolus as a Fundamental Regulator of the p53 Response and a New Target for Cancer Therapy. Biochim. Biophys. Acta, Gene Regul. Mech. 2015, 1849, 821−829. (9) Rubbi, C. P.; Milner, J. Disruption of the Nucleolus Mediates Stabilization of p53 in Response to DNA Damage and Other Stresses. EMBO J. 2003, 22, 6068−6077. (10) Hein, N.; Hannan, K. M.; George, A. J.; Sanij, E.; Hannan, R. D. The Nucleolus: An Emerging Target for Cancer Therapy. Trends Mol. Med. 2013, 19, 643−654. (11) Sirri, V.; Urcuqui-Inchima, S.; Roussel, P.; Hernandez-Verdun, D. Nucleolus: The Fascinating Nuclear Body. Histochem. Cell Biol. 2008, 129, 13−31. (12) Borer, R. A.; Lehner, C. F.; Eppenberger, H. M.; Nigg, E. A. Major Nucleolar Proteins Shuttle between Nucleus and Cytoplasm. Cell 1989, 56, 379−390. (13) Olson, M. O. J.; Dundr, M. Nucleolus:Structure and function. eLS 2015, 1−9. (14) Prieto, J. L.; McStay, B. Pseudo-NORs: A Novel Model for Studying Nucleoli. Biochim. Biophys. Acta, Mol. Cell Res. 2008, 1783, 2116−2123. (15) McLeod, T.; Abdullahi, a; Li, M.; Brogna, S. Recent Studies Implicate the Nucleolus as the Major Site of Nuclear Translation. Biochem. Soc. Trans. 2014, 42, 1224−1228. (16) Ginisty, H.; Sicard, H.; Roger, B.; Bouvet, P. Structure and Functions of Nucleolin. J. Cell Sci. 1999, 112, 761−772. (17) Mitrea, D. M.; Cika, J. A.; Guy, C. S.; Ban, D.; Banerjee, P. R.; Stanley, C. B.; Nourse, A.; Deniz, A. A.; Kriwacki, R. W. Nucleophosmin Integrates within the Nucleolus via Multi-Modal Interactions with Proteins Displaying R-Rich Linear Motifs and rRNA. eLife 2016, 5, 1−33. (18) Pai, C. Y.; Chen, H. K.; Sheu, H. L.; Yeh, N. H. Cell-CycleDependent Alterations of a Highly Phosphorylated Nucleolar Protein p130 Are Associated with Nucleologenesis. J. Cell Sci. 1995, 108, 1911−1920. (19) Puvion-Dutilleul, F.; Bachellerie, J. P.; Puvion, E. Nucleolar Organization of HeLa Cells as Studied by in Situ Hybridization. Chromosoma 1991, 100, 395−409. (20) Martin, R. M.; Ter-Avetisyan, G.; Herce, H. D.; Ludwig, A. K.; Lattig-Tunnemann, G.; Cardoso, M. C. Principles of Protein Targeting to the Nucleolus. Nucleus 2015, 6, 314−325. (21) Li, H.; Kang, Z.; Liu, Y.; Lee, S.-T. Carbon Nanodots: Synthesis, Properties and Applications. J. Mater. Chem. 2012, 22, 24230. (22) Lim, S. Y.; Shen, W.; Gao, Z. Carbon Quantum Dots and Their Applications. Chem. Soc. Rev. 2015, 44, 362−381. (23) Qu, S.; Zhou, D.; Li, D.; Ji, W.; Jing, P.; Han, D.; Liu, L.; Zeng, H.; Shen, D. Toward Efficient Orange Emissive Carbon Nanodots through Conjugated sp2-Domain Controlling and Surface Charges Engineering. Adv. Mater. 2016, 28, 3516−3521. (24) Oberlin, A. Carbonisation and Graphitisation. Carbon 1984, 22, 521−541. (25) Egerton, R. F.; Li, P.; Malac, M. Radiation Damage in the TEM and SEM. Micron 2004, 35, 399−409. E

DOI: 10.1021/acsanm.7b00175 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX