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Carbon Dots for Single-Molecule Imaging of the Nucleolus Syamantak Khan, Navneet Chandra Verma, * Chethana, and Chayan Kanti Nandi ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00175 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018
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Carbon Dots for Single-Molecule Imaging of the Nucleolus Syamantak Khan,*a Navneet C. Verma,a Chethanaa and Chayan K. Nandi,*a a
School of Basic Sciences, Indian Institute of Technology, Mandi. Mandi, Himachal Pradesh, India -175001
Abstract: Carbon dots are newly discovered bright fluorescent biolabeling probes, which non-specifically 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 the organization of 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
interaction with all cellular components.21,22 Therefore, studies towards wavelength tuning and specific biolabeling with carbon dots are highly valuable. Here we synthesized yellow-orange emissive carbon dots, which 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 (TEM), X-ray diffraction (XRD), Raman and infrared (FTIR) spectroscopy. We choose citric acid as a precursor as carbon dots synthesized from citric acid are known to be non-toxic and more biocompatible than most of the commercial fluorescent probes. The carbon dots, with an average diameter less than 10nm, show high crystallinity in selected area electron diffraction (SAED, hexagonal spot pattern), X-ray diffraction (002 Plane) and Raman spectra (D and G band) similar to graphitic carbon (Figure S1).It should be noted here that the formation mechanism of a large ‘graphitic’ structure below 5000 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 as they are formed in a high-temperature pyrolysis process.21,22 The UV-visible absorption spectrum shows a sharp maximum at 542 nm corresponding to π-π* transition and the fluorescence spectra shows an emission maximum at 612nm (Figure S2). The fluorescence excitation spectrum nearly matches the absorbance spectra. 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 XPS data shows the presence of carbon, nitrogen, oxygen, and sodium in the carbon dot. It also confirms the presence of carboxylic groups with a maximum at 289.10eV in the C1s spectra27 (Figure S3). The fluorescence quantum yield of the carbon dots was calculated to be 7.4 % in relative to Rhodamine B (Figure S4).
The 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 nucleolus function can induce a series of pathogenic conditions triggered by errors in ribosomal biogenesis.5 Tumors6 and viruses7 often induce alteration of nucleolar size, organization, and function, which favors the propagation of pathogenesis. The nucleolus also regulates the response of tumor-suppressing genes.8,9As a result, nucleolus has begun to emerge as a potential target for therapeutic applications in various human diseases including cancer.8,10 Imaging of nucleolus remained a challenging task due to 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 nucleolus and nucleoplasm, or even cytoplasm.11,12As a consequence, labeling nucleolus with high specificity is not easy. Electron microscopy reveals three main nucleolar components: the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC), which cannot be resolved by light microscopy.2,11,13Few 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 whole nucleolus) has been identified14–18as labeling target 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, time-consuming and costly. Here we show that fluorescent carbon dots, which are newly discovered inexpensive bright bioimaging probe,21,22can spontaneously accumulate in the nucleolus of HeLa cells enabling a rapid and easy process of nucleolus imaging. Though carbon dots have high cell permeability and low cytotoxicity, two common bottlenecks of these probes are their blue emission (excited by UV light) and non-specific
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The carbon dots were used for labeling HeLa cells (fixed with 10% paraformaldehyde) and studied using a confocal microscope (Nikon) with 560nm laser excitation and 60× oil immersion objective. Figure 1 (and Figure S5) shows carbon dot accumulation in the nucleolus (yellow) with chromatins counterstained with DAPI (blue). The actin filaments of cytoplasm were labeled with Atto 647 (red) 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 noteworthy that, carbon dots do not get excited with other lasers and therefore not visible in other detector channels. The superior imaging contrast can originate from either 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 more multi-exponential 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 accumulates selectively in the nucleolus.
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result in 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 lose nucleolar specificity suggesting that carbon dots indeed bind to RNA. Figure 2 shows carbon dots diffuse out of nucleolus region and disperse throughout the nucleus after degradation of RNA by 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. As the abundance of RNA is commonly observed in most of the cell nucleolus, therefore the carbon dots are expected to possess similar nucleolar affinity in other cells lines too. It should be mentioned here that, though few recent studies have reported nucleolus specific labeling with carbon dots with various cell lines,28–31 the exact origin of RNA specificity is yet not understood. It is known that the RNA is more reactive than DNA due to 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 a chemical bonding. However, it should also be noted that RNA specific fluorophores are very rare (only few styryl dyes) in literature and the binding mechanism is hardly understood. Therefore, with the limited knowledge of the carbon dots’ chemical structure, it would be complicated to predict the binding mechanism at this stage. .Nevertheless, as the carbon dots are highly specific to RNA present in the nucleolus, we extended their prospect for super-resolution imaging.
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Figure 1. 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 Atto647. Inset: Fluorescence intensity decay of free and bound carbon dot. Bottom panel: Images in the individual detector channels with three different laser excitations (401nm, 560nm, and 639nm respectively). Scalebar: 10µm
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Figure 2: RNase digestion test confirms that carbon dots bind to RNA molecules present in the nucleolus. (a) Labeling of native HeLa cells shows carbon dots localize specifically in the nucleolus. (b) Labeling of HeLa cells after digesting with RNase enzyme shows carbon dots localize all over the nucleus with reduced concentration at the nucleolus. Left Panel: Transmission Detector image. Middle Panel: Confocal image. Right panel: Color-map (red to yellow) of the confocal image. Scale bar: 10µm
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 few of them have some natural affinity towards the carbon dot. However, the nucleolus is the place of rRNA synthesis and assembly of ribosomes. Therefore, the specificity towards r-RNA can also
The most popular approach to super-resolution imaging is to localize the position of individual molecules, commonly known as single molecule imaging.32 However, they need a set of strict criteria of optical properties, e.g., high photon count, photostability, on-off switching at the single molecule level.
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Previous studies have proven that carbon dot has superior optical properties at 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 semiconductor quantum dots.33,35 Chizhik et al. have performed stochastic optical fluctuation imaging (SOFI), a type of super-resolution imaging with carbon dots.35,36 In this study, we observed that yelloworange carbon dots also show single particle intensity fluctuation with superior photostability and photon count. Figure 3 a & b shows two typical fluorescent time trace (~80% of the molecules) with intensity fluctuations. A few (~20%) of the time traces also show stable signal and single step photobleaching (Figure 3c). Photon count histogram in Figure 3d shows an exponential distribution with an average 1.6×104 photon per particle. This is comparable to other photoactivatable fluorescent probes like organic dyes, fluorescent proteins, and quantum dots, which typically yield in the order of 103 detected photons per switching event.37 Figure 3e shows survival-time histogram which illustrates the photostability of individual carbon dots.
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(SMLM) algorithm (University of Sussex, UK, 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 the super-resolved (SR) image of the nucleoli. Instead of spherical symmetry, the SR image shows nucleolar ultrastructures and with a reduced average diameter (~500nm). 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&2) in both WF and SR images. Figure 4d shows approximately four 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 SR image. The exact reason for RNA specificity of carbon dots inside nucleolus remains unclear at this point and hence need further investigation. Nevertheless, it is somewhat evident from the SR images that the carbon dots are localized towards 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 towards some nucleolar RNA, which enable us to perform high-resolution imaging of nucleolus.
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Figure 4. Single molecule imaging of carbon dots inside nucleolus. (a) Widefield (WF) diffraction limited image of three nucleoli of HeLa cell. The yellow dotted circle shows the boundary of the nucleolus. (b) Reconstructed and super-resolved image (SR) of three nucleoli. (c) Magnified view of a resolved nucleolus shows greater detail than the Widefield image. (d) Intensity profile along the dotted lines on nucleolus 1 shows four times improvement of FWHM. (e) Intensity profile along the dotted line on nucleolus 2 shows the detail of some well-resolved ultrastructure inside the nucleolus.
To elucidate the detailed localization of carbon dots, we performed single molecule imaging inside nucleolus (Video S12, Figure S6). HeLa cells were partially labeled (to avoid overstaining) with carbon dots without any counterstain for recording single particle blinking. Figure 4a shows typical diffraction limited wide-field (WF) image (100× TIRF objective, Nikon) of HeLa cell nucleus with three nucleoli, which are labeled with carbon dots (excited with 535nm Laser). The nucleoli show nearly spherical symmetry and an average diameter of ~2µm. 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
In conclusion, we present a type of fluorescent carbon dots for specific labeling of 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 QY. Single particle fluorescence displays intensity
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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental Methods, Supporting Figures (PDF)
AUTHOR INFORMATION Corresponding Author *
[email protected], *
[email protected] Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT We acknowledge the AMRC facilities of IIT Mandi for our experiments and Department of Biotechnology (Project No: BT / PR4067 / BRB / 10 / 1128 / 2012) India for the financial support.
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