Introduction

Fluorescence (FL) imaging is one of the most common modalities of clinical imaging [1, 2]. Numerous small molecule dyes (e.g. indocyanine green, fluorescein, methylene blue, and several others) are already approved for clinical use by the United States Food and Drug Administration (US-FDA) and other regulatory bodies [3, 4]. However, small molecule dyes suffer from a short half-life, quenching, and blinking effects in a microsecond (µs) time scale [5, 6]. Therefore, several new fluorescent imaging agents have been developed in the last decade including improved organic small molecules [7,8,9,10], conjugated polymers [11, 12], quantum dots [13,14,15,16], and other hybrid or nanocomposite materials [17,18,19,20,21]. Among these candidates, nanomaterial-based imaging agents in particular quantum dots have been very successful fluorescent probes [22, 23]. These probes are currently used at various stages of clinical trials (e.g. Cornell dots, Feridex I.V., SonoVue, etc.) for specific imaging of a range of diseases [3, 24]. The key advantages of such imaging agents include high biocompatibility, long circulation half-life, ability to target specific disease sites, and significantly improved signal enhancement [25, 26].

More recently, carbon dots (CDs) have become a popular choice as an imaging agent due to their remarkable biocompatibility and proven biodegradation compared to other inorganic semiconductor quantum dots [27,28,29,30]. The fluorescent organic CDs are among the emerging low-cost and promising imaging agents due to their tuneable surface functionality, unique optical properties (e.g. excitation dependent emission), and the ability to use a range of low-cost materials or bio-waste materials as starting precursors for their synthesis [30,31,32].

Based on the synthesis protocols, starting precursor and attachment of linkers or ligands, the size of the CDs and their emitting properties can be effectively tuned [33, 34]. Researchers have created CDs with a wide range of emission wavelength (i.e. colours) [36,37,38,39] that have been utilized in a variety of devices including flexible organic light-emitting diodes [33], environmental sensors [34], bio-targeting and drug delivery [40,41,42,43], cellular and subcellular imaging [44, 45] and diagnostic-imaging [46, 47]. These devices have been applied for environmental, physical, and medical use, while several new applications are now emerging based on their unique optoelectronic properties. To be considered as an efficient FL agent for bioimaging that is superior to common dyes, the CDs must have a high photoluminescent quantum yield (PLQY), be made from an abundant and low-cost starting precursor, and have a high Stokes shift between the excitation and the emission wavelengths. Lili Tong et al. developed green-emitting CDs (i.e. GCDs) with a very high PLQY of 90% for long-term lysosome imaging [48]. However, the precursor used in the synthesis of CDs was very expensive Rose Bengal (US$69 per gram) and the material exhibits a small Stokes shift between excitation and emission (20 nm with excitation at 508 nm and peak emission at 528 nm) wavelengths. Songnan et al. synthesized water-soluble CDs using a one-step microwave reactor-based process with an equal amount of citric acid (3 g) and urea (3 g) as starting precursors. The synthesized CDs showed a good stroke shift of 100 nm (excitation/emission 420/520 nm), with an enhanced fluorescent quantum yield of 40%. Thus, the low cost, biocompatibilty, and distinct PL properties proves these C-dots could potentially be produced on an industrial scale as a new fluorescent ink for versatile applications [49]. Xu et al. used tartaric acid and bran as starting precursors to synthesize green light-emitting CDs [50]. The one-pot solvothermal process produces CDs with 46% PLQY. Pei Yang et al. synthesized nitrogen/sulphur co-doped green–yellow emitting CDs using xylose as starting precursor [51]. They used a microwave reactor to synthesize CDs with PLQY of 42%. With poor PLQY and short or fixed Stokes shift in excitation and emission remains a major challenge. Kateshiya et al. synthesized multi-colored (Blue, Green, and Yellow emitting)-CDs using natural precursor, ripe fruit of Annona squamosal through an acid oxidation process. The prepared hybrid fluorescent nanosensor were surface functionalized with molybdenum trioxide nanoparticles (MoO3 NP) to impart selectivity towards ClO− and were demonstrated for their use as Fl quenching-based sensors to detect ClO− in water [25].

In this work, we have overcome these challenges and synthesized low-cost GCDs with ultra-high PLQY (70%) from inexpensive starting precursors. Due to the unique single-step sulphur and nitrogen doping during synthesis, the GCDs show an excitation-independent emission characteristic. Our work, to date, is the first report to demonstrate excitation-independent green emission from carbon dots prepared using an inexpensive precursor that costs less than AU$0.04 for 1 g. This ultra-high PLQY enables our GCDs to allow for bioimaging in the same spectral range as background autofluorescence, which is not possible with low PLQY conventional fluorophores and other CDs. The prepared GDs have the potential to enable low-cost FL imaging without the need for more expensive IR imaging systems. In addition, the GCDs were highly cytocompatible at concentrations up to 250 µg/mL with RAW264.7 macrophage cells. The GCDs displayed a concentration-dependent FL with the highest emission intensity at 1.2 mg/mL concentration. A similar concentration-dependent FL intensity behaviour was observed upon subcutaneous injection of the GCDs into a fresh mouse cadaver. Interestingly, the optimum excitation wavelength for subcutaneously injected GCDs was 465 nm compared to 420 nm for in vitro assessment due to high background autofluorescence at the latter excitation wavelength.

Materials and methods

Materials

p-Toluenesulfonic acid (PTSA) and ethylenediamine were purchased from merk. Aluminum oxide powder (average particle size of fewer than 1 μm) and ethylenediamine were purchased from Sigma Aldrich. All the reagents were of analytical grade and used as supplied.

Synthesis of CDs

To start, 1.7 g of PTSA was dissolved in 40 mL water. This final solution was loaded in an autoclave with 50 mL of inner Teflon container and 2 mL of ethylenediamine was added to this solution [33, 34]. Please note, the reactor was filled with approximately 80% of its capacity to create high autogenous pressure. The Teflon reactor was sealed in the stainless steel autoclave and heated to 200 °C in an oven for 24 h. Next, the autoclave was naturally cooled to room temperature. The obtained product was filtered using a column packed with aluminum oxide absorbent to remove the undesired by-products. The above purification step was repeated several times to obtain the final solution.

Cytocompatibility of CDs

The cytocompatibility of the CDs was assessed with RAW264.7 cells using our well-established protocol [16]. The cells were cultured in DMEM with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% l-glutamine in an incubator at 37 °C with 5% CO2. Cells were seeded in a 96-well plate with a density of 5000 cells per well overnight before the experiment. Then, the culture medium was replaced with a medium with carbon dots (250, 100, 20, 5, and 1 μg/mL) and cultured for 24 h. At designated time points, cell viability was tested using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) as directed by the manufacturer.

In vitro and ex vivo bioimaging with CDs

The bioimaging capabilities of the CDs were measured using an IVIS® Spectrum optical imaging system (Perkin Elmer, USA) under both in vitro and ex vivo conditions. For in vitro assessment, 20 µL aliquots of CDs at seven different concentrations (250, 375, 625, 1250, 1875, 2187.5, and 2500 µg/mL) in phosphate buffer saline (PBS) were placed on a hydrophobic non-fluorescent black mat then imaged at different combinations of excitation and emission wavelengths. Excitation-dependent fluorescence values were obtained from regions of interest (ROIs) drawn around each aliquot using Living Image software (Perkin Elmer, USA). For ex vivo assessment, 20 µL aliquots of CDs at different concentrations were injected subcutaneously using a 29G needle in freshly euthanized CD-1 mouse cadavers. Approval to use tissues from euthanized mouse was obtained from the University of Queensland (ethics number: ANRFA/TRI/485/18). The mice injected subcutaneously with CDs were imaged at different excitation and emission wavelength combinations like the in vitro assessment to determine the most suitable bioimaging spectral range with the lowest background autofluorescence. Fluorescence intensity was calculated using ROI analysis in Living Image software.

Characterization

Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images of GCDs were collected using a JEOL 2100 TEM (acceleration voltage: 200 kV). For EDX mapping and spectra, a JEOL 2200FS TEM in STEM bright-field mode with a Bruker detector was used.

Absorption spectra

Absorption spectra of GCD were recorded on a Cary 60 G6860A spectrophotometer (Agilent) using water as a suspending medium at concentrations that resulted in absorption below 1 unit.

The Fourier transform infrared (FTIR)

The Fourier transform infrared (FTIR) data were obtained using Bruker ATR using dry powdered GCDs sample.

X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) data were acquired using an Axis Supra (Kratos) with aluminum AlKα (hV = 1486.7 eV). For this, a known amount of GCDs were dropped cast on aluminum foil. The GCD samples were grounded with carbon tape to prevent charging during the measurements. Please note, charge compensation was also used while recording the data.

Photoluminescence emission

Photoluminescence emission of CDs dispersed in water was measured using an Eclipse G9800A (Cary) Fluorescence Spectrophotometer.

Photoluminescence quantum yield measurements

Photoluminescence quantum yield measurements were carried out using the relative PLQY method. Herein, a rhodamine dye with quantum yield of 31% was chosen as the standard reference with known PLQY. Absorbance and FL of standard and GCDs were recorded and PLQY was calculated using Eq. (1) [52].

$$\phi = \phi_{{{\text{Ref}}}} X \frac{{\frac{{\int A_{1} }}{{1 - 10^{{ - {\text{Abs}}}} }}S}}{{\frac{{\int A_{2} }}{{1 - 10^{{ - {\text{Abs}}}} }}R}},$$
(1)

where ɸref = Quantum yield of reference dye, (0.31 in this case); ɸ = Quantum yield of sample, subscript S is for sample and R is for reference sample. Note, the absorbance of both the reference and sample were kept below 0.05 at the measurement excitation wavelength to minimize re-absorption effects in a 10 mm fluorescence cuvette.

Results and discussion

Structural properties of GCDs

Here, we demonstrate the generation of GCDs using an ultra-low-cost chemical precursor, p-toluenesulfonic acid (PTSA). The GCDs were generated by hydrothermal processing of PTSA in presence of ethylenediamine at 200 °C. The key advantages of using PTSA with ethylenediamine for GCDs fabrication include low cost, single-step doping with S=O and nitrogen groups, and ultra-bright green emission with excitation-independent emission characteristics (Fig. 1).

Fig. 1
figure 1

a A schematic of hydrothermal synthesis of GCDs. The initial precursors are mixed in a Teflon container which is filled with 70–80% water to create enough autogenous pressure for the breakdown of precursor and formation of CDS (in a 50 mL reactor). The Teflon reactor is sealed inside stainless steel container to form a pressurized system and kept inside a hot air oven for heating. b TEM of GCDs (scale bar: 100 nm). Inset shows a higher magnification TEM of the same GCDs (scale bar: 20 nm). c HRTEM of GCDs with measurement of the graphitic lattice. d A UV–Visible spectrum of GCDs showing a strong absorption peak around 420 nm. The FL emission spectrum of GCD between an excitation wavelength range of 360–420 nm with minimal effect of the excitation wavelength on the emission wavelength peak. e The FL spectrum of GCDs excited at a higher wavelength from 440 to 500 nm further demonstrating excitation wavelength-independent emission. f FTIR spectrum of GCDs showing characteristic vibration bands for key functional groups imparted because of the precursors used during the synthesis. g Raman spectrum of GCDs deposited on a steel substrate (excitation: 785 nm). The insert shows the Raman spectra of chemical precursor (PTSA) at the same 785 nm excitation. h Time-resolved FL decay measured for GCDs. The inset shows a digital photograph of GCDs solution excited by a 420 nm Laser, where strong green FL can be visually detected

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The prepared GCDs were thoroughly characterized using an array of characterization methods. The TEM images in Fig. 1b show the shape and uniform size distribution of the synthesized GCDs. The inset in Fig. 1b shows a higher magnification image of the same GCDs. The high-resolution TEM presented in Fig. 1c exhibits regular lattice fringes with a fringe width of 0.21 nm, which is very small compared to the graphitic interlayer distance of 0.34 nm. This lattice plane distance points to the presence of (100) diffraction plane of sp2 graphitic carbon, indicating a tight packing of GCDs lattices [31]. In addition to TEM, the size and dispersity of GCDs were also measured using the dynamic light scattering (Fig. S1, Electronic Supplementary Information), which reported a size distribution between 3.6 and 7.3 nm with a polydispersity index of 0.254, which informs of the narrow particle size distribution of the CDs. A narrow size distribution is important for quantum confinement based emitters such as CDs because of a strong dependence of emission wavelength on particle size. A broad particle size distribution is associated with a wider emission spectral band and reduced imaging potential of the CDs [35]. Furthermore, the surface zeta-potential of GCDs determined by the DLS was close to − 18 mV which plays a major role in their colloidal stability. The colloidal stability of GCDs was also explored by measuring the hydrodynamic size of GCDs stored in deionized water (pH 6.8) at room temperature (22–25 °C) over a period of 4 months using DLS. The particle size increased very slightly (Fig. S1), indicating the long-term aqueous storage stability of the prepared GCDs.

Optical and chemical properties of GCDs

The optical properties of GCDs were investigated to understand the associated emission centers. The UV–Visible absorption spectrum of GCDs in Fig. 1d shows strong absorbance at 410 nm. This strong 410 nm absorption peak can be attributed to the S=O molecular entity imparted by the PTSA precursor, since it has sulphonic acid group present in its chemical structure. Similar observations have been made with CDs synthesized using precursors with S=O groups or in presence of sulphuric acid [53, 54]. Furthermore, the FL emission at various excitation wavelengths ranging from 360 to 500 nm is shown in Fig. 1d, e. The CDs display an excitation-independent FL emission with the peak FL intensity when excited at 420 nm. The small dependency of emission even at the higher excitation wavelength (Fig. 1e) of GCDs can be associated with the uniform size distribution and a similar kind of functional group present on the surface with defects being passivated. The photoluminescence quantum yield (PLQY) of these GCDs was close to 70% that has never been demonstrated for carbon dots that display an excitation-independent green emission. More broadly, the high PLQY of the prepared GCDs can be attributed to narrow size distribution which results in small the re-absorption of emission by bigger size particles and the starting precursor with aromatic ring helps in formation of CDs with properly defined structure with minimum side products and impurities. The Absorbance and FL emission spectrum of rhodamine dye used for measuring the relative PLQY of GCDs are provide in Fig. S2. The various studies are summarized in Table S1 (Electronic Supplementary Information). The important role of ligands on the surface of CDs in determining their FL behavior is well established [33]. For the prepared GCDs, the presence of S=O molecular entity, introduced by the starting precursor, was confirmed by FTIR analysis that showed strong vibration at 1047 cm−1 and small stretching vibration peaks between 1300 and 1410 cm−1 that typically correspond to S=O stretching mode (Fig. 1f). The C–H bending vibration and the corresponding C–H stretching dual-bands appear at1015 cm−1, and at 2847 and 2915 cm−1, respectively. A hydroxyl group (O–H) stretching at 330 cm−1 is present due to the surface hydroxyl groups because of the water-based synthesis process. In addition, the nitrogen doping was confirmed by a C–N stretching vibration band at 1115 cm−1.

To study the surface functionalities, the Raman signal of GCDs was recorded (Fig. 1g). The D band located at 1317 cm−1 is a signature of sp3 orbital vibration, whereas the G band at 1582 cm−1 is attributed to sp2 orbital vibration [54]. The ratio of the intensity of D band to G band (ID/IG) provides useful information about sp3/sp2 characteristics of CDs. The ID/IG ratio of 1.2 indicates a high proportion of surface defects in GCDs due to sulfur-containing moiety. The insert in Fig. 1g shows the Raman signal of PTSA precursor. The disappearance of the characteristic Raman peaks of PTSA and appearance of characteristic graphitic D and G band clearly indicating the formation of GCDs has been completed. To investigate the emission centers of GCDs, the PL decay curve under 470 nm pulsed laser (Fig. 1h) was obtained. The PL decay curves of GCDs can be well fitted with a single-component exponential decay model (Fig. 1h), suggesting the presence of a single emission center. The average lifetime of GCDs comes out to be 3.09 ns. Since there is no prolonged emission, indicating the absence of deeper trap states due to defects. The fast σ*–n and π*–n transitions are associated with emissions from functional groups indicating the role of the S=O group in green fluorescence [34].

XPS analysis was conducted to further analyze the elemental bonding states in the surface layer of GCDs, (Fig. 2). The XPS survey spectrum shown in Fig. 2a indicates various chemical components of the GCD, which was comprised mainly of carbon (53.57%) and oxygen (38.53%) with less than 5% amounts of other elements such as nitrogen, which was at 2.45%, sulfur at 3.81%, and sodium at 1.64%. The presence of nitrogen (2.45%), as shown in the XPS, can be attributed to the use of ethylenediamine as a doping agent. The atomic resolution chemical mapping in STEM, confirms that nanoparticles present are carbon with oxygen, nitrogen, and sulfur on its surface (Fig. S3). The chemical structure of PTSA precursor (Fig. 1a) shows that it can serve as for carbon, oxygen, and sulfur content observed in the XPS. Whereas the small amount (1.64%) of sodium (Na1s) is likely an impurity arising from the chemicals or reagents used in the synthesis of GCDs.

Fig. 2
figure 2

Surface properties GCDs investigated using XPS a a survey spectrum with inset table providing the compositional data. b High-resolution C1s XPS spectra of GCDs with three major peaks. c High-resolution S2p XPS spectra of GCDs fitted with two prominent peaks. And d high-resolution N1s XPS spectra of GCDs

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Deconvolution of the high-resolution C1s spectrum of GCD in Fig. 2b affords three peaks. The strongest peak (283.97 eV) can be attributed to C–C bond, while the peak at 288.06 eV corresponds to O–C=O bond. The weakest peak (285.98 eV) can be ascribed to C–O bond. The deconvoluted high-resolution S2p spectrum of GCDs (Fig. 2c) fits two peaks with a stronger peak at 162.32 eV, which can be attributed to the S=O bond and a peak at 163.61 eV from a poly sulfur atom. The presence of this S=O moiety is believed as the reason for the green emission of GCDs. The presence of S=O moiety was also confirmed by FTIR shown in Fig. 1f. Whereas, only a single peak at 399.76 eV can be fitted to the high-resolution N1s spectrum of GCDs (Fig. 2d), which can be attributed to NH2 moiety.

In vitro cytocompatibility of GCDs

The in vitro cell viability of carbon dots was examined using a macrophage cell line RAW 264.7 (RAW cells), which are widely used for determining the cytocompatibility of nanomaterials [17, 56, 57]. The RAW cells grew normally after exposure to carbon dots even up to concentrations as high as 250 µg/mL. Figure S4 shows above 100% cell viability after 24 h exposure to GCDs compared with PBS control measured using MTS assay. As expected, the results showed that the GCDs did not inhibit cell growth, compared with control, indicating good cytocompatibility.

In vitro bioimaging using GCDs

In vitro imaging using GCDs provided essential details of appropriate excitation and emission wavelengths, and concentration-dependent FL behaviour of GCDs. Seven different concentrations of GCDs starting from 250 µg/mL to 2.5 mg/mL were prepared and imaged using IVIS® live animal imaging system at nine different combinations of excitation and emission wavelengths (Fig. 3a–i). After adjusting the image intensity so that the negative control (PBS) showed the zero FL intensity, the FL from the samples was quantified over the spectral region of interest. The analysis showed the maximum in vitro FL at an excitation wavelength of 430 nm and an emission wavelength of 520 nm as shown in the heat map in Fig. 3j.

Fig. 3
figure 3

a–i In vitro IVIS® (imaging of GCDs at different concentrations and different excitation/emission wavelength combinations. Drops (100 µL) of GCDs in PBS were aspirated on a non-fluorescent black paper. The number next to the drop is representative of the concentrations such that 1: PBS, 2: 250 µg/mL, 3: 375 µg/mL, 4: 625 µg/mL, 5: 1250 µg/mL, 6: 1875 µg/mL, 7: 2187.5 µg/mL, and 8: 2500 µg/mL. Note, the order of drops in all the panels from a–i is the same. j A heat map of corrected intensity (i.e. Sample intensity—PBS intensity) was obtained from the in vitro IVIS® FL images in a–i. The FL is presented as radiance efficiency with unit [p/s/cm2/sr]/[µW/cm2]

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Interestingly, an increase in FL was observed for GCDs concentrations of up to 1.25 mg/mL and a drastic decrease in FL for all the tested concentrations above 1.25 mg/mL (i.e. 1.86, 2.19, and 2.5 mg/mL) as presented in Fig. S5. This trend was observed for all the sets of excitation and emission wavelength combinations assessed in this study. These results could potentially be explained by the increased collision of GCDs at concentrations beyond 1.25 mg/mL that leads to FL quenching. This phenomenon has been observed previously with CDs, where a steep decline in FL occurs beyond a concentration and this is attributed to the increased collision of CDs that leads to non-radiative transfer of energy [58, 59]. A detailed mechanistic exploration of concentration-dependent FL bahaviour of CDs produced from citric acid as precursor was carried out in our recent article, where multicolour FL emission from nitrogen-doped CDs was revealed to be dependent on CDs’ concentration [58]. These fundamental understandings of the compositional and concentration relationship with FL behaviour from CDs underpin the work presented in this article. It is worth noting that the surface functional groups of GCDs provide reasonable resistance to fluorescence quenching below 1.25 mg/mL, hence additional linkers were not used to further stabilize the GCDs. Typically, CDs show collision quenching at much lower concentrations (at µg/mL levels) [58]. Moreover, further stabilization will increase the cost of production of such GCDs and add additional steps in the synthesis.

Bioimaging with subcutaneously injected GCDs

After in vitro cytotoxicity measurement and bioimaging of GCDs at different concentrations, GCDs were subcutaneously (SC) injected into a cadaver of freshly euthanized mice. Each mouse was injected with four different concentrations of GCDs and respective controls for fluorescence imaging. First, the best combination of FL excitation and emission wavelength was determined for SC injected GCDs. This important experiment, aided in the selection of the most optimal excitation and emission wavelengths, which were starkly different from that of the in vitro imaging experiment. As observed in Figs. S6 and S7, an excitation wavelength of 465 nm and an emission wavelength of 520 nm provided the highest FL of GCDs with the lowest background autofluorescence from mice skin that was observed for 430 nm excitation wavelength. While the 500 nm excitation neither displayed FL from the GCDs nor a strong background autofluorescence from the skin. These results can be attributed to the excitation-independent FL emission characteristics of the GCDs. Autofluorescence of skin tissue at UV-blue excitation wavelengths and the low excitation light penetration depth is well known and supports our data [60, 61]. It is worth pointing out that despite a low penetration depth of 465 nm in the skin, a strong FL signal from GCDs was observed and can be attributed to their high PLQY. Figure S8 shows FL from different concentrations of GCDs upon SC injection. Similar to the in vitro imaging, an increase in FL up to 1.2 mg/mL concentration of GCDs was observed, which was followed by a decrease with increasing concentrations of GCDs (Figure S5, Supporting Information). As expected, the spot injected with PBS control (Spot 1 in Fig. 4a) did not show any FL. After in vitro cytotoxicity measurement and bioimaging of GCDs at different concentrations, GCDs were subcutaneously (SC) injected into a cadaver of freshly euthanized mice. Each mouse was injected with four different concentrations of GCDs and respective controls for fluorescence imaging. First, the best combination of FL excitation and emission wavelength was determined for SC injected GCDs. This important experiment, aided in the selection of the most optimal excitation and emission wavelengths, which were starkly different from that of the in vitro imaging experiment.

Fig. 4
figure 4

IVIS® imaging of GCDs at different 465 nm excitation and 520 nm emission wavelength combination for a PBS control and three concentrations (250, 375, and 625 µg/mL) of GCDs. b four concentrations (250, 375, and 625 µg/mL) of GCDs. Subcutaneous injection spots are marked with number 1to 8 in. Spot 1 is PBS (i.e. 0 µg/mL), Spot 2 is 250 µg/mL of GCDs, Spot 2 is 375 µg/mL of GCDs, Spot 2 is 650 µg/mL of GCDs, Spot 5 is 1250 µg/mL of GCDs, Spot 6 is 1875 µg/mL of GCDs, Spot 7 is 2187.5 µg/mL of GCDs, and Spot 8 is 2500 µg/mL of GCDs. The FL is presented as radiance efficiency with unit [p/s/cm2/sr]/[µW/cm2]

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Conclusion

In summary, ultra-bright Green Fluorescent Carbon Dots (GCDs) have been synthesized using a high-yielding single-step hydrothermal process. The surface analysis by XPS, reveals the presence of the S=O group on the surface of GCDs, resulting in the excitation-independent green emission. Synthesized GCDs are highly cytocompatible at a dose as high as 250 µg/mL. Furthermore, the steric effects due to the same surface group impart the GCDs resistance to emission quenching below a concentration of 1.2 mg/mL. In vitro imaging demonstrated that GCDs fluorescence across a range of emission wavelengths from multiple excitation wavelengths with the strongest FL signal achieved from the Ex 430 nm/Em 520 nm wavelength combination. Importantly, FL intensity was dose dependent with the peak signals attained at 1.25 mg/mL and drops beyond this concentration due to collision quenching. When subcutaneously injected in freshly euthanized mice, the GCDs also provided strong FL with the maximum signals at the Ex 465 nm/Em 520 nm wavelength combination. The need for a higher excitation wavelength ex-vivo arises from the strong background autofluorescence, which was reduced drastically at 465 nm for the GCDs. The reliable observation of the GCDs fluorescence through the animal tissue highlights its potential utility in biomedical imaging applications.