The Facile Synthesis of Efficient Red‐Emissive Two‐Photon Carbon Dots for Real‐Time Cellular Imaging and High‐Resolution Deep‐Tissue Imaging

In recent years, the development of carbon dot‐based fluorescent nanoparticles for bioimaging applications has attracted the attention of the scientific community. However, many of these systems absorb and emit in the blue–green region of the electromagnetic spectrum, limiting their application in bioimaging. Herein, the facile design and development of highly efficient two‐photon excitable red‐emissive carbon dots are reported and their high performance in bioimaging applications is demonstrated. The importance of aromatic precursors in developing red‐emissive carbon dots is demonstrated. The optimized carbon dots are highly biocompatible and nontoxic, with remarkable photostability in cells under two‐photon near‐infrared excitation. The present study points to the great potential of two‐photon excitable red‐emissive carbon dot as an efficient bioimaging agent for cellular biolabeling, long‐term and real‐time cellular imaging, and high‐resolution deep‐tissue imaging in complex biological systems.


Introduction
Carbon dots (CDs) have been widely investigated for various bioimaging applications due to their superior physicochemical and biological properties [1,2] compared to the traditional inorganic and organic materials.For instance, semiconductor quantum dots and organic dyes suffer from cytotoxicity and photobleaching, respectively. [3]Besides their good biocompatibility and high photostability, CDs have excellent water solubility, ease of synthesis and surface functionalization, excellent two-photon excitation excitation resulting in photobleaching, photodamage, and limited penetration depth due to the light absorption and scattering by biological tissues. [3]This can damage the surrounding cells and, paired with the strong blue-green autofluorescence of biological tissue, limits the application and effectiveness of CDs as bioimaging agents. [9]o overcome these limitations, there has been significant interest in developing two-photon excitable CDs with excitation and emission wavelengths within the red-to-near-infrared (NIR) region.Redshifting the optical properties of CDs to longer wavelengths minimizes the issues associated with bluegreen-emissive CDs and allows for their use as a bioimaging agent within different spectral ranges for cells and tissues. [1,10]wo-photon excitable CDs can be excited by the simultaneous absorption of two lower energy photons, reducing the reliance on high energy photons. [11]This increases their tissue penetration depth while eliminating issues associated with photodamage, photobleaching, and autofluorescence. [11,12]Therefore, it is of great importance to develop a CD, which simultaneously has excitation and emission in NIR region and two-photon excitability, for efficient bioimaging applications.
Currently, a few studies have reported on the development of two-photon excitable red-emissive CDs (TRCD) for biolabeling and bioimaging applications.While some promising results were obtained, there are still some drawbacks.Specifically, the TRCDs suffer from low fluorescence (FL) quantum yield (QY), relative high toxicity, low photo-and chemical stability, and insufficient two-photon absorption (TPA). [13]In addition, the development of these TRCDs tends to be a fortunate synthetic outcome rather than achieved by design, as they lacked a systematic study on how various synthesis parameters would affect physicochemical and biological properties of as-synthesized CDs. [14]Lan et al. hydrothermally synthesized two-photon excitable NIR-emissive CDs at 180 °C for 24 h using polythiophene and diphenyl diselenide as the precursors, and aqueous sodium hydroxide as the solvent. [15]The CDs had a QY of 0.2% and were further used for in vitro and in vivo imaging and photothermal therapy.In another study, red-emissive two-photon CDs with a QY of 26.28% were fabricated through hydrothermal treatment of dopamine and o-phenylenediamine at 200 °C for 8 h. [16]The synthesized CDs were utilized for in vivo bioimaging in nude mice.In a recent study, the use of citric acid and urea as ingredients for the solvothermal synthesis of red-emissive two-photon CDs was investigated, using dimethylformamide (DMF) as the solvent at 160 °C for 6 h (QY = 26% in DMSO). [17]n this study, we developed next-generation TRCDs through our novel synthesis method [6] and a rationalized selection of starting precursors.A comprehensive study was conducted to investigate the importance of aromatic precursors in developing red-emissive CDs.The TRCD developed in this study, D-CD, was found to have a high QY, outstanding photostability in complex biological environments, excellent biocompatibility, TPE ability, and rapid cellular uptake and localization within specific cellular compartments.We also demonstrated the effectiveness of D-CD as a high-resolution bioimaging agent for both cellular and deep tissue applications within complex biological systems, utilizing pig skin as a model due to its structural and physiological similarities to human skin.

Results and Discussion
The previous studies established that red-emissive two-photon CDs can be synthesized under a range of different synthesis conditions, with each possessing distinctive photophysical properties. [14]Therefore, optimizing the synthesis parameters is an important step to ensure the reproducible preparation of CDs with optimal optical properties, safety, and biocompatibility for a range of biological applications.Our recently published study [6] involved optimizing different synthesis and postsynthesis parameters (solvent, heating time, dopant quantity, and particle size distribution range) and investigating their effects on the photophysical and biological properties of CDs.In that study, [6] we demonstrated that CDs synthesized for 8 h in deionized (DI) water, with a nitrogen doping ratio of ascorbic acid arginine mass = 1:1.5,and, with largest particle size distribution of 30-100 kDa, demonstrated optimal photophysical and biological properties.We named these CDs as 30-100 kDa-CD.However, the variation in these parameters only altered the excitation and emission wavelengths of 30-100 kDa-CD from the UV to green region of the visible spectrum (maximum emission wavelength of 450 nm), limiting its wider application as bioimaging agent.
We have recently noticed that the precursor molecular structure may have a direct impact on the photophysical and physicochemical properties of the CDs.This is because the starting material breaks down only partially during the synthesis process, resulting in the retention of some aspects of the initial structure in the synthesized CDs. [14]We therefore hypothesized that inclusion of aromatic precursors would result in the retention of some aromatic structure in the final CDs, leading to more redshifted FL properties.Specifically, this study involved synthesizing CDs using the optimized synthesis conditions obtained from our previous study, with the inclusion of 1,4,5,8-tetraaminoantraquinone as an aromatic precursor alongside the aliphatic ascorbic acid and arginine.The chemical, structural, and photophysical properties of the 30-100 kDa-CD were then compared with the new synthesized CDs.

Synthesis and Characterization of TRCDs
The TRCDs were separately synthesized in the presence of water (to give W-CD), ethanol (E-CD), and DMSO (D-CD).These solvents are commonly used when synthesizing CDs via the hydrothermal/solvothermal method, due to their different physiochemical properties. [11]The as-synthesized TRCDs were filtered using centrifugal filters with molecular weight cutoffs (MWCO) of 30-100 kDa to obtain their optimum size distribution range. [4]The comparison between the three TRCDs shows that the maximum excitation and emission wavelengths of three CDs remained in the orange and red spectral regions, respectively.However, among all samples, D-CD demonstrated the most redshifted excitation and emission wavelengths and highest QY value (Table 1 and Figure S1, Supporting Information).These findings revealed that using DMSO as the solvent results in an efficient hydrothermal synthesis reaction with a higher degree of carbonization compared to water and ethanol.Therefore, D-CD was used for all subsequent studies.Transmission electron microscopy (TEM) images show a narrow size distribution and spherical morphology of D-CD with an average size of 9.03 AE 1.30 nm (Figure 1A,B).Dynamic light scattering (DLS) measurements further confirm the narrow size distribution of D-CD, indicating an average hydrodynamic diameter of %10.2 nm (Figure S2A, Supporting Information).To elucidate the structure of CDs structure, we conducted X-Ray diffraction (XRD) measurements.The XRD patterns for both the 30-100 kDa-CD D-CDs (Figure S2B, Supporting Information) exhibited a broad peak centered at 21.3°, which corresponds to the graphite (002) plane. [6]Based on the XRD measurements, it can be concluded that the CDs consist of an amorphous carbon structure characterized by highly disordered carbon atoms interspersed with occasional particles of a crystalline structure. [6,18]2.Influence of Precursor with Aromatic Structure on TRCD Development

Chemical Structure and Surface Properties
To determine the effects of the precursor molecular structure on the CDs' physicochemical properties, the structural and optical properties of the developed D-CD were investigated and compared with 30-100 kDa-CD.The surface chemistry and chemical functionality of these CDs were analyzed using Fourier-transform infrared (FT-IR) and X-Ray photoelectron spectroscopy (XPS) techniques, respectively.The FT-IR spectra of 30-100 kDa-CD (Figure 1C [18,19] This is further evidenced when comparing the FT-IR spectra of the precursors, 30-100 kDa-CD, and D-CD, highlighting a significantly increased level of interaction due to the formation of a large number of oxygenated and nitro-functional groups and the emergence of new bands within the D-CD structure.The formation of sulfur-functional groups (C-S, C=S, S=O) can be attributed to using DMSO as the solvent.
XPS survey scans were acquired for 30-100 kDa-CD and D-CD to further confirm FT-IR results and identify the CDs' chemical composition.These scans show three and four dominant peaks in 30-100 kDa-CD (corresponding to C 1s, N 1s, O 1s) and D-CD (corresponding to C 1s, N 1s, O 1s, S 2p), respectively (Figure 2 and S4, Supporting Information, and Table 2).The atomic percentage (at%) of these two CDs is reported in Table 2 and demonstrates the presence of more significant amounts of nitrogen and oxygen within the structure of D-CD, compared to 30-100 kDa-CD.Analyzing the highresolution spectra (C 1s, N 1s, O 1s, S 2p) of both samples indicates the introduction of a large number of oxygenated and    D-CD due to the use of an additional aromatic precursor (1,4,5,8-tetraaminoantraquinone).

Optical Properties
The optical characteristics of 30-100 kDa-CD and D-CD were studied using UV-vis absorbance and FL spectroscopies (Figure 3 and S5, Supporting Information).30-100 kDa-CD possess an absorption peak at 336 nm, ascribed to the transition of lone pair electrons from n-nonbonding electron bonds to π*-antibonding electron bonds due to the presence of C=N bonds. [6]The emission peak observed at 450 nm, when excited at the maximum excitation wavelength (λ ex = 360 nm), is likely due to the electron relaxation in C=C and graphitic C-N bonds in the nitrogen-doped graphitic sp 2 hybridized cores.As compared to 30-100 kDa-CD, the absorption spectrum of D-CD demonstrates a shoulder peak at 320 nm and a main peak at 595 nm, confirming the contribution of π-π* transition of the aromatic C=C bond, and π-π* and n-π* transition of the aromatic π system due to the presence of C=N, C=O, and C=S bonds.The FL spectrum of D-CD shows a sharp peak at 700 nm upon excitation at the maximum excitation wavelength (λ ex = 595 nm).The overlap between the absorption and FL spectra of D-CD at %595 nm further indicates the presence of newly formed aromatic structures, resulting in the observed NIR emission (Figure S6, Supporting Information). [20]Our results demonstrate that introducing a precursor with an aromatic molecular structure and high-density sp 2 content can significantly change the chemical, structural, and photophysical characteristics of CDs.This is due to the higher degree of carbonization of the carbon cores, higher oxidation, aromaticity, and greater conjugation of sp 2 carbons in the newly formed CDs structure.Increasing the rate of formation of aromatic rings increases the size of conjugated-π systems within the structure of D-CD, leading to a shift in valence electrons which reduces the HOMO-LUMO energy gaps and causes a redshift in emission wavelengths.Additionally, the greater density of carboxyl and hydroxyl groups correlates to a lower bandgap energy (E bg ) and greater electron donating behavior of D-CD, respectively, which further shifts the emission wavelengths into the NIR region.D-CD displayed a significantly higher QY of 0.37 and an average lifetime of 7.89 ns compared to 30-100 kDa-CD, which has a QY of 0.2 and an average lifetime of 4.41 ns.The enhanced QY and average lifetime in D-CD can be attributed to its greater concentration of the nitrogen dopant and the introduction of a sulfur dopant. [11,21]According to FT-IR/XPS results, these dopants resulted in the formation of new sulfur groups and introduced more nitrogen groups, altering the surface structure and  functional groups distribution of D-CD.These functional groups have a low E gb and high electron donating behavior and would act as excitation energy traps with a concomitant effect of enhancing the FL. [22]We further evaluated the photostability of the D-CD.
Hence, we have clearly demonstrated the importance of precursor selection and consideration of its molecular structure on the optical properties of D-CD.

D-CD for Cellular Bioimaging
In recent years, the development of two-photon excitable fluorescent probes has received significant attention owing to their ability to be excited through simultaneous absorption of two near-to-mid-NIR photons. [14]This minimizes the interference from background species, lowers the risk of photodamage to biological tissues and in turn longer observation time, and increases the penetration depth in tissue. [3]We demonstrated that through single-photon excitation (SPE), D-CD can be excited and emit light in the orange (595 nm) and red (700 nm) regions of the light spectrum, respectively.We therefore sought to examine the TPE ability of D-CD and observed an emission peak at 580 nm upon excitation at 945 nm (Figure S7, Supporting Information).The inconsistency between the sum of the energy of the photons used in SPE and TPE can result in activation of different emission sites within D-CD, thereby causing variations in their maximum excitation/emission wavelengths upon SPE and TPE.To confirm the TPA capability of the D-CD, we explored the variations in FL intensity in relation to excitation laser power.The quadratic relationship between the excitation laser power and FL intensity is depicted in Figure S7B, Supporting Information.This relationship confirms the ability of D-CD to undergo two-photon NIR excitation.The TPA cross section was calculated to be %230 GM (Goeppert-Mayer unit, with 1 GM = 10-50 cm 4 s photon À1 ) for D-CD.
In order to confirm the cellular compatibility of D-CD prior to microscopy studies, we used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) (MTS) assay to determine the cytotoxicity of D-CD in fibroblasts.Our results showed that fibroblast remained viable when cultured for 72 h in the presence of D-CD at concentrations ranging from 0 to 800 μg mL À1 (Figure S8, Supporting Information).These results indicate the potential use of D-CD as a labelling and imaging agent for in vitro imaging of biological cells.
Live fibroblasts incubated with 200 μg mL À1 D-CD for 24 h were initially imaged by SPE confocal microscopy.The differential interference contrast (DIC) images (Figure 4A), FL images (Figure 4B), and the overlay (Figure 4C) show simultaneous cytoplasm and nucleolus labelling of D-CD in live cells (Figure S9 and Video S1, Supporting Information).The localization of D-CD in different cellular organelles may be due to the size and surface charge heterogeneity of D-CD, as well as its interactions with ribosomal RNA, present in both the cytoplasm and nucleolus. [14]Next, we performed TPE FL imaging under 945 nm excitation.As it can be seen in Figure 4A,D-E, TPE FL images demonstrate higher resolution, better signal-to-noise ratio, and less background noise compared to SPE FL images, revealing that D-CD has a great TPE ability in living cells.
The Z-stack images were also analyzed to determine the precise location of D-CD along the Z-axis of the fibroblasts.The cellular uptake and localization of D-CD occurred in the cytoplasm and nucleolus rather than solely adsorbing onto the surface of fibroblasts, indicating that D-CD was able to penetrate through the cell membrane (Figure S9, Supporting Information).
Photostability is essential to ensure the use of D-CD for long-term and real-time bioimaging.The application of commercially available fluorescent labeling agents, such as Alexa 488-streptavidin with %180 s photostability, [23] is limited due to their poor photostability, resulting in lower imaging resolution, signal intensity, and reduced working life.We assessed the photostability of D-CD through exposing them to continuous UV light (500 W, λ ex = 365 nm) irradiation for 3,600 s, during which no FL fluctuations were recorded (Figure S10, Supporting Information).The photostability was further examined by continuously monitoring fibroblasts incubated with D-CD (200 μg mL À1 ).The observations were made using a twophoton confocal FL microscope, paired with a benchtop incubator maintained at 37 °C and 5% CO 2 , over a duration of 25 min (λ ex = 945 nm).The FL intensity of D-CD was reduced by 19% in the first 15 min; however, it remained highly stable with negligible changes when continuously imaged for up to 25 min (Figure 5A-G).The exceptional photostability of D-CD is significantly higher than previously reported fluorescent nanomaterials and commercially available cell agents. [24,25]Therefore, the superior properties of D-CD, such as excellent biocompatibility, low toxicity, two-photon excitation ability, rapid cellular uptake and localization within cellular compartments, and high photostability in complex biological environments, make them an ideal candidate for FL cellular labeling and bioimaging.

D-CD for Deep Pigskin Tissue Imaging
The application of already developed fluorescent nanomaterials and molecules for deep-tissue imaging has been limited due to their excitation and emission wavelength lying within the 300-400 nm range.This causes strong tissue autofluorescence from biological tissue, which can significantly decrease the signal-to-noise ratio and reduce contrast, affecting the sensitivity and accuracy of tissue imaging.Using single-photon UV-vis light as the excitation source causes strong scattering and refraction of the light, limiting the penetration depth.Further, the risk of photodamage to the cells and tissues is increased, and the observation time is shortened.In this study, the deep-tissue penetration depth of D-CD was examined by injecting it into pigskin tissue through the transdermal route and washing it with phosphate-buffered saline (PBS) buffer 5 times prior to imaging (Figure 6A).Two-photon excitation FL imaging was performed to investigate the capability of D-CD for deep-tissue imaging (λ ex = 945 nm, λ em = 400-800 nm).D-CD demonstrated excellent penetration and distribution within pigskin tissue in a short period, which may be due to their small size (9.03 nm, Figure 1A,  B).Analyzing the Z-stack FL images found that D-CD was able to image the tissue for a penetration depth of up to 455 μm.Compared to recently developed two-photon excitable bluegreen-emissive CDs, [3,26] D-CD showed remarkably higher tissue penetration depth, spatiotemporal resolution, and signal-to-noise ratio as well as minimal autofluorescence, tissue light scattering, and tissue damage (Figure 6B and S11, Supporting Information).These results indicate the potential use of D-CD as a labelling and imaging agent for in vivo monitoring of biological tissues.
Thus, this study successfully demonstrated the promising physicochemical and biological properties of D-CD and their application in cell labelling, long-term and real-time cellular imaging, and high-resolution deep-tissue imaging.D-CD has the potential to replace commercially available fluorescent nanoparticles and bioimaging agents due to its superior photophysical and physicochemical properties.

Conclusion
This study illustrates the successful development of novel two-photon excitable red-emissive CDs (D-CD) through systematic optimization of the hydrothermal synthesis method and rational selection of precursors.Compared with previously synthesized CDs which only used aliphatic structured precursors (30-100 kDa-CD), the integration of a precursor with an aromatic structure resulted in a greater degree of carbonization, higher oxidation and aromaticity, and greater size of conjugated-π systems within the structure of the D-CD.These chemical and/or structural changes resulted in a significant redshift in the excitation and emission wavelengths of D-CD.Our findings indicate that D-CD possesses optimal optical properties and can be excited using both SPE (λ ex = 595 nm) and TPE (λ ex = 945 nm) with emission peaks at 700 and 580 nm, respectively.Further, D-CD possesses a high QY (0.37), negligible cytotoxicity (%100% cell viability after 3 days of treatment), excellent photostability and real-time monitoring ability of cellular compartments (up to 25 min).We showed that D-CD can be utilized as a high-performance agent for simultaneous cytoplasm and nucleolus labeling in live fibroblasts.D-CD can also be used for high-resolution deep-tissue imaging applications, as a tissue penetration depth of up to 455 μm was obtained in pigskin tissue, as well as minimal autofluorescence and light scattering.Therefore, this study indicates the significant potential of D-CD as an agent for biolabeling, and long-term and real-time monitoring of biological events in vitro and deep in vivo.

Experimental Section
Materials: All chemicals used were of analytical grade and required no purification prior to usage.L-Ascorbic acid, L-arginine, ethanol, DMSO, and 1,4,5,8-tetraaminoanthraquinone (Disperse Blue 1) were purchased from Sigma-Aldrich (Australia).Dulbecco's Modified Eagle's Medium (DMEM), TrypsinLE Express, and PBS were purchased from Gibco Life Technologies (USA).Fetal calf serum (FCS), penicillin, and streptomycin were purchased from ThermoFisher Scientific (USA).MTS was purchased from Progema (USA).Primary human dermal fibroblasts from the foreskin, herein referred to as fibroblasts, were kindly donated by Prof. Rebecca Mason's group at the University of Sydney.DI water was used in all studies.
Preparation of TRCDs: TRCDs were prepared using the optimized synthesis conditions obtained from our previous study, [6] with the difference that Disperse Blue 1 as an additional precursor with aromatic structure was included into synthesis process.Briefly, L-ascorbic acid (0.5 g), L-arginine (0.75 g), and Disperse Blue 1 (0.5 mg) were mixed in 50 mL DMSO followed by ultrasonication to achieve a homogeneous mixture.The solution was transferred into a 100 mL Teflon-lined stainless-steel autoclave chamber with a 50% filling ratio and heated at 180 °C for 8 h (Figure 7), and a dark blue solution was obtained (Figure 3F).The initial purification was performed by passing the solution through a 0.22 μm cellulose acetate Millipore filter (Millipore, USA) to remove the large or agglomerated particles.Next, the CDs were separated by size by centrifugation through dialysis tubes with different MWCO to obtain CDs with particle size distribution in the range of 30-100 kDa, herein referred to as D-CD.The sample was dried using the rotary evaporator (Heidolph, Germany) to produce a 2 mg mL À1 D-CD solution by adding water.The solution was stored at 4 °C for further use.A similar process was used for the synthesis of other samples, except that the solvent was replaced with water and ethanol to prepare W-CD and E-CD, respectively.The photophysical and physicochemical properties of the developed TRCDs were compared with the previously synthesized CDs under optimized synthesis condition using ascorbic acid and arginine as precursors (30-100 kDa-CD), to demonstrate the influence of the inclusion of precursors with aromatic structure in developing red-emissive CDs.
Characterization: TEM images were captured by JEOL JEM-2100, operating at 200 kV.DLS measurements were performed using Malvern Zetasizer Nano ZS90 (UK).FT-IR spectra were obtained on a Bruker Tensor 27 FT-IR spectrometer (USA).XPS data were obtained using a Kratos Axis Nova spectrometer (Kratos Analytical, UK), equipped with a monochromatized aluminum X-Ray source (Al Kα 1486.6 eV) operating at 10 mA and 15 kV (150 kW).Survey and high-resolution spectra were acquired at detector pass energies of 160 and 20 eV, respectively.The XPS data were analyzed with Thermo Avantage processing software (v5.9902).UV-vis absorption and FL spectroscopy were performed using a Varioskan Lux spectrometer (Thermo Fisher Scientific, Singapore).A Leica SP5 II multiphoton confocal microscope (USA) equipped with a femtosecond pulsed infrared laser and benchtop incubator (5%, 37 °C) was used for measuring FL lifetime, investigating the TPA, studying emission behavior of synthesized CDs, as well as live imaging of fibroblasts and deep pigskin tissue imaging.The fluorescent images were processed using Leica Application Suite X and image analysis ImageJ.
QY Measurement and TPA Cross-Sectional Measurements: FL QY was determined by plotting the absorbance values and integrated FL intensities of rhodamine 6 G as the standard (QY of 95% in EtOH) and the samples at the same wavelength. [6,27]The gradients of the linear fits of these lines were input into the following formula where the subscript R refers to the reference rhodamine 6 G, while no subscript refers to the CD sample; m is the gradient of the linear fit of absorbance against integrated FL intensity; and n is the refractive index of the solvent which for Q was ethanol and for CDs in water.
The two-photon FL intensity of a fluorophore depends on its emission QY and TPA cross section (σ 2p ).By using fluorescein (FLN) as the reference sample (σ FLN = 37 GM at 780 nm), [28] the TPA cross section of D-CD (σ CD ) was calculated at 780 nm excitation wavelength.We measured the two-photon emission intensities of D-CD (F CD ) and fluorescein (F FLN ) at known concentrations (C CD and C FLN ) and the TPA of our probe was estimated as follows [11,28,29] σ Cell Culture, Viability, and Bioimaging: Fibroblasts (passage 10) were cultured in ventilated T-175 tissue culture flasks in a complete medium containing DMEM, supplemented with 10% v/v FCS, 100 U mL À1 penicillin, and 100 μg mL À1 streptomycin, herein referred to as supplemented DMEM, and incubated at 37 °C in a 5% CO 2 environment.The medium was refreshed every 3 days until 80-90% confluence was reached.
MTS assay was used to determine cell viability.Fibroblasts were incubated with increasing concentrations of D-CD.Fibroblasts were seeded in 96 well plates at 10 000 cells well À1 density and incubated at 37 °C in a 5% CO 2 for 24 h.Next, the medium was removed and replaced with 200 μL of DMEM medium containing CDs of different concentrations (100, 200, 400, and 800 μg mL À1 ).The control cells were supplemented with DMEM only.Fibroblasts viability was assessed using CellTiter 96 AQueous One Solution Reagent (Promega) according to the manufacturer's instructions.Briefly, 100 μL of MTS solution was added to each well and incubated for 4 h at 37 °C under 5% CO 2 .The absorbance of the plate was then measured at 490 nm using a Multiskan Sky Microplate Spectrophotometer (Thermo Fisher Scientific, USA).The cell viability (%) was calculated relative to the control averages as per the formula Cell viability ¼ ½A ½A control Â 100 (3) where [A] and [A] control is the average absorbance of the control and sample wells, respectively.The mean and standard error of the mean of these cell viability percentages were calculated in Origin 2018, and SEM was plotted on column graphs as error bars.FL live-cell imaging of the fibroblasts incubated with D-CD was performed to investigate its ability as a labelling agent for cellular imaging.Fibroblasts were seeded onto 35 mm glass-bottom dishes at a density of 100 000 cells per dish and incubated for 24 h at 37 °C in 5% CO 2 .After 24 h, supplemented DMEM was removed from dishes and replaced with medium containing CDs (200 μg mL À1 ) and left to incubate for 24 h at 37 °C in 5% CO 2 to allow for the uptake of D-CD by fibroblasts.After incubation, cells were washed three times with 1.0 mL of PBS (10 mM, pH 7.4).The incubated cells with D-CD were immediately observed by a twophoton confocal FL microscope equipped with a benchtop incubator (Leica TCS SP5 II, USA) at 37 °C in 5% CO 2 .
For tissue imaging, pigskin was cut with a thickness of %4000 μm.Next, D-CD (200 μg mL À1 ) was transdermally injected into pigskin tissue and washed with PBS buffer five times prior to imaging.Two-photon confocal FL microscope (Leica TCS SP5 II, USA) was used to perform deep pigskin tissue imaging.
) displayed peaks corresponding to N-H (3400 cm À1 ), O-H (3260, 1399, 1356 cm À1 ), C-H (1459 cm À1 ), C=N (1770, 1684 cm À1 ), C=C/N-H (1597 cm À1 ), and C-N (1175, 1102, 1050 cm À1 ).The FT-IR spectra of D-CD (Figure1C) show the formation of O-H/N-H (3260 cm À1 ), C-H (2920, 2850, 1470 cm À1 ), C=N (1774 cm À1 ), C=C/N-H (1651 cm À1 ), C=O/S=O (1575 cm À1 ), C-C (1415 cm À1 ), C-N (1320 cm À1 ), C-S (1178 cm À1 ), N-O (1106 cm À1 ), C-O (1015 cm À1 ), and C=S (950 cm À1 ).The comparison between the FT-IR spectra of both CDs and the precursors (FigureS3, Supporting Information) demonstrates a blueshift and reduced intensity in most D-CD bands, along with the presence of new functional groups such as C=O, C=S, C-S, N-O, C-O, and S=O.The presence of C=O (1575 cm À1 ) and C-O (1015 cm À1 ) bands in the FT-IR spectra of both D-CD and Disperse Blue 1 highlights the occurrence of notable chemical interactions and bonding throughout the hydrothermal synthesis procedure.These variations are reflective of major chemical and/or structural changes that may have occurred due to the interactions between the aromatic and aliphatic structures present in the precursors.In addition, the presence of a strong stretching vibration band (O-H/N-H), the introduction of a large number of oxygenated and nitrofunctional groups (carbonyl (C=O), ether (C-O), nitro (N-O)), and the formation of new bands at 1415 cm À1 (C-C) and 1470 cm À1 (C-H) indicate the development and growth of new polyaromatic structures and carbon domains in D-CD compared to 30-100 kDa-CD.

Figure 1 .
Figure 1.Morphological and surface characterization of synthesized CDs.A,B) TEM image and particle size distribution of D-CD, respectively (scale bar represents 100 nm).C) FT-IR spectra of 30-100 kDa-CD and D-CD (newly formed functional groups on D-CD are indicated).

λ
Sample λ ex maximum [nm] a) λ em maximum [nm] b) QY [%] c) ex , excitation wavelength; b) λ em , emission wavelength; c) QY, quantum yield.nitrogen functional groups into the structure of D-CD as well as the presence of new functional groups (C-H, graphitic N, C-OH, COOH, C-S, S-H, S-C, S=O) (Table S1 and S2, Supporting Information).These results support the findings obtained from the FT-IR results and reveal the formation of new polyaromatic structures and carbon domains within

Figure 2 .
Figure 2. Chemical composition of synthesized CDs.A,B) XPS full scan spectrum, C,D) C 1s spectra, E,F) N 1s spectra, and G,H) O 1s spectra of 30-100 kDa-CD and D-CD, respectively.

Figure 3 .
Figure 3. Optical performance of synthesized CDs.Absorption and FL emission spectra of A) 30-100 kDa-CD and B) D-CD.ABS, absorption; FL, fluorescence emission.C-E) Photograph of 30-100 kDa-CD aqueous solution in daylight under λ ex = 405 nm and λ ex = 680 nm, respectively.F-H) Photograph of D-CD aqueous solution in daylight under λ ex = 405 nm and λ ex = 680 nm, respectively.

Figure 4 .
Figure 4. D-CD as high-performance cellular labelling agent.A) DIC image and B,D) SPE (λ ex = 458 nm) and TPE (λ ex = 945 nm) FL images, and C,E) overlay of DIC and FL images of fibroblast incubated with 200 μg mL À1 D-CD for 24 h.The images are false color (scale: 15 μm).

Figure 5 .
Figure 5. D-CD as labelling agent for long-term and real-time bioimaging.Real-time two-photon FL images of fibroblast incubated with 200 μg mL À1 D-CD.A-F) Time-lapse confocal FL images of D-CD in fibroblasts were acquired every 1 min (λ ex = 945 nm) under continuous laser irradiation for 25 min.The images are false color (scale bar: 45 μm).G) Average corrected cell FL of fibroblast obtained from (A) to (F).

Figure 6 .
Figure 6.D-CD for rapid and noninvasive deep-tissue imaging.A) schematic of the setup to examine the D-CD penetration depth in pigskin tissue.B) Two-photon-excited Z-stack FL images of pigskin tissue incubated with D-CD (200 μg mL À1 ) from 0 to 455 μm; λ ex = 945 nm, λ em = 400-800 nm.Scale bar: 100 μm.

Figure 7 .
Figure 7. Synthetic strategy of TRCD.Hydrothermal/solvothermal synthesis diagram of D-CD.The solvent was replaced with water and ethanol to prepare W-CD and E-CD, respectively.

Table 1 .
Summary of optical properties of synthesized CD samples.

Table 2 .
Atomic percentage of CDs from XPS.