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Preparation of Carbon Dots for Cellular Imaging by Molecular Aggregation of Cellulolytic Enzyme Lignin Na Niu, Zhuoming Ma, Fei He, Shujun Li, Jian Li, Shou-Xin Liu, and Piaoping Yang Langmuir, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Preparation of Carbon Dots for Cellular Imaging by Molecular Aggregation of Cellulolytic Enzyme Lignin ∥

Na Niu†,‡,§, Zhuoming Ma†,§, Fei He , Shujun Li*,†, Jian Li†, Shouxin Liu†, Piaoping Yang*, †

∥

Key Laboratory of Bio-based Material Science and Technology of Ministry of

Education, Northeast Forestry University, Harbin 150040, P.R. China ‡

Colledge of Science, Northeast Forestry University, Harbin 150040, P.R. China

∥

Key Laboratory of Superlight Materials and Surface Technology, Ministry of

Education, College of Materials Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, P.R. China

ACS Paragon Plus Environment

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ABSTRACT: Carbon dots, which are less than 10 nm in diameter, have been widely investigated because of their unique luminescence properties and potential for use in bioimaging. In the present work, natural carbon dots (L-CDs) were obtained by molecular aggregation, using ethanol-extracted cellulolytic enzyme lignin. The whole process for the preparation of L-CDs was green and simple to operate and did not use toxic chemical reagents or harsh conditions. The newly prepared L-CDs emitted multicolour photoluminescence following one- and two-photon excitation. The L-CDs also showed good cellular biocompatibility, which is crucial for biological applications. One- and two-photon cell imaging studies demonstrated the potential of L-CDs for bioimaging.

KEYWORDS: molecular aggregation, lignin, carbon dots, fluorescent, cellular imaging


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INTRODUCTION Bio-imaging is the cornerstone of research in both cell biology and developmental biology. Although a wide variety of fluorescent materials, including organic dyes, semiconductor quantum dots and upconverting nanoparticles, have been developed for use in biological imaging,1–6 most of these materials have poor photostability and/or poor biological compatibility.7–9 Recently, carbon dots (CDs), a new class of fluorescent nanomaterials with diameters less than 10 nm, have demonstrated great potential for applications in biological imaging, catalysis, and sensing.10–15 Compared with traditional organic dyes or semiconductor quantum dots, the CDs have significantly improved photostability and biological compatibility.16,17 Moreover, in addition to their very distinctive one-photon fluorescence properties, CDs can also be used for two-photon fluorescence imaging because of their absorption of near-infrared (NIR) light (800–900 nm).18–20 CDs are now recognized as a new class of high performance one- and two-photon fluorescence imaging agents for cancer cells. Until now, several methods, including laser ablation,21 arc discharge,22 microwave irradiation,23,24 electrochemical oxidation25 and hydrothermal carbonization,26 have been developed for the fabrication of CDs. Carbonization required for formation of the large conjugated carbon cores of CDs has always been triggered by heating, electrochemical techniques, microwave irradiation or treatment with acids. Thus more mild and eco-friendly processes for the preparation of CDs are still desirable. The ability to prepare CDs from cheap and sustainable carbon sources is another important consideration. And in this respect, biomass materials are suitable resources since they


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are green and cheap carbon precursors.27–35 Lignin is a class of complex organic polymers found in plants, which is also one of the most underutilized natural resources. Sufficient commercial applications for this material have yet to be identified. Syntheses of activated carbon materials and carbon fibres from sulfonated, alkali and other kind of lignin have been developed to explore it’s potential applications.36–39 Some fundamental studies investigating the molecular structure, J-type π-π aggregation induced molecular aggregation in organic solvent and aggregation-induced fluorescence emission of these materials have also been reported.40,41 Nevertheless, CEL, a by-product of fermentation to produce ethanol, has not been put to significant use and has not been well studied. Only one work has been reported for the synthesis of CDs using lignin through hydrothermal method,42 which in our opinion, dose not fully concern the advantage of lignin as the carbon resource of CDs. From a chemical perspective, CEL contains similar basic subunits and linkages compared to sulfonated or alkali lignin, but is much closer in structure to natural lignin, making it more suitable for green applications.43 Different types of lignin all have abundant oxygen-containing groups, substituted aromatic moieties and alkyl chains. Ideally, they should share similar physicochemical properties, such as π-π aggregation-induced molecular aggregation and aggregation-induced fluorescence emission. If CEL forms molecular aggregates in solvent, these molecular aggregates should thus be ideal materials for fluorescent CDs because of aggregation-induced emission. Neither heating nor other harsh conditions would be needed to form these CDs and they would provide a valuable application for underutilized CEL.


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Inspired by this, we now propose a simple and green molecular aggregation method for the preparation of CDs (referred to herein as L-CDs) from CEL. CEL with a narrow molecular weight distribution was firstly gained by ethanol extraction and centrifugation of raw fermentation residue, and π-π aggregation-induced molecular aggregation then took place in the ethanol solution. The molecular aggregates were subsequently purified by dialysis to provide the L-CDs. The structure of the newly synthesized L-CDs was investigated using nuclear magnetic resonance (NMR) spectroscopy,

X-ray

diffraction

(XRD)

spectroscopy,

transmission

electron

microscopy (TEM), Atomic Force Microscope (AFM), Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Cell viability experiments showed that the L-CDs had good biocompatibility. Confocal laser scanning microscopy (CLSM), using both one- and two-photon excitation, showed that the L-CDs were efficiently taken up by HeLa cells. Methods for the preparation and demonstration of luminescence properties of L-CDs are shown in Scheme 1. In the present study, we have developed an extremely simple and environmentally friendly method for the fabrication of CDs and, in doing so, have identified a potential method for converting CEL into a value-added product.

EXPERIMENTAL SECTION Materials The CEL used in this work was obtained from the residue that remains after fermentation of corn stalks to produce ethanol.44 During this process, most of the carbohydrates in the corn stalks are converted to ethanol, yet the lignin is basically


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unaltered. We have previously shown45 that this CEL comprises up to 62.18% of the fermentation residue, which also contains unreacted carbohydrates and ash. Compared with other industrial lignin, CEL contains abundant functional groups, including phenyl groups, phenolic hydroxyl groups and ether linkages46. All other reagents used in this work were analytical grade and were used without further purification. Preparation of L-CDs The raw fermentation residue was ground to a powder (< 80 mesh) and dissolved in anhydrous ethanol (~25 g/L).After stirring for 6 h at room temperature using a magnetic stirrer, the suspension was allowed to settle for about 30 min to precipitate insoluble substances. The supernatant was collected and centrifuged at 16000 g for 30 min to remove fine precipitates. The supernatant was separated, allowed to stand for several hours to allow formation of particles and then dialyzed with ethanol using dialysis tubing (8–14 KD molecular weight cut-off). After about 10 h, the solution of L-CDs that formed the dialysate was vacuum dried (0.1 MPa) at 40 ℃ for 24 h to provide the L-CDs as a powder suitable for further analysis. The L-CDs were re-dispersed in deionized water using ultrasonic irradiation and centrifugation. Characterization of L-CDs The structure of the L-CDs was examined by TEM and high-resolution TEM using a JEM 2100 transmission electron microscope (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 200 kV. The diameter distribution data from the TEM image were acquired using DigitalMicrograph software 3.4. Atomic force microscopy (AFM) images were obtained by Agilent 5400 AFM system (Agilent Technologies, Inc.,


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Santa Clara, CA, USA). The elemental composition and chemical bonding configurations of the L-CDs were determined by XPS, performed using a PHI5700 spectrometer (Physical Electronics, Inc., Chanhassen, MN, USA) equipped with an Al K-alpha X-ray source (1486.6 eV). Molecular weight analysis of the samples was conducted using an Agilent 1100 gel permeation chromatography (GPC) instrument (Agilent Technologies, Inc., Santa Clara, CA, USA), using polystyrene standards. The sample was dissolved in THF (1 mg/mL) and the flow rate of the mobile phase (THF) was set at 1 mL/min. FT-IR analysis of the samples was accomplished using a Nicolet 6700 Frontier system (Thermo Fisher Scientific, Inc., Madison, Wisconsin, USA), operating in the frequency range 500–4000 cm-1. The zeta potential was obtained by ZetaSizer Nano S90 (Malvern Instruments Ltd., United Kingdom). Raman spectra of the samples were recorded using a JY HR800 laser raman spectrometer (Horiba Jobin Yvon SAS, Paris, France), with a 458 nm laser beam. XRD spectra of the samples were obtained using a D/max-2200VPCpowder X-ray diffractometer (Rigaku Corp., Tokyo, Japan) with Cukα radiation (40 kV, 40 mA). 1H and 13C NMR spectra of L-CDs dissolved in methanol-d4 were recorded using a Bruker-Avance 400 MHz spectrometer (Bruker Corp., Karlsruhe, Germany), operating at a frequency of 100 MHz. UV–vis absorption spectra were recorded over the range 200–800 nm using a TU–1901 ultraviolet-visible double beam spectrophotometer (Persee General Instrument Co., Ltd., Beijing, China). Photoluminescence (PL) measurements were performed using a FLS980 fluorescence spectrophotometer (Edinburgh Instruments, Inc., Livingston, United Kingdom), equipped with a 150 W xenon lamp as the


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excitation source. A PL decay curve was recorded at room temperature using the time correlated double photon counting technique for L-CD transitions, with excitation at 320 nm. Two-photon emission spectra were also obtained using an FLS980 fluorescence spectrophotometer (Edinburgh Instruments, Inc., Livingston, United Kingdom) with an 808 nm LD as the excitation source, recording from 400 to 900 nm. The quantum yield of L-CDs was gained in a FLS980 fluorescence spectrophotometer (Edinburgh Instruments, Inc., Livingston, United Kingdom). The emission and excitation lights were scattered and collected in an integrating sphere and detected with a photomultiplier with detection range of 200 – 1010 nm. As for the L-CDs that dispersed in water, pure water was used as the reference. And as for the solid L-CDs sample, a piece made of BaSO4 (same materials of the integrating sphere) was used as the reference. The quantum yield is calculated with a formula: QY = Psample/(Sreferences – Ssample), in which the Psample is emission intensity, Sreferences and Ssample represent the scattered light intensities of the reference and sample. PL intensity of L-CDs in different pH values were also been taken in FLS980 fluorescence spectrophotometer using HCl and NaOH to adjust the pH value. All measurements were performed at room temperature. Cell viability measurement The effects of the newly prepared L-CDs on cell viability were examined in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in L929 fibroblasts. The L929 fibroblasts were seeded onto a 96-well plate, with 6000–7000 cells in 200 µL of culture medium per well. The plate was then incubated


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at 37 ℃ for 24 h in the presence of 5% CO2 to allow the cells to attach to the wells. The cells were then exposed to L-CDs at different concentrations (0, 12.5, 25, 50, 100, 200, 400 and 800 µg) and incubated for a further 24 h at 37 ℃ in the presence of 5% CO2. MTT solution (20 µL, 5 mg/mL) was then added to each well and, after incubation at 37 ℃ for 4 h, the absorbance of each well was measured using a microplate reader, with 490 nm as the detection wavelength. The average readings and standard deviations were based on four samples and all tests were performed in triplicate. Cell viability was calculated using the following equation: Cell viability (%) = Atest/Acontrol, where Atest is the average cell viability in the presence of L-CDs and Acontrol is the average cell viability in the absence of L-CDs (control experiment). Celluar uptakes and imaging Cellular uptake of L-CDs was examined using a confocal laser scanning microscope (CLSM, Leica SP8). HeLa cells were plated onto 6-well culture plates (8000–10000 cells per well) and a clean coverslip was placed on each well. The cells were then grown overnight as a monolayer and incubated with L-CDs (100 µg/mL) at 37 ℃ for different lengths of time. The cells were then rinsed three times with phosphate buffered saline (PBS). The nuclei were stained by treatment with 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) solution (Molecular Probes, 20 µg/mL in PBS, 1 mL/well) for 10 min. The cells were then rinsed three times with PBS, fixed with 2.5% formaldehyde (1 mL/well) at 37 ℃ for 10 min, and rinsed again three times with PBS. The cover slips were placed on a glass microscope slide and the samples were examined using the CLSM.


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RESULTS AND DISCUSSION Physiochemical characterization TEM revealed that the L-CDs were well dispersed and almost spherical (Figure 1a–c), with a narrow calculated size distribution (Figure 1d). Measurements of more than 130 particles were averaged to calculate the average particle size, which was found to be about 2.4 nm. All of the data indicate that the L-CDs have a very narrow size distribution. The TEMs showed obvious lattice fringes (Figure 1c), with an interplanar distance between adjacent lattice planes of 0.23 nm. Since CEL has an amorphous structure and no harsh conditions were used during the preparation of the L-CDs, the lattice fringes are most likely caused by the ordered arrangement imposed by π-π aggregation. In support of this hypothesis, AFM image (Figure S1) and GPC (Figure S2) analysis were also been provided. The relevant Abbott-Firestone curve displays that the L-CDs have a relative narrow height distribution of 0.6 to 3.0 nm, with an average height value of about 1.4 nm. The relative smaller height value than dimension can be contributed to the molecular aggregation induced CDs. And the small size is more beneficial for the application as fluorescent bioimaging. The GPC showed that the molecular weight of the L-CDs was less than 1 KD, which is more characteristic

of

molecular

aggregation

driven

by

π-π

aggregation

than

macromolecular lignin. XRD demonstrated that the L-CDs contained crystalline, as well as amorphous, structures. The broad peak at 21.3° in the XRD patterns of most of the samples (Figure S3a) indicates that the L-CDs have an amorphous structure. No crystalline structure can be gained may be due to the small number of single benzene


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rings and the weak interaction forces between these single benzene units among the whole amorphous structure of CEL. The Raman spectrum of the L-CDs (Figure S3b) shows peaks at 1347 cm-1 and 1592 cm-1, corresponding to the D-band and G-band of the material. The ratio of intensities of the D-band and G-band (ID/IG) is usually used to estimate the ratio of sp3/sp2 carbon atoms, which indicates the degree of disorder. In this case, the ID/IG ratio of the L-CDs was 0.57. This result is in agreement with the basic chemical structure of lignin and also confirms the coexistence of amorphous and crystalline structures in the L-CDs.47-49 The chemical composition of the L-CDs was characterized using XPS. The XPS survey spectrum shows two peaks at 285.1 and 533.1 eV (Figure 2a), which can be attributed to C1s (77.2%) and O1s (22.8%). The high resolution C1s and O1s spectra (Figure 2b and 2c) provide detail about different types of chemical bonds. The C1s spectrum could be divided into four peaks at 284.3 eV, 285.8 eV, 287.7 eV and 289.0 eV, corresponding to C-H/C-C/C=C, C-O, C=O and -COOR bonds, respectively. The O1s spectrum could be divided into three peaks at 531.3, 532.4 and 533.3 eV, corresponding to O-C=O/Ar-O-Ar, C-O/C-O-C and Ar-OH bonds, respectively. The FT-IR spectrum (Figure 2d) also helped to elucidate the surface groups present on the L-CDs. The broad peak at 3300 cm-1 resulted from the stretching vibration of O-H groups. The peaks at 2932 and 1460 cm-1 were attributed to asymmetric and symmetric vibrations of methyl or methylene groups.50 The peak at 1650 cm-1 corresponded to stretching vibrations of conjugated C=O bonds and the peaks at 1330 and 1215 cm-1 corresponded to stretching vibrations of aromatic C-O bonds. The three


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peaks at 1600, 1515 and 1425 cm-1 were associated with stretching vibrations of C-C bonds in the aromatic skeleton. The XPS and FT-IR spectra thus revealed that the L-CDs contain aromatic structures and are rich in oxygen-containing functional groups. The presence of carboxylate groups can make the L-CDs surface negatively charged in aqueous solution, which was confirmed by measured zeta-potential value averaged as –29.88 mV. The 1H and 13C NMR spectra of the L-CDs are shown in Figure 3. In the 1H NMR spectrum, signals due to methyl and methylene protons were observed at < 2.4 ppm (Figure 3a). Signals at 4.91 and 3.26 ppm were attributed to the CH3 and OH groups of residual methanol in the deuterated solvent. The broad signal at 3.4–4.0 ppm was attributed to methoxy groups in the syringyl and guaiacyl units. The weak signals at 5.35 and 2.67 ppm were attributed to H-α in the β-5 and β-β structures of lignin. The broad signals at 6.00–8.00 ppm are characteristic of aromatic protons,51 suggesting that an aromatic ring structure is present in the L-CDs. In the 13C NMR spectrum, the peaks for carbon atoms in saturated aliphatic side chains were present at < 40.0 ppm (Figure 3b). The signals corresponding to the methoxy group and methanol were observed at 56–57 ppm and 52–42 ppm, respectively. The signal at 50–75 ppm was assigned to carbon atoms in the propane side chains (α, β, γ) in the ether structures (α/β-O-4) and in the linkage bond (β-1). The peaks at ~110–150 ppm were assigned to aromatic carbon atoms, including carbon atoms (C-2/3/4/5/6) in the syringyl, guaiacyl and p-hydroxyphenyl units.52 The NMR data suggest that the L-CDs were primarily composed of low molecular weight CEL.


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Optical characterization The optical characterization showed that the L-CDs have favourable optical properties. Following excitation using an 800 nm laser, emission peaks occurred mainly at ~440 nm (Figure 4a). It is possible that, at certain wavelengths, the L-CDs emit anti-Stokes type emission, leading to emission peaks at shorter wavelengths than the excitation wavelength. The PL excitation (left) and emission (right) spectra are shown in Figure 4b. The emission spectrum was obtained by monitoring the excitation from incident light at 320 nm, which resulted in a broad emission peak with a maximum at ~400 nm. The UV-vis absorption spectrum (Figure S4) shows a broad peak at 280–500 nm, which was attributed to the typical absorptions of aromatic π systems and n-p* transitions of carbonyl and other oxygen-containing groups.53 Figure 4c shows the positions of the CIE (Commission Internationale de L'Eclairage 1931 chromaticity) coordinates calculated from the one- and two-photon emission spectra shown in Figure 4a and 4b. The L-CDs emitted bright blue-green light under both one- and two-photon excitation. The chromaticity coordinates were (x = 0.185, y = 0.241) and (x = 0.204, y = 0.201), respectively. The emission caused by irradiation with either 320 nm UV light or an 800 nm laser could be seen with the naked eye and was photographed using a digital camera (Figure 4c, inset). A typical decay curve for the luminescence of the L-CDs at 400 nm (λex = 320 nm) is shown in Figure 4d. The curve can be fitted to a double exponential function as I(t) = A1exp (-t1/τ) + A2exp (-t2/τ), where τ is the 1/e lifetime. The luminescence lifetime was calculated to be 6.827 µs. The quantum yields (QY) of the L-CDs in water and in solid state have been


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calculated as 1.68% and 2.47%, respectively. The higher QY of solid state L-CDs may due to the aggregation induced fluorescence. Since the L-CDs can absorb light over a broad range of wavelengths, PL emission spectra were obtained using different excitation wavelengths. When the excitation wavelength was increased from 280 nm to 500 nm, the emission wavelength gradually red-shifted from 390 nm to 560 nm, accompanied by a decrease in intensity (Figure 5a). This phenomenon has been previously reported with other carbon dots21,37. The L-CDs produced different emission colours, including blue, green and red, when excited at 320, 400 and 480 nm (Figure 5b). The excellent multicolour photoluminescent properties of L-CDs offer great potential for bioimaging applications. The PL intensity of the L-CDs in different pH values can were also changed (Figure S5). The PL intensity and position show differences when the L-CDs solution is acidity or alkalinity. When the pH value is 1 to 2, the intensity of the fluorescence decreased. When the pH value is 9 to 12, the PL intensity also decreased as well as the peak position showed redshift. When the pH value is 3 to 9, the L-CDs exhibit relative same PL spectra and the highest intensity, which is relatively stable. The pH-dependent emission might be caused by pH-sensitive strengthening or weakening of π-π aggregation, leading to changes in emission wavelength or intensity. The influences of ionic strength and temperature on the photoluminescence intensity of L-CDs were also investigated to understand the fluorescence stability in environment. As shown in Figure S6 and S7, the stability of as-prepared L-CDs was pretty well under different ionic strength and temperature conditions, indicating that


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can be applied in different environment. Cellular imaging MTT assays showed that the newly synthesised L-CDs have excellent biocompatibility (Figure 6). L929 fibroblasts remained viable when cultured for 24 h at 37 ℃ in the presence of increasing concentrations of L-CDs. Cell viability was greater than 90% even at an L-CDs concentration of 800 µg/L, indicating excellent biocompatibility. CLSM images (Figure 7) of HeLa cells incubated with L-CDs for different lengths of time at 37 ℃ were used to monitor cellular uptake. Images on the left hand side show cell nuclei stained with DAPI, images in the middle show L-CDs and images on the right hand side show these two images superimposed. In the first 30 min, only a few L-CDs were taken up by the HeLa cells (Figure 7a–c). After incubation for 1 h (Figure 7d–f) or 3 h (Figure 7g–i), more L-CDs had crossed the cell membrane and were localized in the cytoplasm. Inset in Figure 7h showed that the fluorescence intensity increased as time increased, proving the L-CDs were taken up efficiently by the HeLa cells. Some cell nuclei can be seen in Figure 7e and Figure 7h, indicating that the L-CDs can also enter into the nucleus because of their small size. Furthermore, the cross section fluorescence images of HeLa cell incubated with L-CDs for 3h were taken (Figure S8). It can be seen that the cell fluorescence is more strongly at the x and y cross section, indicating that the L-CDs has been successfully entered the HeLa cells. Green one-photon fluorescence emission (Figure 8a) and blue-green two-photon fluorescence emission (Figure 8b) observed in the inverted fluorescence microscopy


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images of HeLa cells demonstrate the robust cellular uptake of L-CDs. Combined with the luminescence imaging effect of the DAPI to the nucleus (Figure 8c), the imaging effect of the L-CDs to the HeLa cells by the one- or two-photon way can be further confirmed. The whole imaging effect to the cells can be observed from the overlay of the DAPI and one- or two-photon imaging images (Figure 8d, e), as well as the overlay of these three imaging results (Figure 8f). This dual-mode imaging can greatly expand the range of applications for a particular material. Based on the results described here, it can be inferred that the new L-CDs have low cytotoxicity, good cell permeability and versatile imaging effects, making them very suitable for bioimaging.

CONCLUSIONS In the present study, L-CDs were prepared from CEL using a green, simple and easy-to-operate molecular aggregation method. No harsh conditions or toxic chemical reagents were involved during the whole preparation, separation and purification process. The newly synthesised L-CDs were of uniform size, with an average diameter of approximately 2.4 nm. Structural analysis showed that the L-CDs were essentially composed of low molecular weight lignin, which contains aromatic rings and is rich in oxygen-containing functional groups. Following excitation at 320 nm, the L-CDs emitted a broad peak with maximum emission at about 400 nm and a luminescence lifetime of 6.827 µs. The L-CDs absorbed light over a broad range of wavelengths and the emission wavelength was gradually red-shifted as the excitation wavelength was increased. The PL stability of the L-CDs was relatively good under different pH, ionic strength and temperature. Following excitation at 800 nm, a


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two-photon emission was observed at about 440 nm. The L-CDs exhibit interesting one- and two-photon fluorescence and excellent biocompatibility, suggesting that they would be useful for cell imaging. CLSM images and inverted fluorescence microscope images obtained using both one- and two-photon excitation. Our newly developed method thus provides an efficient process for the fabrication of CDs that are suitable for bioimaging applications. π-π Aggregation-induced formation of CDs could also be used to prepare CDs from other biomass materials that contain benzene units.

ASSOCIATED CONTENT Supporting Information The AFM analysis, GPC curve, XRD spectra, Raman spectrum, UV–vis absorption spectra, PL emission spectra in different pH value, ionic strength, and temperature, as well as the CLSM fluorescence images of different cross section are given. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail:[email protected]; [email protected] Author Contributions §

Na Niu and Zhuoming Ma Contributed equally to this work.

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT


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This work was supported by the National Key Research and Development Program of China (2016YFD0600806), Fundamental Research Funds for the Central Universities (2572015BX13), Heilongjiang Postdoctoral Fund (LBH-Z16009), and China Postdoctoral Science Foundation (2016M591501). We are grateful for the fundings.


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Scheme 1. Diagram showing preparation and luminescence of L-CDs.


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Figure 1. (a–c) TEM images at different magnification; (d) Size distribution graph calculated using data from Fig. 1a Inset in panel c is corresponding high resolution TEM image of single L-CDs.


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Figure 2. (a) XPS survey spectrum, (b) XPS C1S peaks and fitting curves, (c) XPS O1S peaks and fitting curves and (d) FT-IR spectrum of L-CDs.


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Figure 3. (a) 1H and (b)

13

C NMR spectra of L-CDs, with peak assignments. S

indicates carbon atoms in syringyl residues (aromatic units with two methoxy groups), G indicates carbon atoms in guaiacyl residues (aromatic units with one methoxy group), ne indicates carbon atoms in non-etherified arylglycerol β-aryl ethers and e indicates carbon atoms in etherified arylglycerol β-aryl ethers.)


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Figure 4. (a) Two-photon fluorescence spectrum upon excitation with 800 nm laser and (b) one-photon emission and excitation spectra of L-CDs; (c) CIE chromaticity diagram and photograph of L-CDs showing emission colours; (d) fluorescence decay curve of L-CDs irradiated at 320 nm.


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Figure 5. (a) PL emission spectra of L-CDs with different excitation wavelengths (corresponding normalized spectra are shown in inset); (b) CIE chromaticity diagram and corresponding luminescent images of L-CDs upon excitation at 320 nm (point A), 400 nm (point B) and 480 nm (point C).


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Figure 6. In vitro L929 fibroblast cells viability after incubating with L-CDs with different concentration for 24 h.


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Figure 7. CLSM fluorescence images of HeLa cells stained with DAPI and incubated with L-CDs for (a, b, c) 0.5 h, (d, e, f) 1 h, and (g, h, l) 3 h at 37 ℃. Left hand panels show nuclei stained with DAPI, middle panels show L-CDs and right hand panels show these two images superimposed. Scale bars for all images are 50 µm. Inset in (h) is the intensities of main signals at different time points showing the L-CDs fluorescence.


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Figure 8. Inverted fluorescence microscope images of HeLa cells incubated with DAPI for 10 min and with L-CDs for 3 h. (a) One-photon luminescence image, (b) two-photon luminescence image, (c) fluorescence images of DAPI, (d) overlay of one-photon image with DAPI image, (e) overlay of two-photon image with DAPI image and (f) overlay of all three images . Scale bars for all images are 50 µm.


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