Carbon quantum dots for fluorescence imaging of zebrafish

Thank you for visiting nature.com. The browser version you are using has limited support for CSS. For the best experience, we recommend that you use a newer browser (or turn off compatibility mode in Internet Explorer). At the same time, in order to ensure continuous support, we display the website without styles and JavaScript.
Carbon quantum dots (C-QD) are becoming an ideal substitute for metal-based QD and dye probes due to their high biocompatibility, low toxicity, easy preparation and unique photophysical properties. Here, we describe the fluorescent bioimaging of zebrafish using C-QDs as probes from the perspectives of C-QDs preparation, zebrafish breeding, embryo harvesting, and introduction of C-QDs into embryos and larvae by immersion and microinjection . The pleochroism of C-QD was verified by imaging zebrafish embryos. The distribution of C-QD in zebrafish embryos and larvae can be successfully observed from their fluorescence emission. Zebrafish was used as a model to test the biological toxicity of C-QD, and C-QD did not interfere with the development of zebrafish embryos. All the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probes. The absorption, distribution, metabolism and excretion pathways (ADME) of C-QDs in zebrafish are revealed by their distribution. Our work provides useful information for researchers interested in using zebrafish as a model and the application of C-QD. The operation-related zebrafish is suitable for studying the toxicity, adverse effects, transport and biocompatibility of nanomaterials, and for drug screening using zebrafish as a model.
Nanomaterials provide many considerable opportunities for bioimaging and clinical diagnosis. Semiconductor quantum dots (QD) are molecular-sized nanocrystals with a size less than 10 nm. Due to its powerful quantum confinement effect, quantum dots have a variety of unique photoelectric properties, such as size and wavelength-dependent luminescence and low photobleaching 2, 3, 4. QDs-based imaging combines these characteristics to improve the advantages of fluorescence imaging, including real-time, accurate and in-vivo observation, with high sensitivity, fast response, low cost and no radiation. However, due to the use of heavy metals, metal-based quantum dots (M-QDs) are associated with long-term toxicity and environmental issues5,6. Obtaining M-QD requires complicated procedures, and their aggregation is usually observed after long-term storage7.
Carbon quantum dots (C-QDs) are quasi-spherical carbon materials with a diameter of less than 10 nm8. C-QD does not have the toxic effects seen with heavy metals because C-QD contains a large amount of carbon and relatively little oxygen, hydrogen and nitrogen9,10. Carbon exists in the form of crystalline graphite, which is the core of C-QD, while oxygen and hydrogen form hydroxyl and carboxyl groups, which contribute to the functionalization of C-QD 8 and improve its hydrophilicity. C-QD also exhibits wavelength-dependent luminescence and low photobleaching. In addition, C-QD has good storage and light stability11. After 1 year of storage under ambient conditions, good stability of the fluorescence intensity of C-QDs was observed12. Therefore, considering the low toxicity and unique photophysical and chemical properties of C-QD, they are becoming an ideal substitute for M-QD.
C-QD is prepared by chemical oxidation of graphite 13, soot 14, and carbonized products with strong acid or oxidant through a top-down procedure [8,15]. Bottom-up methods are diverse, because various molecular precursors can be hydrothermally treated to obtain C-QDs8. Although strong acids and oxidants are not required, passivators provide the possibility to obtain surface passivation and/or heteroatom-doped C-QDs, and have improved luminescence8. Using ethylenediaminetetraacetic acid (EDTA) as precursor 16, a C-QD yield of 78% can be obtained. The combination of glucose and various passivating agents has been widely used to prepare C-QD17,18 with enhanced fluorescence, high yield, simple operation, low cost and high biocompatibility. Here, we provide detailed information on the preparation of C-QD using glucose and ethylenediamine as precursors and passivators 19, 20, 21, 22, 23, and their imaging applications in important model organisms, zebrafish.
Zebrafish are important model organisms because of their rapid embryonic development and short generation time24. The embryo placement time can be controlled by adjusting the light-dark cycle 25. Taking care of zebrafish and embryos is also very simple. Zebrafish has been used for in vivo imaging, behavioral testing, and compound screening26,27. The zebrafish special edition “Methods” was released in three areas in 2013, discussing new technologies using zebrafish, including genomics and epigenomics, regenerative medicine and human disease modeling, and zebrafish-related imaging technologies27 . Importantly, zebrafish are highly homologous to mammals. About 70% of human genes are homologous to zebrafish, 28,29. Therefore, modeling human diseases using zebrafish as a model becomes an important and rapid strategy. . As an important milestone for use as a model organism, the zebrafish genome was reported on 201328,29.
Embryo development is an important model for studying toxicity, system damage in the body, and the transport and biocompatibility of nanoparticles and drugs3,30. Based on these development results, possible risks to human health and the environment are proposed. Compared with other vertebrate model systems (such as mice and rats), zebrafish embryos are unique in these studies31,32. The embryonic development of zebrafish is completed quickly within 120 hours, and its developmental stage has good characteristics. The special external development of zebrafish embryos helps to directly visually detect the phenotypes of pathological embryonic death and dysplasia. In addition, zebrafish embryos are transparent, and it is easy to ignore the transport and influence of nanoparticles and real-time drug screening31,32. The results based on embryo development proved dose-dependent biocompatibility, the initial steps into the embryo, the potential applications and adverse effects of nanomaterials, and drug screening [3,33,34]. For these types of research, the introduction of foreign species into embryos is crucial.
Zebrafish has been widely used in biological and biochemical research, such as fluorescent protein expression 35, visualization of blood flow in the heart 36, gene knockout and functional cardiac imaging 37, study of mutants with bone calcification defects 38, and evaluation of nanoparticles Oxidative toxicity. The zebrafish primordial germ cells (PGC) are monitored to check their morphology during early development40. Embryogenesis in zebrafish has been extensively studied through in vivo imaging41. Here, we describe zebrafish culture and operations involving zebrafish fluorescence imaging using C-QDs as probes. It explains in detail how to introduce C-QD into zebrafish embryos and larvae through microinjection and immersion.
By simply immersing the embryo in a solution of C-QDs, C-QDs can penetrate the yolk membrane and germ into the embryo. The fluorescence of C-QDs observed their distribution in zebrafish embryos and larvae, and verified the potential of C-QDs as probes. Microinjection procedures have been used to produce transgenic zebrafish after injection of DNA, gene knockdown after injection of antisense morpholino, study of infectious diseases after injection of microorganisms, and study of tumor progression after injection of cancer cells42. Describes the procedure of microinjection of C-QD into zebrafish embryos for fluorescent biological imaging. After some fine-tuning, other nanomaterials or drugs can be used to adapt the microinjection procedure to the zebrafish embryo model. The cultivation and handling of zebrafish is simple and convenient, while C-QD has biocompatibility and low toxicity. This article uses C-QDs as a probe to perform fluorescence bioimaging of zebrafish, which is an example that can be applied to basic life science research and drug screening.
Since its first discovery in 200443, extensive research has been conducted on the preparation and photoelectric properties of C-QD (such as fluorescence, electrochemiluminescence and catalysis)44. Their excitation-dependent emission characteristics allow C-QDs to perform multicolor cell imaging44. Sun’s Group45 realized two-photon imaging with C-QD as a probe. Through the hydrothermal procedure, the preparation of C-QD with glucose as the precursor is relatively simple. In addition, ethylenediamine as a passivating agent can significantly enhance fluorescence emission46. Figure 1 shows the transmission electron microscope (TEM) image and size distribution of C-QD prepared using glucose and ethylenediamine as precursors. The size of C-QD is mainly distributed in the range of 1-3 nm, with an average diameter of 1.89 nm (Figure 1B), which confirms the quantum confinement effect of C-QD on the nanoscale. The absorption and fluorescence spectra of C-QD at different excitation wavelengths are shown in Figure 1C. The normalized emission spectrum of C-QD in the inset of Figure 1C clearly illustrates the excitation-related emission, which provides the possibility of using C-QD as a probe for multicolor imaging.
Transmission electron microscope (TEM) image (A) and size distribution (B) of C-QD. (C) Absorption and fluorescence spectra of C-QD with different excitation wavelengths. Inset: Normalized fluorescence spectrum. Scale bar, 20 nm.
X-ray photoelectron spectroscopy (XPS) analysis showed the composition of the synthesized C-QD. As shown in Figure 2A, C, N and O were found in the C-QD of the XPS spectrum. The C1s peak is resolved into three components, 284.6, 285.8, and 287.4 eV, respectively, representing sp2C-sp2C, N-sp2C and CO/C=O bonds 21, 47, 48 (Figure 2B). The high-resolution spectrum of N1s (Figure 2C) shows the presence of pyridine (399.2 eV) and pyrrole (401.2 eV) N atoms [21,48], indicating that C-QD has been successfully doped with nitrogen atoms. Figure 2C reveals that the O1s peak can be decomposed into two components centered at 530.7 and 531.3 eV, representing the presence of C=O and C-OH/COC groups 49,50. XPS results showed that the surface of the synthesized C-QD was functionalized with multiple oxygen and nitrogen groups through the reaction between glucose and ethylenediamine.
Zebrafish has been widely used in biological and biochemical research40. We chose zebrafish as the model because it has been widely used in medical and chemical research. The zebrafish embryo is also an important model for studying toxicity, system damage in vivo, and the transport and biocompatibility of nanoparticles and drugs3,30. As shown in Figure 3, the multi-color in vivo fluorescence imaging using C-QDs as a probe was verified. Figure 3 shows the bright-field and multi-color fluorescence images of embryos soaked with C-QDs. The difference in brightness between the yolk and the internal quality of the embryo in the fluorescence image shows that C-QD has a different affinity for these tissues (Figure 3B D). Therefore, C-QD enters the embryo by simply immersing it through the chorion and germ, and is mainly deposited in the yolk sac due to its small size. As the concentration of C-QDs increases, the fluorescence images of the embryos become brighter (Figure 3B D), while their brightfield images are indistinguishable from each other (Figure 3A). The blue fluorescence image of the embryo can be observed with ultraviolet radiation (Figure 3B), while the green and red images can be observed with blue and green illumination (Figure 3C, D). The fluorescence of C-QDs can observe their distribution in zebrafish embryos and verify the practicality of C-QDs as imaging probes.
Brightfield (A) and multi-color fluorescence [(B) blue, (C) green and (D) red) images of zebrafish embryos after soaking in C-QDs solutions of different concentrations for 3 hours.
Images acquired under strong light show the yolk sac (ys) and the internal quality of the embryo (ime). A 10x eyepiece and a 4x objective lens are used. Scale bar, 1.0 mm.
After being immersed in C-QD, fluorescence imaging of zebrafish embryos allows visualization of embryo development, from single cells to larvae (Figure 4). From the fluorescence images of embryos immersed in 2.5 mg mL-1 C-QD solution at different periods (bottom row in Figure 4D F), C-QDs are mainly deposited in the yolk sac at 24 hpf ((Figure 4D), It is redistributed to the trunk of the larva at 48 hpf (the yellow arrow in Fig. 4E, F). This redistribution of C-QD during embryonic development reveals that C-QD has different tissue affinities. With increasing incubation time Increase, the embryo becomes darker and darker. At 60 hpf, the fluorescence emission of C-QDs in the embryo almost disappears. We assume that the digestive system removes some C-QD from the embryo, as shown by the bright intestine at 48 hpf (Figure 4E). By comparing the bright field images of embryos cultured in C-QDs solution (upper row of Figure 4) for 3 h and control (upper row of Figure S1) at different periods, we found that C-QDs did not It can interfere with embryo development, which proves the low toxicity and high biocompatibility of C-QD. Due to its stable fluorescence emission and biocompatibility, C-QD is used to reveal biological phenomena related to zebrafish embryo development. Progress in time and space.
Brightfield (upper) and fluorescence (lower) images of zebrafish embryos after immersing 2.5 mg mL-1 C-QDs solution for 3 hours at the time point:
(A) 3, (B) 6, (C) 12, (D) 24, (E) 48, (F) 60 h  pf. A 10x eyepiece and a 4x objective lens are used. Scale bar, 1.0 mm.
The microinjection of C-QD is used to illustrate the procedure of introducing foreign species directly into zebrafish embryos. The bright field and fluorescence images of the embryos microinjected with C-QD are shown in Figure S2. As the concentration of C-QD increases, the fluorescent images of embryos become brighter (bottom row of Figure S2), while their brightfield images are not significantly different (top row of Figure S2). The brightness of the embryo showed that the C-QDs had been successfully introduced into the embryo by microinjection, and the embryo was still alive. Therefore, microinjection is a strategy to introduce these materials directly into embryos.
Microinjection procedures have been used to generate transgenic zebrafish after injection of DNA, gene knockdown after injection of antisense morpholino, study of infectious diseases after injection of microorganisms, and study of tumor progression after injection of cancer cells42. The procedure of microinjecting C-QD into zebrafish embryos can also be used in the above research, such as other nanomaterials or drugs based on zebrafish embryos. Interestingly, the distribution of C-QD in zebrafish embryos is different, and there are different methods of introduction: immersion or microinjection. This phenomenon may be crucial for the further medical and chemical applications of C-QD.
Zebrafish larvae were used as a model to validate the imaging application and tissue distribution of C-QD in vivo. Figure 5 shows the images of the whole body and amplified parts of zebrafish larvae after being soaked in C-QDs solutions of different concentrations for 10 hours. As the concentration of C-QD in the concentration-dependent model increases, zebrafish larvae exposed to C-QDs become brighter, indicating that C-QDs have been successfully introduced into larvae. After C-QD enters the larva through swallowing and skin absorption 51, 52, they selectively gather in the head, yolk sac, and tail, showing the tissue-dependent affinity of C-QD (Figure 5). The brightness of the dorsal aorta indicates that C-QD has entered the circulatory system (Figure 5D), which is important for the transport of C-QD in zebrafish. The eye is the brightest part of the zebrafish’s head, and its brightness increases with the increase of the concentration of C-QDs, and the lens is easily distinguished from the eyeball (Figure 5B). This indicates that C-QD can cross the blood-eye barrier and enter the eye. The C-QD in the yolk sac mainly accumulates in the intestine, indicating that C-QD enters the digestive system and can be eliminated from the body (Figure 5C). Some C-QD was removed from zebrafish larvae by metabolism, which was confirmed by the bright gut (Figure 5A). C-QDs preferentially accumulate around zebrafish (Figure 5B, D), indicating that skin absorption is an important way for C-QDs to enter zebrafish. Therefore, the outline of the zebrafish is illustrated by the fluorescence emitted by C-QD.
After being immersed in different C-QDs solutions for 10 h, bright field (upper) images and fluorescence (lower) images of zebrafish larvae (A) whole body, (B) head, (C) yolk sac and (D) tail concentration. The magnified image shows (B) the eye and lens, (C) the yolk sac and intestine, and (D) the blood vessels in the tail, with fluorescent images. Use 10x eyepieces for (A, B, C and D). (A) Use 4 times objective lens, (B, C and D) use 10 times objective lens. The scale bar, (A) is 1.0 mm, (B), (C) and (D) are 500 ¦Ìm.
Therefore, the absorption, distribution, metabolism and excretion (ADME) pathways of C-QDs in zebrafish are revealed through their distribution. C-QD enters the zebrafish body 52 through swallowing and skin absorption, and is partially excreted through the intestine. Some C-QD enters the cardiovascular system and is transferred throughout the body, which is confirmed in the brightness of blood vessels and tail tissues. Therefore, C-QD is a biocompatible probe that has no obvious quenching effect and is suitable for in vivo imaging. Zebrafish have a high degree of homology with mammals28,29, so the results obtained from zebrafish can be used to simulate the biological effects of other higher animals.
C-QD fluorescence can also be used to illustrate zebrafish morphology by laser scanning confocal imaging. As shown in Figure 6, C-QD gathered on the yolk sac and eyeball of the larva. Confocal images on different scanning planes clearly show the cyst-like structure of the yolk sac. It was also found that C-QD accumulates on the eyeball and enters the lens, which means that C-QD can penetrate the blood-eye barrier and enter the eye. The results of the confocal image are in good agreement with the results of Figure 5. The darkness of the midbrain in zebrafish larvae indicates that C-QD cannot cross the blood-brain barrier (BBB)  and enter the brain. All results confirm that C-QD has the potential for in vivo imaging because they are easily transported through the cardiovascular and digestive systems. Since the eye development and morphology of zebrafish are similar to those of other vertebrates, selective accumulation of C-QD in the eye area of  other vertebrates may also occur, indicating the potential imaging application of C-QD. In addition, the dark appearance inside the zebrafish head indicates that C-QD cannot cross the BBB and enter the brain. This shows that C-QD can be used for eye-related imaging, and the possibility of brain damage is very low.
Confocal images of zebrafish (72 hpf larvae immersed in 2.50 mg mL-1 C-QD for 10 h) on different scanning planes.
The lens, eyes, yolk sac and midbrain are clearly visible. A 10x eyepiece and a 10x objective lens are used. Scale bar, 250¦Ìm.
Zebrafish embryos and larvae were used as models to verify the in vivo biotoxicity and biocompatibility of C-QD synthesized after immersion/microinjection. As shown in Figure S3A, after microinjection of 1.5 mg mL-1 C-QDs solution within 0~3 hpf, the survival rate of zebrafish embryos was higher than 80% at 24 hpf, and at 2.5 mg, the survival rate dropped to About 55% mL-1 of C-QDs solution. The survival rate of embryos immersed in low-concentration C-QDs solution (0.5, 1.0, 1.5 mg mL-1) at 0~3 hpf is higher than 80% at 24 hpf, while embryos immersed at 2.5 mg mL" The survival rate was reduced to about 60% with 1 C-QD solution (Figure S3B). Figure S4 shows the survival rate of zebrafish larvae at 84 hpf after soaking in the C-QDs solution for 10 hours. Similar to the control group, the survival rate of larvae soaked in low-concentration C-QDs solution (0.156, 0.313, 0.625 mg mL-1) was higher than 95%. After soaking in 1.25 and 2.5 mg mL-1 C-QDs solutions for 10 hours, the survival rates of zebrafish larvae were higher than 85% and 55%, respectively. Using zebrafish as a model, the toxicity of maghemite@SiO2 rattle-shaped microspheres was studied, and it was found that 200¦ÌgmL-1 of maghemite@SiO2 caused severe deformities in zebrafish53. Therefore, the toxicity of C-QDs is lower than that of maghemite@SiO2 microspheres. Compared with metal-based quantum dots and other nanoparticles 53,54,55,56, C-QD has a higher tolerance to zebrafish embryos and larvae.
In addition, as shown in Figure 1.5, the zebrafish larvae grow normally after being immersed in 1.5 mg mL-1 C-QDs solution. The deformity of the experimental group was very small, almost the same as the control group. The result also confirmed that our C-QD has good biocompatibility. As the incubation time increased, the fluorescence of C-QDs in zebrafish larvae became weaker and weaker (Figure S5B). The C-QD of the head and tail is removed faster than the yolk sac. The intestine (yellow arrow in Figure S5B) is always the brightest part of the larva, which further confirms the conclusion that C-QD is removed from the larva through metabolism. Therefore, C-QD is removed from the larvae and does not affect the development of the larvae.
The conclusion is that in preparing C-QDs, zebrafish breeding, embryo harvesting, and introducing C-QDs into embryos and larvae by immersion and microinjection, fluorescence imaging of zebrafish using C-QDs as probes is reported. C-QDs fluorescence can observe their distribution in zebrafish embryos and larvae, and verify the use of C-QDs as imaging probes. Zebrafish was also used as a model to verify the characteristics of C-QDs such as multicolor, low toxicity and high biocompatibility. The multicolor fluorescence of C-QDs remains in the zebrafish embryo and can be used for multicolor imaging in vivo. It clearly illustrates the toxicity of zebrafish and its influence on embryonic development and biodistribution of C-QDs. C-QDs can enter zebrafish embryos and larvae by being immersed and concentrated in different parts, which indicates their tissue affinity for zebrafish. Both microinjection and immersion are used to introduce C-QDs into zebrafish embryos, which confirms that this operation is a general strategy for introducing different species into zebrafish for the study of other drugs and nanomaterials. Zebrafish fluorescence bioimaging with C-QDs as a probe provides an example that can be used in basic life science research, toxicity testing and drug screening using zebrafish as a model.
Glucose was purchased from Amresco in Shanghai, China. Phenol red solution, paraffin oil, 1-phenyl-2-thiourea, and 3-aminobenzoate methanesulfonate were purchased from Sigma-Aldrich in Shanghai, China. Ethylenediamine was purchased from Tianjin Chemical Reagent Wholesale Company in Tianjin, China. The high-purity nitrogen was obtained from the Liufang High-Tech Gas Plant in Tianjin, China. All solutions were prepared using ultrapure water (18.25M¦¸cm) from the Aquapro ultrapure water system in Chongqing, China.
The transmission electron microscope (TEM) image was performed using Tecnai G2 F20 from FEI Company in the United States, and it was operated at an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) analysis is performed by a Kratos Axis Ultra DLD spectrometer equipped with a monochromatic AlK¦Á X-ray source (h¦Í1486.6 eV), hybrid (magnetic/electrostatic) optics, multi-channel plate and delay line detector . The UV-visible absorption spectrum was recorded with a UV-2450 spectrophotometer from Shimadzu Corporation. The fluorescence spectrum of C-QD was performed by FL-4500 fluorescence spectrometer from Hitachi, Japan. Under the Leica 200 M 2000 microscope, the DP71 digital camera of Olympus of Japan was installed on the BX51 fluorescence microscope and Olympus DP71 to obtain bright field and fluorescence images. The micro-pulse pressure injector used in the experiment is American ASI, MPPI-3.
The excitation-related emission was observed when the fluorescence image was collected, which verified the polychromatic nature of C-QD. When collecting fluorescent images of zebrafish and their embryos, the sensitivity (ISO 400) can be maintained to reduce equipment interference. Before acquiring fluorescence images, first acquire brightfield images to record the morphology of zebrafish embryos or larvae. The control sample is used to optimize the exposure time and obtain almost invisible fluorescence images. This process is repeated every time an image is acquired at a different time.
Add 40 mg glucose, 10 mL ultrapure water and 100 ¦ÌL ethylenediamine to the inner lining (30 mL) of a Teflon-lined autoclave vessel (Figure S6A), and then put the inner lining in an ultrasonic cleaner and Perform ultrasonic treatment and stand for 10 minutes to obtain a colorless and transparent solution. The lining is sealed in a container and heated at 200¡ãC for 4 hours, and then naturally cooled to ambient temperature (20¡ãC-30¡ãC) (Figure S6B). The obtained brown solution was centrifuged at 12000 rpm for 10 minutes. The supernatant was then collected and lyophilized for 28 hours to obtain a brown C-QDs paste, which can be dissolved again for later use. The step-by-step process for preparing C-QD can be found in SI.
Zebrafish are cultured in circulating water in a 10 L aquarium (use NaCl and NaHCO3 and pH 7.0-7.2 to control the salinity at about 450-500¦Ìscm-1, add 10% fresh deionized water every day) at 28.5¡ãC Under the conditions, a 10/14-hour dark/bright cycle (light up at 7:00 AM-9:00 PM). In an aquarium of 10 L per day, feed them 3 ml of freshly hatched live brine shrimp at 8:00 AM, 12:00 noon and 5:00 PM every day (from 3.5 g brine shrimp embryos in 1 g of water). Out). (Note: Zebrafish and their embryos are sensitive to pH and temperature, so please ensure that the pH of the medium is 7.0 7.2 and culture at 28.5¡ãC; in a 10 L aquarium, the number of adult zebrafish should be less than 10.) All experimental protocols using animals were approved by the Institutional Animal Care Committee of Nankai University. The method is carried out in accordance with approved guidelines.
Choose one adult female zebrafish and two adult male zebrafish, and place them on different sides of a 1.5 L breeding cage at 5:30 PM with a dividing line (Figure S7A and B). Remove the dividing line from the breeding cage at 8:30 am the next morning (Figure S7C). After half an hour, the embryos were harvested from the breeding cage through a filter and fed with E3 medium, while removing unfertilized embryos and debris (Figure S7D). (Note: Do not disturb the zebrafish during spawning, so as not to affect their spawning.) Zebrafish can be reorganized in a larger aquarium to produce more embryos. The step-by-step process for zebrafish rearing and embryo harvesting can be found in SI.
To prepare the microinjection needle, use a microloader pipette to pull 2  ¦ÌL of 25% phenol red solution into the needle. Insert the needle into the micro syringe and seal it. Turn on the air source and the micro syringe. Step on the foot pedal and monitor the droplet diameter of the phenol red solution, while trimming the needle under the microscope and adjusting the injection pressure as needed. The droplet diameter of the phenol red solution in paraffin oil is used to calculate the volume of a single injection. The phenol red solution beads with a diameter of 0.12 mm are 1 nL (the volume required for a single microinjection) of phenol red solution (Figure S8A). (Note: The ideal injection volume is approximately 10% of the embryo volume, for example 1 nL is used in this study). Hold the needle with a pair of sharp tweezers and place the needle in the proper position so that the needle can inject 1 nL of C-QDs solution into the embryo and provide a consistent solution bead size. The step-by-step procedure for preparing the microinjection needle can be found in SI.
Nanoparticles can enter the embryo through endocytosis. First, immersion was used to introduce C-QDs into zebrafish embryos to study the effects on embryonic development and imaging applications of C-QDs. Dissolve an appropriate amount of C-QDs in E3 medium to prepare 5 mL C-QDs solutions of different concentrations. Add 5 ml of C-QDs solution to each well of a six-well flat-bottom cell culture plate (Figure 7A). Twenty zebrafish embryos were placed in each well of the cell culture plate and soaked in 5 mL of medium containing different concentrations of C-QD (Figure 7B). Before soaking, make sure that the embryo has not developed beyond the four-cell stage, which is 1 hour after fertilization (hpf). Ideally, these embryos should be at the single-cell stage (0.5 hpf) to ensure that C-QD can penetrate into the embryo and be dispersed throughout the embryo. After 3 hours, the embryos were washed three times with E3 medium to remove excess C-QD. The embryos were cultured in E3 medium at 28.5¡ãC with a dark/light cycle of 10/14-h. The culture medium was replaced with E3 medium at 8:30 AM and 5:30 PM every day, and the E3 medium contained 0.003 wt per day. %PTU to prevent pigmentation and mediate visualization.
(A, B) Different concentrations of embryos soaked in C-QDs solution: 0.5, 1, 1.5, 2.5 and 5 mg mL-1. (C, D) Acquire images of zebrafish embryos. (E) Brightfield (upper) and fluorescence (lower) images of zebrafish embryos after soaking in 2.5 mg mL-1 C-QD. Scale bar, 1.0 mm.
Embryos soaked with different concentrations of C-QD were placed on the concave surface of a single concave glass slide through a dropper and immersed in E3 medium (Figure 7C). Place the glass slide under the fluorescence microscope and place the embryo in the proper position for fluorescence imaging using the fluorescence microscope (Figure 7D). Brightfield and fluorescence images of zebrafish embryos were obtained using a 10x eyepiece and a 4x objective lens (Figure 7E). Images of embryos with different concentrations of C-QD were collected at 3, 6, 12, 24, 48 and 60 hpf. The step-by-step process of introducing C-QD into zebrafish embryos by immersion can be found in SI.
Microinjection has been used to produce transgenic zebrafish after injection of DNA, gene knockdown after injection of antisense morpholino, study of infectious diseases after injection of microorganisms, and study of tumor progression after injection of cancer cells43. The microinjection of C-QD is used to illustrate the operation steps of the microinjector prepared in Figure S8. Dissolve 10 mg C-QDs in 2 mL ultrapure water to obtain 5 mg mL-1 C-QDs solution, and dilute with ultrapure water to 2.5, 1.5, 1.0, and 0.5 mg mL-1 (Fig. 8A). Arrange the embryos on the side of the slide by pipetting to form a single column (Figure 8B). Pull the 2¦ÌLC-QDs solution into the needle previously drawn with a micropipette. After piercing the chorionic surface and entering the yolk needle, by stepping on the foot pedal, 1 nL (bead diameter of 0.12 mm) of C-QDs solution is injected into the embryo through a micro syringe (Figure 8C). The solution should be injected into the yolk quickly and accurately so that the embryos can survive and grow. Air bubbles should be avoided during the injection, as they may kill the embryo. Before microinjection, make sure that the embryo has not developed beyond the four-cell stage (1 hpf). Ideally, the embryo should be at the single-cell stage (0.5 hpf) to ensure that C-QD is dispersed throughout the embryo. After completing a row of embryos, immediately transfer the injected embryos into a clean Petri dish with mild E3 medium, and inject the embryos with the injected Petri dish labeled with C-QD concentration. Embryos were cultured in E3 medium at 28.5¡ãC with a 10/14-h dark/light cycle. The culture medium was replaced with E3 medium at 8:30 AM and 5:30 PM every day, and the E3 medium contained 0.003 wt. %PTU to prevent pigmentation and mediate visualization. The step-by-step process of introducing C-QD into zebrafish embryos by microinjection can be found in SI.
(A) C-QDs solutions of different concentrations for microinjection experiments. (B) The embryos are neatly arranged next to the glass slide in the Petri dish (inset: enlarged embryo image). (C) The C-QD is microinjected into the embryo under the microscope, while the embryo and the needle tip are enlarged. Scale bar: 4.0 mm.
The rapid development of zebrafish provides an opportunity to study the distribution of nanomaterials, drug screening and study the toxicity of nanomaterials. The imaging of zebrafish larvae using C-QD as a probe illustrates the operation and procedures. Cultivate zebrafish embryos in E3 medium at 28.5¡ãC with a 10/14-h dark/photoperiod until the larvae hatch (about 72 hpf). 2 mL of C-QDs solutions with different concentrations were prepared (Figure 9A). Add 2 mL of culture medium containing different concentrations of C-QD and 5-7 zebrafish larvae to each of the 6 wells of the 24-well cell flat-bottom culture plate (Figure 9B). The larvae were cultured in E3 medium at 28.5¡ãC for 10/14-h dark/light cycle. After 10 hours, the C-QDs solution was replaced with E3 medium, and the larvae were washed three times with E3 medium with a dropper to remove excess C-QDs.
(A, B) Culture 5 to 7 zebrafish larvae (72 hpf) in C-QDs solution at different concentrations in each of the 6 wells of the 24-well flat-bottom culture plate. (C) Soak zebrafish larvae with 0.016% 3-aminobenzoate methanesulfonate solution to anesthetize them. (D) The zebrafish larvae are placed on the concave surface of a single concave glass slide to keep the 3-aminobenzoate methanesulfonate solution immersed in the larvae. (E) Acquire an image of zebrafish larvae. (F) Bright field (upper) and fluorescence image (lower) of zebrafish larvae immersed in 5 mg mL-1 C-QDs. Scale bar, 1.0 mm.
Citation method: Kang, Y.-F. etc. Carbon quantum dots for fluorescence imaging of zebrafish. Science Rep. 5, 11835; doi: 10.1038/srep11835 (2015).
Wang, Y., Li, Z., Wang, J., Li, J. & Lin, Y. Graphene and graphene oxide: biofunctionalization and its application in biotechnology. Trends in biotechnology. 29, 205 212 (2011).
Mazumder, S., Dey, R., Mitra, MK, Mukherjee, S. & Das, GC Comment: Bio-functionalized quantum dots in biology and medicine. J. Nano. 2009, 1-17 (2009).
Lee, KJ, Nallathamby, PD, Browning, LM, Osgood, CJ & Xu, XHN In vivo imaging of the transport and biocompatibility of single silver nanoparticles in the early development of zebrafish embryos. Acs Nano 1, 133-143 (2007).
Jaiswal, JK, Mattoussi, H., Mauro, JM & Simon, SM use quantum dot bioconjugates for long-term color imaging of living cells. Nat Biotechnology. 21, 47 51 (2003).
Zhang, YQ, etc. One-pot synthesis of N-doped carbon dots with adjustable luminescence characteristics. J. Mater. Chemistry 22, 16714 16718 (2012).
Derfus, AM, Chan, WCW and Bhatia, SN explore the cytotoxicity of semiconductor quantum dots. Nanolet. 4, 11-18 (2004).
Bera D., Qian L., Tseng T.-K. & Holloway, PH quantum dots and their multimodal applications: a review. Material 3, 2260 2345 (2010).
Baker, SN and Baker, GA Luminous carbon nanodots: the emerging nanolight. Angew. Chemical Interpretation Ed 49, 6726 6744 (2010).
Liu, CJ, etc. Carbon dot-based PEI passivation enhances gene nanocarriers for gene delivery and biological imaging. biomaterials. 33, 3604 3613 (2012).
Zhou Jianguo et al. An electrochemical approach to blue light-emitting nanocrystals from multi-walled carbon nanotubes (MWCNT). J. Am Chemical Soc. 129, 744 745 (2007).
Ding, C., Zhu, A. & Tian,  Y. C-point functional surface engineering for fluorescent biosensing and in vivo bioimaging. Cumulative chemical Res. 47, 20-30 (2014).
Wang, J., Wang, CF & Chen, S. Amphiphilic egg-derived carbon dots: rapid plasma manufacturing, pyrolysis process and multi-color printing patterns. Angew. Chemical Interpretation Ed 51, 9297-9301 (2012).
Zhao, QL, etc. Easily prepare fluorescent carbon nanocrystals with low cytotoxicity through the electrooxidation of graphite. Chemical Community 44, 5116-5118 (2008).
Liu, HP, Ye, T. & Mao, CD Fluorescent carbon nanoparticles derived from candle soot. Angew. Chemical Interpretation Ed 46, 6473 6475 (2007).
Ray, SC, Saha, A., Jana, NR & Sarkar, R. “Fluorescent carbon nanoparticles: synthesis, characterization and bioimaging applications”. J. Physical Chemistry C 113, 18546-18551 (2009).
When, QQ etc. High-yield, high-solubility nitrogen-doped carbon dots: formation, fluorescence mechanism and imaging applications. RSC Progress 4, 1563 to 1566 (2014).
Wei, WL, etc. Non-enzymatic diffusion reaction: a general way to produce nitrogen-doped carbon dots with adjustable multicolor luminescence displays. Science Rep. 4, 3564 (2014).
Li, HT etc. One-step ultrasonic synthesis of water-soluble carbon nanoparticles with excellent photoluminescence properties. Carbon 49, 605 609 (2011).
Qu, Q., Zhu, A., Shao, X., Shi, G. & Tian,  Y. Develop carbon quantum dot-based fluorescent Cu2 + probes suitable for live cell imaging. Chemical Community 48, 5473-5475 (2012).
Xu, Y. Wait. Nitrogen-doped carbon dots: a simple and universal preparation method, photoluminescence research and imaging applications. Chemistry Euro. J.19, 2276-2283 (2013).
Dong, Y. Wait. Carbon-based dots are co-doped with nitrogen and sulfur to achieve high quantum yield and emission independent of excitation. Angew. Chemical Interpretation Ed 52, 7800-7804 (2013).
Liu, R. Wait. An aqueous approach to multicolor photoluminescent carbon dots using silica balls as the carrier. Angew. Chemical Interpretation Ed 121, 4668 4671 (2009).
Yang, ST, etc. Carbon dots for optical imaging in the body. J. Am Chemical Soc. 131, 11308-11309 (2009).
Ko, SK, Chen, X., Yoon, J. & Shin, I. Zebrafish are good vertebrate models for molecular imaging using fluorescent probes. Chemical Soc. Rev. 40, 2120 2130 (2011).
Westerfield, M. Zebrafish Book. Zebrafish (Danio rerio), Edinburgh Laboratory Use Guide. No. 4 (University of Oregon Press, Eugene; 2000).
Weber, T. & Köster, R. A genetic tool for multicolor imaging of zebrafish larvae. Method 62, 279 291 (2013).
Howe, KC, etc. Zebrafish reference genome sequence and its relationship with the human genome. Nature 496, 498 503 (2013).
Kettleborough, RNW, etc. Genome-wide systematic analysis of zebrafish protein coding gene function. Nature 496, 494 499 (2013).
Liu, Y. Wait. A gradual method for evaluating zebrafish’s sensitivity to nanoparticle toxicity. Integer Biology 4, 285-291 (2012).
Wang, Y., Seebald, JL, Szeto, DP and Irudayaraj, J. Biocompatibility and biodistribution of surface-enhanced Raman scattering nanoprobes in zebrafish embryos: in vivo and multiplex imaging. Acs Nano 4, 4039 4053 (2010).
Kimmel, CB, Ballard, WW, Kimmel, SR, Ullmann, B. & Schilling, embryonic developmental stages of TF zebrafish. Developer Dynam. 203, 253 310 (1995).
Wu, M. Wait. The glycosyl-modified diporphyrin is used for in vitro and in vivo fluorescence imaging. Chembiochem 14, 979 986 (2013).
Nallathamby, PD, Lee, KJ & Xu, XHN designed stable and uniform single nanoparticle photonics for in vivo dynamic imaging of zebrafish embryo fluid nanoenvironment. Acs Nano 2, 1371 1380 (2008).
Liu, M. Wait. In vivo three-dimensional dual-wavelength photoacoustic tomography of far red fluorescent protein E2-dark red expressed in adult zebrafish. Biomedical Science. select. Express 4, 1846 1855 (2013).
Park, J. Wait. High-frequency photoacoustic imaging is used to visualize the blood flow of the zebrafish heart in vivo. select. Express 21, 14636-14642 (2013).
Umemoto, N. Wait. Fluorescence-based methods for zebrafish gene knockout and functional cardiac imaging. Lol biotech. 55, 131 142 (2013).
Xi, Y., Chen, D., Sun, L., Li, Y. & Li, L. Characterization of zebrafish mutants with bone calcification defects during development. Biochemical. Biography Res. Co.440, 132 136 (2013).
Ramesh, R. Wait. Changes in antioxidant enzymes and DNA damage in SiO2 nanoparticles exposed to zebrafish. surroundings. Monit. Evaluation. 185, 5873 5881 (2013).
Deniz Koç, N. & Y¨¹ce, R. Optical and electron microscopy studies of primordial germ cells in zebrafish (Danio rerio). Biology Res. 45, 331 336 (2012).
Spain, HP etc. Robot injection of zebrafish embryos for high-throughput screening of disease models. Method 62, 246 254 (2013).
Xu, X. Wait. Electrophoresis analysis and purification of fluorescent single-walled carbon nanotube fragments. J. Am Chemical Soc. 126, 12736-12737 (2004).
Xu, Y. Wait. Reducing carbon dots and carbon oxide dots: research on photoluminescence and electrochemiluminescence for specific applications. Chemistry Euro J.19, 6282-6288 (2013).
Xu, Y. Wait. The carbon quantum dots designed by 13C can be used for dual reactions of magnetic resonance and fluorescence in vivo. Analyst 139, 5134-5139 (2014).
Teng, X. etc. Konjac flour is used to synthesize nitrogen-doped carbon dots through the “on-off” fluorescent green of Fe3 + and L-lysine for biological imaging. J. Mater. Chemistry B 2, 4631 4639 (2014).
Ma, Z., Ming, H., Huang, H., Liu, Y. & Kang, Z. One-step ultrasonic synthesis of N-doped fluorescent carbon dots in glucose and their visible light-sensitive photocatalytic ability. NewJ. Chemistry 36, 861 864 (2012).
Wang, W. Wait. Simple synthesis of water-soluble and biocompatible fluorescent nitrogen-doped carbon dots for cell imaging. Analyst 139, 1692 1696 (2014).
Zhang, R. & Chen, W. Nitrogen-doped carbon quantum dots: easy to synthesize and use as “off” fluorescent probes to detect Hg2 + ions. Biosen. Bioelectronics. 55, 83 90 (2014).
Handy, R., Henry, T., Scown, T., Johnston, B. & Tyler, C. Nanoparticles manufactured: their uptake and effect on fish-mechanism analysis. Ecotoxicology 17, 396 409 (2008).
Reddy, ER, Banote, RK, Chatti, K., Kulkarni, P. & Rajadurai, MS use fluorescent organic nanoparticles to perform selective multicolor imaging of zebrafish muscle fibers. Chembiochem 13, 1889-1894 (2012).
Liu, Y. Wait. A gradual method for evaluating zebrafish’s sensitivity to nanoparticle toxicity. Integer Biology 4, 285-291 (2012).
He, N. Wait. To explore the toxicity of bismuth-asparagine coordination polymers on the early development of zebrafish embryos. Chemical Res. poison. 26, 89-95 (2013).
Yeo, MK & Kang, M. The effect of CuxTiOy nanoparticles on biological toxicity during zebrafish embryogenesis. Korean Journal of Chemistry. 26, 711 718 (2009).
Kim, MS, etc. Citrate-functionalized TiO2 nanoparticles were used to study the effect of particle size on zebrafish embryo toxicity. Analyst. 139, 964 972 (2014).
This work was supported by the China National Basic Research Program (973 Program, No. 2011CB707703), the National Natural Science Foundation of China (No. 21375064 and 81301080), the China National Key Technology Research and Development Program (No. 2012BAI08B06), and the research fund. Higher Education Doctorate Program (No. 20130031110016).
Analytical Science Research Center, School of Chemistry, Nankai University, Tianjin Key Laboratory of Biosensing and Molecular Recognition, State Key Laboratory of Medicinal Chemical Biology and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071
Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, School of Medicine, Nankai University, Tianjin 300071, China
XBY conceived and designed this research. XBY supervised this work. YFK, YHL, YWF, YX and XMW conducted experiments. XBY, YFK and YHL analyzed the data. XBY and YFK wrote this paper. All authors discussed the results and commented on the manuscript, and the manuscript reflects the contributions of all authors.
This work has been licensed under the Creative Commons Attribution 4.0 International License Agreement. The pictures or other third-party materials in this article are included in the Creative Commons license of the article, unless otherwise stated in the credit line; otherwise, if the material is not included in the Creative Commons license, the user will need to obtain the license holder¡¯s Only permission to copy the material. To view a copy of this license, please visit https://creativecommons.org/licenses/by/4.0/
Kang Yufen, Li Yuhua, Fang Yuwen. Wait. Carbon quantum dots for fluorescence imaging of zebrafish. Sci Rep 5, 11835 (2015). https://doi.org/10.1038/srep11835
By submitting a comment, you agree to abide by our terms and community guidelines. If you find abusive or inconsistent behavior with our terms or guidelines, please mark it as inappropriate.