English

• 0.   Introduction

  Carbon dots (CDs) have gained interest due to their unique fluorescence properties, good biocompatibility, great aqueous stability, and facile synthesis. Thanks to their peculiar properties, CDs have shown great application in environmental detection^[1-3], biomedicine^[4-8], optoelectronic devices^[9-11], photocatalysis^[12-13], information encryption/anticounterfeiting^[14-15], and cancer treatment^[16-17]. Compared to other carbon sources, biomass carbon sources are ecofriendly natural items that offer numerous benefits for making CDs, including being cheap, accessible, green, and abundant^[18-28]. Additionally, low - value biomass waste may be transformed into valuable and practical materials by the manufacturing of CDs from natural biomass^[29-30]. Moreover, biomass containing heteroatoms is the best raw material for preparing CDs, in contrast to the CDs of manufactured carbon sources that require the addition of external heteroatoms^[31-33]. Therefore, various biomass has been used as carbon sources to prepare CDs recently.

  In terms of carbon source, biomass CDs can be roughly divided into vegetables and fruit^[18-22], Chinese medicine drinks^[23-25], plants^{[9, 26]}, seeds^[27-28], waste utilization, and so on^{[14, 29-30]}. Liu et al. have reported that carrot CDs, which offer excellent biocompatibility, low cytotoxicity, and good cellular imaging capability for HaCaT cells^[20]. Kim et al. have synthesized CDs with blue, green, and yellow fluorescent colors under hydrothermal conditions by using mango as a carbon source, derived through the controlled carbonization method by adding different concentrations of H₂SO₄ or H₃ PO₄ to the reaction solvent^[21]. Based on in vitro imaging and in vivo biodistribution results, biocompatible CDs have shown potential in biological fields. Wang et al. extracted CDs from Tsingtao beer, which showed excellent stability when exposed to strong acid, high salt, and laser irradiation environment^[25]. The results of the cytotoxicity study showed that the cell viability was not significantly inhibited even with a concentration as high as 12.5 mg·mL^-1. Therefore, the CDs can be directly applied in live MCF - 7, MDA - MB - 231, and BT - 549 human breast cancer cell imaging. Recently, Yu et al. synthesized highquality fluorescent CDs by hydrothermal treatment of food - grade oatmeal without any surface passivation^[28]. The obtained CDs emit strong blue fluorescence, and the fluorescence quantum yield is up to 37.40% at 360 nm excitation. Due to the combined advantages of green synthesis, high stability, and biocompatibility, CDs also show great potential for Hela cell imaging. However, we noticed that the majority of cancer cells are used in CD fluorescence imaging, such as lung cancer cells^[21], breast^[25], and cervical^[28], and relatively few studies of stem cells or normal cells are studied^[4]. As a potential cell imaging agent, its ability to image different types of cells must be examined to prove its universality. On the other hand, the vegetables and fruits are seasonal, which is not easy to preserve, so it is hard to ensure their repeatability as carbon sources due to the influence of climate in different regions. For plant - based and waste - utilizing CDs, the shortcomings are the same. The repeatability of carbon sources is difficult to guarantee, so the repeatability of synthetic CDs is conceivable. This poses particular challenges for future mass production and is not conducive to practical applications. On the contrary, if the seeds were selected as the carbon source, the disadvantage mentioned above can be overcome. Consequently, there is still wide space to develop green, inexpensive, and reproducible seed carbon sources for cell imaging.

  Because of this, black rice, which is cheap and easy to obtain, was chosen as the carbon source to synthesize CDs with excellent fluorescence properties and stability by hydrothermal method. Three typical cells of the normal cells (human oral keratinocyte, Hok), cancer cells (cervical cancer, Hela), and Bone marrow mesenchymal stem cells (BMSC, MC3T3) were selected for cell imaging. The results demonstrate that as-synthesized CDs using black rice possess good biocompatibility and can serve as a potential candidate for organic dyes or semiconductor quantum dots for cell imaging.

  1.   Experimental

  1.1   Reagents and instruments

  The edible black rice was purchased from a local supermarket and washed with deionized water for further use. Fe(NO₃)₃·9H₂O, MgCl₂·6H₂O, SrCO₃, MnSO₄· H₂O, Co(NO₃)₂·4H₂O, CrCl₃·6H₂O, CuSO₄·5H₂O, BaCl₂, AgNO₃, CaCl₂·2H₂O, KCl, ZnCl₂, La(NO₃)₃· 9H₂O, and LiNO₃ were purchased from Aladin Ltd. (Shanghai, China) and Beijing Chemical Corp. Fetal bovine serum (FBS), penicillin mixture, phosphate buffer saline (PBS), Roswell park memorial institute 1640, Trypsin, and cell counting kit - 8 (CCK - 8) were purchased from Amresco, USA. Hok cells were provided by the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science.

  Transmission electron microscopy (TEM, JEOL, JEM - 2100) measurements were carried out using an accelerating voltage of 200 kV. The samples for TEM characterization were prepared by coating the given volume of CD dispersion on a carbon - coated copper grid. With a Bruker D8 powder diffractometer, powder X - ray diffraction (PXRD) patterns were obtained for CDs with a Cu Kα radiation (λ=0.154 06 nm) at a scanning rate of 5 (°)·min^-1 from 10° to 80°, operating at 40 kV and 20 mA. The X - ray photoelectron spectroscopy (XPS) experiments were carried out on a Thermo ESCALAB Ⅺ with a monochromatic Al Kα X - ray source at a pressure of 5×10^-8 Pa. UV-Vis spectra were gained by a UV spectrophotometer (TU-1950) with one pair of 10 mm quartz cells. Fourier transform infrared (FT - IR) spectra were performed on an FT - IR spectrophotometer (Thermo-Fisher, Nicolet iS50) in a range of 400 - 4 000 cm^-1. Fluorescent emission spectra of the as - prepared CDs were recorded by a Horiba Jobin Yvon Fluorolog 3 - 22 fluorescence spectrophotometer. The absolute quantum yield (QY) uses an integrating sphere and a steady - state/transient fluorescence spectrometer (FLS1000, Edinburgh Instruments). The fluorescence lifetime decay profile was studied on the same instrument as fluorescence QY using a Time - Correlated Single Photon Counting (TCSPC) Spectrometer. An inverted fluorescent microscope was used for the cell imaging experiments (IX51, OLYMPUS).

  1.2   Preparation of black rice CDs

  The CDs were prepared by hydrothermal process. Black rice purchased from the local market was washed with purified water three times. The washed black rice was placed in an oven drying and then crushed into powder with a wall breaker. Black rice flour and water were mixed in a certain mass ratio and sealed in a Teflon container, which was then autoclaved at 180 ℃ for 12 h. After completion of the reaction, it cooled naturally to room temperature. The dark brown solution was obtained and purified by centrifugation and dialysis, which were intended to remove the reaction residue and by - products. During the dialysis, the water was replaced every 6 h. Finally, the obtained CDs were freeze - dried to form a brown powder. The whole synthesis process did not require any organic solvent, which was highly environment - friendly. The synthesis method of black rice CDs with different mass ratios and different temperatures was the same as above.

  1.3   Stability of black rice CDs

  To evaluate the stability, desired amounts of CDs were dissolved in solutions with a pH range from 3 to 11. The fluorescence intensities were then measured by fluorescence spectrophotometer, with the excitation and emission wavelength at 468 and 520 nm, respectively. Similarly, the effect of different metal ions and concentrations of NaCl solutions was also studied under the same conditions. In addition, the fluorescence properties of CDs obtained at different temperatures were investigated. The CDs powder sample was placed on a variable temperature stage connected to the spectrometer, mounted, and covered with a wafer cover glass. The fluorescence spectrum at 468 nm was measured after reaching the set temperature.

  1.4   Cytotoxicity

  The cytotoxicity of CDs was evaluated by using the CCK-8 assay. We seeded Hok in 96 -well plates at a density of 1×10⁴ mL^-1. After 24 h of incubation, the medium was replaced by the CDs with concentrations of 25, 50, 100, 200, 400, and 800 mg·mL^-1 and further incubated for 24 h. After the designated time intervals, the wells were washed thrice with PBS, and 100 μL of freshly prepared CCK - 8 (0.5 mg·mL^-1) solution in a culture medium was added to each well. After 2 h of incubation, the CCK - 8 medium solution was carefully removed. Dimethyl sulfoxide (100 μL) was then added to each well, and the plate was gently shaken for 20 min. The absorbance at 520 nm was recorded by the microplate reader (model SPECTRA, MAX PLUS 384). Cell viability was expressed by the ratio of absorbance of the cells incubated with CDs to that of the cells incubated with culture medium only.

  1.5   Cell imaging test

  Considering the excellent fluorescence properties and good stability of black rice CDs, we applied them to cell imaging. Hok cells were seeded in 96 - well plates at a density of 1×10⁴ per well and cultured at 37 ℃ for 24 h. We mixed the fluorescent suspension of filtered CDs (0.2 mg·mL^-1) with 1 mL of Hok cell culture medium and then added it to the wells. For fluorescence imaging, the cells were washed thoroughly three times with PBS after an incubation of 24 h. Afterward, fluorescence imaging of cells was carried out using a fluorescence microscope.

  2.   Results and discussion

  2.1   Microstructural characterization

  All the samples of CDs to be used for characterizing and testing were prepared by hydrothermal methods in a ratio of 1 g of black rice (br) flour to 20 g of ultrapure water (m_br∶m_H2O=1∶20). It is known that the size of CDs is not only related to their fluorescence properties but also an important parameter as a cell imaging agent. TEM images revealed that the obtained CDs had uniform size distribution in a range of 12 -30 nm, with an average size of 20 nm (Fig. 1a), which was much smaller than that of oatmeal CDs^[28]. This size allows CDs to be rapidly metabolized out of the body, ensuring the safety of future clinical applications. At the same time, no discernible lattice fringes were observed on the high - resolution TEM, indicating the amorphous characteristics of the prepared CDs, which is consistent with the XRD results. A broad diffraction peak at 2θ=21.5° was observed in XRD patterns shown in Fig. 1b, suggesting an amorphous nature. To identify the organic functional groups on CDs, the FT-IR spectra of the black rice and obtained CDs were carried out, as shown in Fig. 1c. It is not difficult to find that both the black rice and CDs exhibit characteristic absorption bands of —OH stretching vibration at 3 262 cm^-1[30], C—H stretching vibration at 2 920 cm^-1, and C— N stretching vibration at 1 393 cm^-1, C=C stretching vibration at 1 654 cm^-1[20], and C—O stretching vibration at 1 026 cm^-1[30]. These results show that the CDs and carbon source (black rice) display many common characteristics. Still, we found that the absorption peak at 3 262 cm^-1 was significantly reduced, indicating that a dehydration reaction occurred during the synthesis of CDs. The absorption peaks at 2 920, 1 393, and 1 026 cm^-1 indicate that carbonization has happened again. With the increase in temperature, CDs were further polymerized and were finally obtained CDs. XPS was also used to further investigate the elements and chemical structures of CDs. The XPS spectrum of the CDs suggests the presence of carbon, nitrogen, and oxygen (Fig. 1d). The C1s high-resolution spectrum is shown in Fig. 1e. The peaks at 283.1, 284.5, 285.5, and 286.5 eV correspond to the C—C, C=C, C—O, and C=O, respectively^[20]. N1s spectrum of CDs could be split into three peaks centered at 397.7, 398.8, and 400.4 eV, which are probably ascribed to C—N—C, N— (C)₃, and N —H functional groups, respectively (Fig. 1f)^{[12, 19]}. XPS and FT-IR results show that the CDs surface is rich in —COOH and —OH functional groups, which promote the dispersibility of CDs in water.

  Figure 1

  

  Figure 1.  (a) TEM images of CDs; (b) XRD pattern of CDs; (c) FT-IR spectra of the black rice and CDs; (d) XPS survey spectrum, (e) high-resolution spectrum of C1s, and (f) high-resolution spectrum of N1s for CDs

  Inset: particle size distribution histogram

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  2.2   Optical properties

  To explore the optical properties of the prepared CDs, UV - Vis and photoluminescence spectra have been conducted. We first measured the fluorescence spectra of CDs synthesized with different m_br∶m_H2O values of black rice. It can be seen that the fluorescence intensity of the synthesized CDs gradually increased with increasing mass of water before 1∶80 (Fig. 2a) and decreased with increasing mass of water after 1∶80. The reason is simply explained below. When the m_br∶m_H2O was larger, part of the fluorescence was absorbed by the substance itself, resulting in a decrease in fluorescence intensity. At the same time, when the m_br∶m_H2O was too small, the number of chromophores involved in emitting fluorescence in black rice CDs was smaller, so the fluorescence emission intensity was smaller. Therefore, the fluorescence intensity of the synthesized CDs reached the maximum when the m_br∶m_H2O was 1∶80. Meanwhile, we observed that with the decrease of m_br∶m_H2O, the maximum emission peaks shifted to the lower wavelength (from 550 to 520 nm), which may be due to the increase of the band gap caused by the quantum size effect. So, we decided that the best m_br∶m_H2O was 1∶80. Fig. 2b from left to right are the UV - Vis absorption spectrum (black), the excitation (blue), and emission (red) spectra of CDs (1∶80). The strong UV-Vis absorption band at 284 nm could be attributed to the π-π* transition of the C=C bond in the graphite carbon, which is consistent with the results of CDs synthesized from bee pollen^[13]. The UV-Vis absorption and emission spectra of CDs synthesized at different temperatures were also measured, as shown in Fig.S1 (Supporting information). We observed that both the peak of the absorption spectrum and the emission spectrum of obtained CDs reached a maximum at 180 ℃. The strongest absorption peak was located at 284 nm, which corresponds to the chromophore of CDs, thus helping to enhance the fluorescence of CDs. Moreover, the greater the intensity of the absorbed light, the greater the number of particles excited to a high energy level, and the stronger the fluorescence produced by the downward transition. The luminescence life of CDs synthesized at different temperatures was also measured, and the computed results of the average fluorescence lifetimes are 6.91 ns at 160 ℃, 5.53 ns at 180 ℃, and 5.91 ns at 220 ℃, respectively (Fig. S2). Molecules with large conjugated double bonds easily emit fluorescence, which has a high quantum efficiency of π - π* transitions and a short lifetime (10^-9 -10^-7 s). The greater the conjugation effect, the higher the fluorescence efficiency. According to the UV-Vis absorption spectra, emission spectra, and fluorescence lifetime of CDs synthesized at different temperatures, we conclude that the best reaction temperature of CDs was 180 ℃. To sum up, the optimum synthesis conditions are set as 1∶80, 180 ℃, and 12 h. In addition, the strongest emission peak occurred around 520 nm, which corresponds to the optimal excitation wavelength of 468 nm, as shown in Fig. 2b.

  Figure 2

  

  Figure 2.  (a) Emission spectra of CDs synthesized by black rice with different m_br∶m_H2O values; (b) UV-Vis absorption, excitation, and emission spectra for CDs

  Inset: photographs of CDs under daylight (left) and UV light (right)

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  To further investigate the wavelength-dependence feature of CDs, the fluorescence emission spectra of CDs solution was recorded by changing the excitation wavelengths. Here, we selected the CDs synthesized with the m_br∶m_H2O values of 1∶80 and 1∶20 to study, and the results are shown in Fig. 3 and S3. It was found that when the excitation wavelengths changed from 410 to 470 nm, the position of the emission peak barely moved, which kept at about 520 and 536 nm, respectively. Meanwhile, the fluorescence intensity of CDs gradually increased with increasing excitation wavelength, as shown in Fig. 3a and S3a. This part of fluorescence characteristics mainly comes from the carbonyl and amino functional groups containing double bonds on CDs. The difference in energy levels from the fixed ground state to the excited state leads to the fact that the emission wavelength does not change with the change in excitation wavelength^[28]. Fluorescence intensity decreased dramatically as the excitation wavelength increased from 470 to 510 nm, indicating a weak dependence on the excitation wavelengths, as shown in Fig. 3b and S3b. Although up to date, it is not entirely clear what causes fluorescence emission from CDs, we believe that the wavelength - dependent phenomenon may arise from the different distribution of emissive energy traps on their surfaces^[34-35].

  Figure 3

  

  Figure 3.  (a) Excitation-independent spectra and (b) excitation-dependent spectra of CDs with a m_br∶m_H2O value of 1∶80

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  2.3   Stability

  We assessed the fluorescence stability of CDs aqueous solution in order to determine if CDs could be used as a fluorescent biomarker in a real biological environment. As shown in Fig. 4a, when the pH value was increased from 3 to 10, the fluorescence intensity of the CDs solution at 468 nm only displayed minor fluctuations. A physiological environment often has a pH value of 7-8, which is advantageous for their bioimaging applications. To investigate the durability of CDs in high ionic strength settings, the fluorescence spectra of CDs were also recorded in NaCl solutions of various concentrations. As demonstrated in Fig. 4b, there was only a slight decrease in fluorescence intensity even at a high NaCl concentration of 1 mol·L^-1, which also proves that CDs have excellent stability. We also studied the influence of temperature on the fluorescence of CDs between 20 and 100 ℃. According to the illustration in Fig. 4c, it was apparent that the fluorescence intensity decreased at higher temperatures (100 ℃), while the fluorescence declined by only 5% when the temperature ranged from 30 to 40 ℃. Considering that the normal temperature range of the human body is between 35 and 38 ℃, the fluorescence changes of CDs caused by temperature can be ignored when applied to human cell imaging. In addition, the fluorescence of CDs has also been quenched through redox reactions or electron transfer mechanisms involving heavy metal ions, such as Hg²⁺ and Cu²⁺. Accordingly, we studied the fluorescence characteristics of CDs solution in the presence of several metal ions commonly found in living cells, as shown in Fig. 4d. The concentration of all metal ions was maintained at 50 μmol· L^-1. A slight quenching of fluorescence intensity (< 15%) was observed when La²⁺ ions were added to CDs aqueous solution, while the effect on the fluorescence intensity of CDs aqueous solution was relatively obvious for Ag⁺ and Cr³⁺ ions. However, for some other metal ions (Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Hg²⁺, K⁺, Li⁺, Mg²⁺, Mn²⁺, Na⁺, Sr²⁺, and Zn²⁺), more than 60% of the fluorescence intensity of CDs solution was still retained. The metal ion-induced fluorescence quenching and its sensitivity to different ions are influenced by the structure of CDs which are associated with the synthesis methods and the carbon sources. As we know, there are abundant macromolecules in biological systems, such as RNA, DNA, cysteine (Cys), serine (Ser), homocysteine (Hcy), leucine (Leu), glutathione (GSH), valine (Val), aspartic acid (Asp), tyrosine (Tyr), tryptophan (Trp), alanine (Ala), methionine (Met), threonine (Thr), glycine (Gly), isoleucine (Ile), arginine (Arg), lysine (Lys). The necessary condition for being a fluorescent marker is to be free from their interference. To this end, we give intuitive evidence after adding different macromolecular materials, and the experimental results are shown in Fig.S4. We found that the addition of the same concentration of the above macromolecular materials had little effect on the fluorescence of CDs. The above results show that CDs synthesized with black rice as a carbon source have excellent stability and biocompatibility, which is suitable as fluorescent probes for intracellular labeling.

  Figure 4

  

  Figure 4.  (a) Normalized fluorescence intensities of CDs under different pH values; (b) Normalized fluorescence intensities of CDs with different NaCl concentrations; (c) I/I₀ of CDs at different temperatures; (d) I/I₀ of CDs after adding different metal ions

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  2.4   Cell imaging

  The biocompatibility of CDs allows them to have many applications in biology, including bioimaging, drug delivery, and disease treatment. In this work, the biocompatibility of CDs was evaluated by CCK-8 assay. Hok cells were incubated with different concentrations of CDs in 96-well plates, and the cellular viability was determined using a microplate reader at 520 nm. Fig. S5 exhibited the cellular viabilities of Hok cells treated by CDs, with a concentration of 0-400 μg·mL^-1. It can be seen that when the concentration of CDs was increased to 200 μg·mL^-1, more than 88% of Hok cells were still alive after incubation for 24 h, indicating that the cytotoxicity of CDs was very low. The experimental results are comparable to those reported in the literature^{[22, 36]}. Therefore, black rice CDs derived from natural sources are safe, which ensures them being potentially used for bioimaging and other biological applications. After that, CDs were evaluated for their bioimaging capabilities. Hok cells were incubated with CDs (200 μg·mL^-1) for 12 h and observed by an inverted fluorescence microscope. According to Fig. 5, CDs did not significantly alter or damage cell morphology, confirming their low toxicity. Using blue, green, and red channels to excite and observe the cells, it was discovered that the cells only emitted red and green fluorescent signals. Fluorescence signals of green and red could be explained by the fluorescence spectra of CDs, which are excited by wavelengths above 470 nm (Fig. 3b). Moreover, it is clear that the green and red fluorescence is mostly detected in the cytoplasm of cells rather than in the nucleus. It shows that the CDs pass through the cell membrane successfully, and retain their fluorescence properties.

  Figure 5

  

  Figure 5.  Bright field images of Hok cells: (a) untreated with CDs, (b) treated with CDs; Fluorescence images under different excitation wavelengths: (c) 488 nm, (d) 543 nm

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  For MC3T3 cell imaging, we found that the cells incubated with CDs (200 μg·mL^-1) also emitted bright green and red fluorescence similar to Hok cells, as shown in Fig. 6b and 6c. From the bright-field and overplayed fluorescence images (Fig. 6a and 6d), the fluorescence signals originated from the perinuclear region of the cytosol. Also, no morphological damage to the MC3T3 cells appeared after being labeled with CDs, which suggests that the cells are live as before. Besides, to further illustrate the potential of CDs as a cell imaging agent, we selected fennel and black rice CDs for comparison and extended the incubation time to 32 h in HeLa cell imaging. As shown in Fig. S6, black rice CDs had better cell imaging capabilities than fennel CDs. Compared with Hok and MC3T3 cells, the fluorescence imaging ability of Hela cells was weaker, which is due to the concentration of CDs being only half of the first two cells. Clearly, these results show that the CDs synthesized using black rice are biocompatible and can serve as a potential candidate for organic dyes and semiconductor quantum dots in cell imaging.

  Figure 6

  

  Figure 6.  Cellular imaging of MC3T3 cells: (a) bright field; (b) 488 nm; (c) 543 nm; (d) overlap of the corresponding bright-field images and fluorescence images

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  3.   Conclusions

  In summary, a cheap black rice carbon source was used to synthesize CDs through a green hydrothermal process. The fluorescence intensity of the CDs was not sensitive to changes in temperature or pH or NaCl solution, and more interestingly, the CDs were immune to many metal ions. The obtained CDs exhibited low cytotoxicity at a higher concentration (400 μg·mL^-1) during the cell viability experiment against the Hok cells. These results indicate that CDs prepared from black rice have good stability and biocompatibility, and can be used as an excellent imaging agent for cell labeling in vivo. Meanwhile, the CDs showed dual fluorescence properties of excitation independence and dependence. Overall, the CDs synthesized from black rice could exhibit bright green and red fluorescence in Hok, MC3T3, and Hela cells with excellent biocompatibility, which might be potentially applied as bioimaging agents for early disease detection and rapid screening.

  Supporting information is available at http://www.wjhxxb.cn

  
  Acknowledgments: The authors would like to acknowledge the support from the National Natural Science Foundation of China (Grant No.11704274), the Natural Science Foundation of Shanxi Province (Grant No. 201901D111267), and the Leading Talents Program of Shanxi Province (Grant No.20190042).
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  Figure 1  (a) TEM images of CDs; (b) XRD pattern of CDs; (c) FT-IR spectra of the black rice and CDs; (d) XPS survey spectrum, (e) high-resolution spectrum of C1s, and (f) high-resolution spectrum of N1s for CDs

  Inset: particle size distribution histogram

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  Figure 2  (a) Emission spectra of CDs synthesized by black rice with different m_br∶m_H2O values; (b) UV-Vis absorption, excitation, and emission spectra for CDs

  Inset: photographs of CDs under daylight (left) and UV light (right)

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  Figure 3  (a) Excitation-independent spectra and (b) excitation-dependent spectra of CDs with a m_br∶m_H2O value of 1∶80

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  Figure 4  (a) Normalized fluorescence intensities of CDs under different pH values; (b) Normalized fluorescence intensities of CDs with different NaCl concentrations; (c) I/I₀ of CDs at different temperatures; (d) I/I₀ of CDs after adding different metal ions

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  Figure 5  Bright field images of Hok cells: (a) untreated with CDs, (b) treated with CDs; Fluorescence images under different excitation wavelengths: (c) 488 nm, (d) 543 nm

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  Figure 6  Cellular imaging of MC3T3 cells: (a) bright field; (b) 488 nm; (c) 543 nm; (d) overlap of the corresponding bright-field images and fluorescence images

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