# Fluorescent Carbon Dot-Supported Imaging-Based Biomedicine: A Comprehensive Review

Authors: Le Minh Tu Phan; Sungbo Cho
Journal: Bioinorganic Chemistry and Applications
 (2022)

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## Abstract

Carbon dots (CDs) provide distinctive advantages of strong fluorescence, good photostability, high water solubility, and outstanding biocompatibility, and thus are widely exploited as potential imaging agents forin vitro and in vivo bioimaging. Imaging is absolutely necessary when discovering the structure and function of cells, detecting biomarkers in diagnosis, tracking the progress of ongoing disease, treating various tumors, and monitoring therapeutic efficacy, making it an important approach in modern biomedicine. Numerous investigations of CDs have been intensively studied for utilization in bioimaging-supported medical sciences. However, there is still no article highlighting the potential importance of CD-based bioimaging to support various biomedical applications. Herein, we summarize the development of CDs as fluorescence (FL) nanoprobes with different FL colors for potential bioimaging-based applications in living cells, tissue, and organisms, including the bioimaging of various cell types and targets, bioimaging-supported sensing of metal ions and biomolecules, and FL imaging-guided tumor therapy. Current CD-based microscopic techniques and their advantages are also highlighted. This review discusses the significance of advanced CD-supported imaging-based in vitro and in vivo investigations, suggests the potential of CD-based imaging for biomedicine, and encourages the effective selection and development of superior probes and platforms for further biomedical applications.


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## Body

## 1. Introduction

Bioimaging plays a significant role in understanding the structure and biological processes of biomolecules, living cells, tissues, organs, and organisms [1]. Imaging has rapidly become an essential approach in biomedical applications due to its possibility to improve the prevention, detection, and treatment of disease. The powerful bioimaging techniques of fluorescence microscopy (FM) make it a prerequisite for taking advantage of many medical applications in preclinical studies and clinical interventions, by providing excellent imaging of anatomical structures and molecular imaging of specific biomarkers, as well as functional imaging of physiological activities [2, 3]. Compared to small fluorescent molecules (rhodamine, fluorescein, …) or semiconductor quantum dots, highly fluorescent (FL) carbon dots (CDs) that exhibit more advanced properties are currently being developed for various applications owing to their unique properties, such as multicolor emissions, excellent photostability and biocompatibility, and easy surface functionalization [4]. They can be easily synthesized in a one-step process using microwave or hydrothermal methods with environmental friendliness and affordability. With non-toxicity, high water solubility, strong fluorescence, and two-photon excitation (TPE) capability, CDs are broadly utilized in live-cell imaging, catalysis, electronics, biosensing, targeted drug delivery, and imaging-guided biomedical applications [5]. Taking advantage of the strong fluorescence of CDs has accelerated the intensive investigation of the biomedical sciences, especially in bioimaging-supported fields, providing significant benefits in effective bioimaging-supported medicine. The combination between CDs and advanced imaging technologies might increase the precision and accuracy of biomedical applications.Various reports in the literature have summarized the synthesis, properties, and applications of CDs, in the biosensing and theragnostic fields [5–8] to emphasize the applicable potential of CDs for biomedical applications. These reviews broadly discussed the applications of CDs including bioimaging, sensing, and therapy, without deeply focusing on bioimaging-based medicine. Imaging in modern biomedicine is especially beneficial procedure in the interest of diagnosing, monitoring, or treating disease, making it extremely significant in medical fields. Therefore, an overview of bioimaging-supported biomedical sciences that focuses on the excellent bioimaging properties of CDs is needed to explore the potential significance of CDs in the field of imaging-supported medical applications. Hence, this review emphasizes the exploitation of current CD-based FL imaging as powerful nanoprobes for advanced biomedical applications that include pristine bioimaging, bioimaging-supported sensing platforms, and bioimaging-guided therapy. The carbon dot-supported microscopic techniques are highlighted, along with their advantages for effective bioimaging purposes. The latest research on the CD-based bioimaging-supported biomedical applications is also comprehensively reviewed. In particular, the imaging of different species targets (cells, organelles, bacteria, fungi, animals), the monitoring of various metal ions and biomolecules in live cells and tissue, and the imaging-guided therapeutics of tumors (Figure 1). This review provides an interesting focus on advanced innovation for further in vitro and in vivo investigation based on bioimaging to develop superior platforms for efficient healthcare study. The challenges and perspectives are also discussed for considering the future of these CD-supported imaging-based biomedical sciences.Figure 1
Diverse imaging-supported biomedical applications using fluorescent carbon dots include the FL imaging of various biotargets (different targeted cells, tissues, fungi, bacteria, and animals), FL imaging-supported sensors (metal ions, biomolecules, pH, thermal, polarity), and FL imaging-guided therapy (chemotherapy, photothermal therapy, photodynamic therapy, and combined therapy).


## 2. Current Carbon Dot-Supported Microscopic Techniques

Microscopic imaging has paved the way for a revolution as a major technique for the visualization and understanding of the structure and physiology of cells and tissues that provides unprecedented insights into the biological processes related to physiological and disease processes, as well as supports various biomedical applications [9, 10]. There are two types of FM techniques that can or cannot overcome the resolution limit where the objects become unresolvable if the distance between the two-point objects is less than half of the wavelength of the excitation source, namely the diffraction-limited and super-resolution microscopy techniques, respectively. Diffraction-limited microscopy techniques consist of epifluorescence illumination microscopy, confocal laser scanning microscopy (CLSM), total internal reflection FM, and light-sheet FM, while super-resolution microscopy techniques include photoactivated localization microscopy, stochastic optical reconstruction microscopy, and ground-state depletion, followed by individual return microscopy, stimulated emission by depletion microscopy, structured illumination microscopy, binding-activation localization microscopy, and scanning near-field optical microscopy. Microscopic techniques are optimally selected depending on the spatial organization of the biological entity, temporal dynamics, and susceptibility to phototoxicity [11, 12]. For specific CD-supported microscopic techniques, scientists have mainly focused on the use of optical and electron microscopy techniques that include FM, CLSM, and single-photon and two-photon excitation microscopy for in vitro studies, and in vivo fluorescence imaging systems for in vivo studies, as shown in Table 1, which techniques are mainly applied in the fields of cell biology or biomedical sciences [115]. These FM techniques have been widely exploited using FL CDs for biomedical applications that include the imaging of cells and tissues, imaging-based sensing in vitro and in vivo, and imaging-guided therapy. CLSM obtains superior quality of FL images by improving the signal-to-noise ratio compared to widefield microscopy, two-photon excitation microscopy enables imagery of high depths, which is unachievable by single-photon excitation microscopy [94, 116–118]. Taking advantage of CDs, the CD-supported FM has been successfully exploited as an ideal candidate for diverse biomedical applications, such as providing active physiological processes through the imaging of species in live cells or tissues, as well as support for therapeutic applications to disease.Table 1
In vitro and in vivo bioimaging applications using FL CDs.
Precursors/FormsFL ColorApplied Ex/Em (nm)MicroscopicApplicationBiotargetRef.Ethylenediamine (EDA) into yeast extract solutionGreen405/–CLSMBacterial viability evaluationE. coli, S. aureus[13]Si-QAC, glycerolBlue360/473CLSMGram-type identificationS. aureus, M. luteus, B. subtilis[14]Gram-positive antibacterialPluronic F-127, 4-chlorophenylboronic acidBlue380/400CLSMDetection of glucose-containing bacteriaE. coli, S. aureus[15]Tulsi leavesBlue/Yellow/Red360/435FMImaging of bacteriaE. coli, B. subtilis[16]Choline bicarbonate, oleic acidBlue–red300–500/416–556FMImaging of bacteriaE. coli, Vibrio owensii, Bacillus cereus, and Vibrio alginolyticus[17]Roasted gramsBlue–red320–500/475–610FMImaging of bacteriaE. coli[18]Beer yeastBlue405/–CLSMBacterial viability evaluationE. coli[19]Green488/–Imaging of dead bacteriaRed555/–Thiourea, TAEBlue380/430–470FMImaging of bacteria, cellsE. coli, Pseudomonas, Staphylococcus, Klebsiella, and hBEC[20]Green450/510–560Red505/585–6403-Aminophenylboronic acid, CABlue360/420CLSMImaging of cellsHela[21]CollagenBlue370/–Two-photon CLSMImaging of cellsRL-14[22]Green490/–Red550/–CA, urea, cupric chlorideBlue405/–CLSMImaging of cellsMCF-7[23]Green488/–Red543/–CA, PEIBlue405/–CLSMImaging of cells3T3[24]Green488/–EDA, H3PO4Blue405/422CLSMImaging of cellsSiHa[25]Green488/500Yellow515/570Red543/650Aconitic acid, EDABlue360/450FMImaging of folate receptor-overexpressed cellsHela, SMMC-7721[26]Sucrose, EDA, and H3PO4Blue402/–CLSMImaging of cellsHepG2 cells[27]Green488/–Red543/–ATP, LaCl3Blue350/460CLSMImaging of cells, tissueA549, nude mice[28]Green488/500Red543/650D-Arginine, 4-MethylumbelliferoneBlue378/452CLSMImaging of cellsMC3T3[29]Resorcinol, EDABlue230/326CLSMImaging of cellsOVCAR-3, MCF-7[30]Green230/443YellowRedWatermelon juice, ethanolBlue355/439CLSMImaging of cellsHepG2[31]Crab shellsBlue360/450CLSMImaging of cellsHela[32]Diammonium hydrogen citrate, PEG-400Blue370/432CLSMImaging of cellsBGC-823, CT26.WT[33]CA, dethylene glycol bis (3-aminopropyl) etherBlue300/440FMImaging of tissueGlioma[34]Lily bulbsBlue370/448FMImaging of cellsA549[35]Succinic acid, glycerolBlue280–480/410–525FMImaging of cellsKKU213[36]GreenMetformin hydrochloride, glucose anhydrousBlue480/–FMImaging of cellsCT26.WT[37]Green560/–Red720/–Urea, PEGBlue405/–CLSMImaging of cellsL929[38]Green488/–Red543/–SaffronGreen-blue400/485FMImaging of cells (rat)Olfactory mucosa cells[39]Bone marrow cellsAlendronate sodiumBlue405/–FMImaging of cellsBT474[40]Green488/–EDA, starchBlue405/420–510CLSMImaging of cellsBHK[41]Green405/490–560Red405/590–750AppleBlue380/490FMImaging of cellsMDA-MB-231, A549, and HEK-293[42]HongcaitaiBlue405/–CLSMImaging of cellsHepG2[43]Green488/–Red559/–CA, melamineBlue405/–CLSMImaging of cellsHepG2[44]Green488/–Red559/–CA, p-phenylenediamine, boraxBlue315/–FMImaging of cellsHepG2[45]Green445/–Yellow485/–Red515/–Tartaric acid, ureaBlue–CLSMImaging of cellsMDA-MB-231[46]GreenRedAcidic cotton linter wasteBlue390/446FMImaging of cellsH2452, HUVEC[47]Green482/532Red586/646Quince fruit-480/518FMImaging of cellsHT-29[48]1,6-hexanediamineBlue410/–CLSMImaging of cellsMCF-7[49]Dihydrochloride, dimethyl sulfoxideGreen460/–Yellow480/–Orange530/–Citrus fruit peelsGreen330–385/–FMImaging of cellsMCF-7[50]Yellow450–480/–Red510–550/–Gum olibanum resinGreen420–495/525FMImaging of cellsB16F10[51]Adenosine disodium triphosphateRed543/–CLSMImaging of cellsHeLa[52]CA, EDA, arginineBlue330–380/–FMImaging of cellsNIH 3T3, HEK 293, Hela, and MCF-7[53]Green460–495/–Red530–550/–Thiourea, CA, D-Glucosamine hydrochloride, and triethylamineGreen368/505FMImaging of cellsHepG2, HL-7702[54]Food wasteBlue360/435FMImaging of cellsHCT116[55]SugarcaneBlue405/410–460CLSMImaging of cellsDU145, MCF-7, and A375[56]Green488/–500–530Yellow515/–530–570Red559/–575–675Hyperbranched polysiloxaneBlue320–380/440FMImaging of cellsMouse osteoblast[57]Prunus cerasifera fruitsBlue405/450FMImaging of cellsHepG2[58]Green488/–Ginkgo fruitsBlue405/–CLSMImaging of cellsHeLa[59]Green488/–Triethylenetetramine, phenylenediamineGreen360/510CLSMImaging of cellular RNAHEp-2[60]m-phenylenediamine, FAGreen405/480–580CLSMImaging of cell nucleolusHeLa[61]CA, PEIBlue405/–CLSMImaging of cellsHEK293[62]Green488/–Red561/–Chitosan, EDA, mercaptosuccinic acid, and CD-RBGreen532/550CLSMImaging of mitochondrial-targeted cellsMCF-7[63]Polyacrylic acid, EDABlue356/520FMImaging of cellsEM-6[64]CDs@SiO2GreenImaging of tissuesOnion bulb epidermal tissuePEI, 4-formylphenylboronic acidBlue360/462FMImaging of cellsHela[65]CEL powderBlue320/400CLSMImaging of cellsHela[66]Green400/–Red480/–CA,Red488/678CLSMImaging of EGFR-overexpressed cellsHCC827[67]5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP)MDA-MB-231CA, guanidine carbonateGreen800/480Two-photon FMImaging of cellsHela[68]Imaging of tissuesRat liver tissueCarbon powder, ammoniaBlue405/–FMImaging of cellsHeLa[69]Green488/–Red563/–CA CDs-PEI-MLGreen405/420–580CLSMImaging of lysosome-targeted cellsHela[70]Starch, L-tryptophan, and CD-MIPGlcABlue371/452CLSMImaging of hyaluronan-targeted cellsHaCaT, HeLa[71]Green445/500p-phenylenediamine, aspartic acidYellow410/535CLSMImaging of cellsMIDA-MB-231, MIDA-MB-68, and RPE1[72]Na2EDTA, GdCl3, YbCl3, and L-arginineBlue340/418FMImaging of cellsHeLa[73]DL-malic acidCyan405/–CLSMImaging of cellsTrout gill epithelial cells.[74]Purple488/–Green514/–Red561/–Carbon glassy, PEG-200Green405/420–637CLSMImaging of cellsHT29, A549[75]CA, EDA, GdCl3, and YbCl3Blue405/450CLSMImaging of cellsHela, NIH-3T3, and CHO[76]Adenosine triphosphate, graphene oxideGreen488/–CLSMImaging of cellsA549[77]Red543/–Imaging of bacteriaS. aureusCA, PEI, GdCl3, and DTPABlue354/445FMImaging of cellsHeLa[78]CA, thiourea, and 3-amino phenyl boronic acidBlue405/–CLSMImaging of cellsHepG2, L929[79]Green488/–Red546/–ZIF-8Green405/503CLSMImaging of cellsHeLa[80]p-phenylenediamineOrange514/633CLSMImaging of tissuesArabidopsis thaliana leaf[81]PEI600, poly(tetrafluoroethylene)Blue348/460CLSMImaging of cellsHeLa, 7702[82]Polythiophene phenylpropionic acidRed543/610CLSMImaging of cellsHeLa[83]Cds@AuL-asparagineBlue370/449CLSMImaging of cellsHeLa[84]CA, urea, and laurylamineBlue360/430CLSMImaging of lysosome-targeted, endoplasmic reticulum-targeted cellsMCF-7[85]Bamboo leavesGreen488/–CLSMImaging of cellsHeLa[86]CDs-CBBA-DoxImaging-guided chemotherapyp-phenylenediamine, phosphoric acid, and Mn(OAC)2Red530/600CLSMImaging of cellsB16F1, HeLa[87]o-phenylenediamineYellow, Green465/570FMImaging of cellsHepG2[88]CA, EDA, and ginsenoside ReBlue360–380/–FMImaging of cellsA375[89]Green460–480/–Red510–530/–AEAPMSGreen473/–FMMeasurement of cellsMacrophages[90]GNR@SiO2@CDm-PhenylenediamineBlue360/444CLSMImaging of cellsMCF-7[91]o-PhenylenediamineYellow450/533p-PhenylenediamineOrange530/574Poly(vinylpyrrolidone), L-cysteineBlue350–400/420–470CLSMImaging of cellsTramp C1[92]Green460–490/520Red510–550/590Platanus biomassBlue360/476CLSMImaging of cellsHeLa, L02, and macrophages[93]Cyanobacteria powderGreen405/–CLSMImaging of cellsPC12[94]Diammonium hydrogen citrate, ureaGreen420/537CLSMImaging of cellsHeLa[95]CA, EDABlue405/–CLSMImaging of cellsCNE-1, HEK-293T[96]Green488/–Red640/–L-cystine, o-phenylenediamineOrange560/595CLSMImaging of cells293T[97]Red560/628Protamine sulfate, PEGBlue405/–FMImaging of cellsHEK-293[98]Green488/–Red594/–Methyl red, EDABlue–/470CLSMImaging of fungiC. gloeosporioides[99]Green–/511Yellow–/530Acacia concinnaBlue405/–CLSMImaging of fungiPenicillium sp.[100]Green488/–Red561/–Manilkara zapotaBlue350/443CLSMImaging of bacteriaE. coli[101]Green420/515Imaging of fungiAspergillus aculeatus, Fomitopsis sp.Yellow440/563L-cysteine, m-PDGreen488/>515FMImaging of nucleolusMGC-803, Mel-RM, H7, HeLa, and SKOV3[102]Cancer/normal cell differentiationGES-1, FB, and HEM-dCA, ureaYellow542/612CLSMImaging of nucleolusHeLa cells[103]Carbon NPs, p-phenylenediamineGreen488/500–560CLSMImaging of nucleolus dsDNAHeLa, A549[104]Red543/570–650Imaging of nucleolus ssRNAC. elegansdsDNA/ssRNA differentiationTris(hydroxymethyl)aminomethane, betaine hydrochlorideRed520/600In vivo imaging systemImaging of stem cellsHuman mesenchymal stromal cells - mice[105]P. acidus fruitBlue400/–FMIn vivo imaging of nematodeC. elegans[106]Green470/–Red550/–p-phenylenediamine PDA@N-CDs(Mn)Red550/620In vivo imaging systemIn vivo imaging of tumorA549 tumor-bearing mice[107]p-phenylenediamine, NiCl2Ni−pPCDsRed520/605CLSMImaging of nucleolus-targeted cellsA549[108]In vivo imaging of tumorIn vivo imaging of tumorZebrafishU14 tumor-bearing miceCoal tar CDs-liposomeOrange535/605CLSMImaging of cellsHeLa[109]In vivo imaging systemIn vivo imaging of tumorHeLa tumor-bearing miceCarbon nanopowder, EDA, and glycerinGreen400/486FMImaging of boneZebrafish[110]Aniline, EDA, ureaGreen410/510FMIn vivo imagingZebrafish[111]CV acetate, wax-like PEG sample, SiC powdersRed520/620In vivo imaging systemIn vivo imaging of tumorTumor-bearing mice[112]Wheat straw and bamboo residuesBlue420/–CLSMImaging of cellsSP2/0[113]Red540/–In vivo imaging systemIn vivo imaging of tumorSmmc-7721 tumor-bearing miceCA, H3PO4, EDAGreen340/430FMImaging of cellsRAW 264.7[114]Red450/500In vivo imaging systemIn vivo imaging of tumorSGC7901 tumor-bearing BALB/c mice


## 3. Fluorescent Carbon Dots for Bioimaging Applications

Table1 summarizes the latest research of various CDs based on the precursors of synthesis, applied color, and excitation/emission wavelengths for bioimaging of various targets such as cells, organelles, bacteria, fungi, and animal models. Case studies of CD-based bioimaging are then discussed in this section, according to biotarget.


### 3.1. Fluorescent Carbon Dots for the Bioimaging of Cells and Tissues

Various multicolor FL CDs have been widely fabricated and applied as excellent nanoprobes to effectively image cells and tissues. By simply mixing glucose as a carbon source, EDA as an N-dopant, and phosphoric acid as P-dopant, water-soluble FL nitrogen and phosphorus dual-doped carbon dots (NP-Cdots) of about 3.5 nm in diameter were rapidly synthesized by the acid-base neutralization spontaneous heat, and they exhibited multicolor fluorescence, with the optimal excitation and emission wavelengths at (403 and 502) nm, respectively. These biocompatible NP-Cdots were utilized as a multicolor FL agent for the intracellular imaging of human cervical carcinoma SiHa cells. The multicolor fluorescence of cells stained with NP-Cdots presented blue, green, yellow, and red Em under laser stimulation at 405, 488, 515, and 543 nm, respectively, showing internalization and good decentralization into the cytoplasm of SiHa cells, along with the entering of some NP-Cdots into the cell nucleolus. These results indicate the excellent ability of these NP-Cdots to permeate the cell membrane for the application inin vitro cellular imaging (Figure 2(a)) [25]. The double rare-earth-doped (gadolinium, ytterbium) carbon dots (Gd/Yb@CDs) of 5.26 nm in diameter were fabricated via a one-step hydrothermal process at 200°C for 10 h using Na2EDTA and L-arginine as raw materials, and GdCl3 and YbCl3 as dopants. These Gd/Yb@CDs exhibited Ex-dependent Em, with the strongest Em at 418 nm when excited at 340 nm, superhigh photostability without change of fluorescence intensity for 2 h, and superior biocompatibility without toxicity to HeLa cells up to 1 mg/mL of Gd/Yb@CDs. The in vitro HeLa cells and in vivo nude mice treated with Gd/Yb@CDs displayed significant blue fluorescence with very weak interference from the autofluorescence of cells [73].Figure 2
In vitro FL imaging of various targets using CDs. (a) CLSM images of CDs-treated SiHa cells. (A) Bright-field image, (B)–(E) multicolor FL images from blue, green, yellow, and red under excitation/emission wavelengths of ((405/422 ± 25), (488/500 ± 25), (515/570 ± 25), and (543/650 ± 25)) nm, respectively, adapted with permission from Ref. [25]. (b) Washfree selective nucleus imaging of Hela cells through hydrogen-bonding-induced emission, adapted with permission from Ref. [61]. (c) Selective lysosomal imaging of live Hela cells confirmed by well-overlapped image of CDs (collected at (420–580) nm) and Lyso-Tracker Red (commercial lysosomal probe, collected at (650–800) nm) in Hela cells under 405 nm excitation, adapted with permission from Ref. [70]. (d) Mitochondrial imaging of MCF-7 cells incubated with CDs (Ex: 488 nm), and then MitoTracker (Ex: 638 nm), ER-Tracker (Ex: 552 nm), Golgi-Tracker (Ex: 552 nm), or LysoTracker (Ex: 552 nm), confirmed by the coincident overlay between CDs green FL and MitoTracker red FL, but not ER-Tracker, Golgi-Tracker, or LysoTracker, adapted with permission from Ref. [63]. (e) Red-emissive FL imaging of different plant tissues through the selective cellulose-binding of CDs, adapted with permission from Ref. [81]. (f) CLSM images of the CDs FL monitoring of S. aureus and E. coli at various incubation periods, adapted with permission from Ref. [15]. (g) Differentiation of gram-positive S. aureus bacteria from gram-negative E. coli bacteria using CLSM images of a mixture of CDs-treated bacteria under 405 nm excitation, adapted with permission from Ref. [14]. (h) CLSM images of C. gloeosporioides fungus labeled by different color CDs, adapted with permission from Ref. [99].Various o-, m-, or p-phenylenediamines (pDA) with triethylenetetramine as precursors were used to synthesize the FL carbon dots with different colors for the imaging application of cellular ribonucleic acids (RNA). However, only m-phenylenediamines formed carbon dots (m-CDs) with a size of 2.75 nm, and maximum green Em at 510 nm under Ex at 360 nm demonstrated excellent ability to specifically bind to the cellular RNA of HEp-2 cells, allowing successful long-term real-time monitoring of RNA dynamics during cell apoptosis, mitosis, and proliferation. The RNA affinity of m-CDs is associated with the isoquinoline moieties and amines on the surface of m-CDs that bind to RNA throughπ–π stacking and electrostatic bonding, respectively. Furthermore, investigations of the in vivo zebrafish larvae body showed major accumulation in the lens, pronephros, intestine, and vessels, suggesting the efficient excretion of m-CDs after 48 h of incubation. Hence, m-CDs as smart nanoprobes for cellular RNA imaging could be valuable for the development of visualized screening of RNA [60]. Interestingly, hydrogen-bonding-induced Em CDs (HBIE-CDs) formed by a hydrothermal method using m-phenylenediamine (mDA) and folic acid (FA) as precursors displayed 460 nm of blue Em in nonhydrogen-bonding solution and 535 nm of green Em in hydrogen-bonding solution due to the inhibition of nonirradiation by a H-bond network. The HBIE-CDs possessed strong affinity toward nucleic acid as the donor of H-bond without interference, dramatically increasing 6-fold green fluorescence after binding with the DNA or RNA of Hela cells. After incubation of Hela cells with HBIE-CDs for 5 min, cells without washing exhibited strong green fluorescence in the nucleus after binding with DNA and RNA and almost no background fluorescence in the cytoplasm, which was almost similar to cells with PBS washing, allowing continuous monitoring of the cell nucleus without interruption (Figure 2(b)) [61]. Furthermore, monitoring the dynamics of DNA and RNA structures in live cells is essential for tracking cell behavior. However, the difficulty in fabricating a the nanoprobe that can distinguish between double-stranded DNA and single-stranded RNA, and cross multiple membrane barriers from the cell to the tissue level is still a challenge. Thus, a cationic carbon quantum dot of 2–3 nm in diameter and zeta potential at +20 mV synthesized from conductive carbon nanoparticles and p-phenylenediamine was successfully developed to address this challenge. The CD exhibited green fluorescence at 510 nm under 400 nm Ex, but significant fluorescence enhancement was obtained with the existence of DNA, up to 8-fold, due to the CD insertion into the grooves of dsDNA that restricted the rotation of the linkage bonds between the pDA and the CD core. In contrast to DNA, the presence of RNA induces the shift of Em fluorescence to red at around 620 nm under 540 nm Ex, because it leads to the formation of CD aggregates to make the strong fluorescent transit from green to red. Upon treatment of CD, the nuclei of a total of 12 cell types emitted strong green fluorescence under 488 nm Ex, while strong red fluorescence was observed in the cytoplasm under 542 nm Ex, due to the CD staining of DNA and RNA, respectively, indicating its ability to cross both the plasma and nuclear membranes to dye the target DNA and RNA. The CD’s ability to enter the intestinal pathway was confirmed through the greater concentration of CD in intestinal wall cells using the imaging of wireworms. Further, it was successfully applied to monitor DNA and RNA during cell division at different cell cycles, including prophase, metaphase, telophase, and interphase. CD’s selectivity towards DNA and RNA, and the excellent ability to penetrate through various biological barriers in cells and live organisms, make CD an efficient agent for basic cell biology, embryology, and clinical diagnosis [104].Lysosomes, as another subcellular organelle, also play critical roles in protein degradation, cell signal transduction, plasma membrane repair, homeostasis, and autophagy, making the development of a specific probe for the imaging of lysosomes extremely necessary to understand the dysfunction of lysosomes that is related to various diseases, such as Alzheimer’s disease, neurodegenerative diseases, and cancer. Through the conjugation of the morpholine group (ML) as lysosome targeting to CDs formed from polyethyleneimine (PEI) and citric acid (CA) as precursors, CDs-PEI-ML were successfully fabricated to act as a long-term tracking probe of lysosomes for cell imaging with excellent properties that include strong blue Em, high photostability, adequate biocompatibility, and high selectivity toward lysosomes. Imaging of the lysosomal localization of CDs-PEI-ML in live Hela cells was described that CDs-PEI-ML were translocated into lysosomes via an endocytic pathway, and accumulated in acidic lysosomes with less diffusion into the cytoplasm due to the presence of lysosome target group morpholine on the surface of CDs-PEI-ML. This CDs-PEI-ML exhibited high specificity in lysosomal imaging, presenting an alternative FL probe for lysosome labeling and imaging (Figure2(c)) [70].Mitochondrion as a vital energy-supplying organelle, plays an important role in cellular functions, such as the generation of ATP and reactive oxygen species (ROS), regulation of transmembrane potentials, and initiation of apoptosis and autophagy, making the dysfunction of the mitochondrial responsible for various diseases, such as Alzheimer’s disease, Parkinson’s disease, diabetes, and cancer. The green FL CDs synthesized using chitosan, ethylenediamine, and mercaptosuccinic acid as precursors, showed intrinsic mitochondrial targeting ability to visualize mitochondria for bioimaging. These CDs quickly entered cells and specifically targeted mitochondria through energy-dependent, caveolae-mediated endocytosis, exhibiting superior features that included multicolor fluorescence, good photostability, and long-term imaging capability for at least 24 h, compared to MitoTrackers (commercial mitochondrial probes), representing a good FL nanoprobe for mitochondrial imaging that could be an alternative to commercial probes (Figure2(d)) [63].As another targeting species in cellular bioimaging, hyaluronan (HA), a linear long-chain polymer, is intimately associated with human tumor progression by stimulating growth, survival, and angiogenesis within primary tumors or their metastases. About 3.2 nm N-doped CDs using starch as a carbon source and L-tryptophan as nitrogen dopants were coupled with molecularly imprinted polymer (MIP) and glucuronic acid (GlcA) as an epitope of hyaluronan to form CD-MIPGlcA that was employed to probe HA cancer biomarkers. CD-MIPGlcA with (3–10) nm diameter emitted the green maximum Em at 500 nm under 445 nm Ex with high selectivity to HA, showing about 4 times higher FL intensity on HeLa cells than that on HaCaT cells, suggesting the ability of CD-MIPGlcA to differentiate between cancer cells and normal cells. The possibility of CD-MIPGlcA in HA-targeting FL nanoprobes indicates how powerful these tools are for the imaging of tumor biomarkers [71].A desirable goal in bioimaging applications is to discriminate against interference from autofluorescence. However, it is still a challenge to develop room temperature (RT) phosphorescence (RTP) probes that feature ultralong Em lifetimes. CD-based silica composites (CDs@SiO2) with long Em lifetimes of 1.64 s were successfully fabricated for ultralong lifetime phosphorescence imaging of cells and tissue at RT using polyacrylic acid and EDA as CD precursors. CDs@SiO2 emitted blue fluorescence and the afterglow green Em maximum at 520 nm under an Ex of 356 nm. Although due to strong endogenous autofluorescence at 365 nm irradiation, it was difficult to distinguish between CDs@SiO2-untreated and treated onion bulb epidermal tissues, strong green phosphorescence of the CDs@SiO2-treated group in EM-6 mouse breast carcinoma cells and onion bulb epidermal tissue was observed after turning off the irradiation, highlighting the successful phosphorescence imaging of CDs@SiO2 in bioimaging in cells or plant and animal tissues by eliminating the autofluorescence interference. These CDs@SiO2 could serve as excellent nanoprobes for bioimaging with an efficient ability to discriminate against tissue autofluorescence, suggesting the potential for ultralong-lived RTP reporters [64]. In addition, a CD-grafted cellulose hybrid phosphor with a high affinity to bind cellulose was prepared for effective fluorescence mapping of cellulosic plant cell walls. These reddish-orange emissive CDs exhibited strong fluorescence at 633 nm with a 44% high quantum yield and high photostability. Various model plant tissues, including onion bulb epidermal tissue, the root of mung bean sprout, and the stem and leaf of Arabidopsis thaliana, were employed to demonstrate the bioimaging capacity of these CDs. CDs can readily have absorbed in plant tissues and bind to a cellulose-rich structure with deep penetration to 55.5 μm depth in the stem of Arabidopsis thaliana, 177.2 μm in the root of mung bean sprout, and 136.3 μm in onion bulb epidermal tissue, respectively, which could address the limitation of FL dyes in their difficulty in visualizing deeper tissue layers of ≥ (50 to 100) μm. These CDs could serve as useful probes for the study of cell wall structure in living plant cells (Figure 2(e)) [81].Undoubtedly, CDs exhibit excellent performance for bioimaging applications that can enhance the understanding of cells or tissues. CDs can also be applied as nanoprobes for the recognition of cancerous cells from normal cells. However, each specific mechanism of interaction between CDs and targets in cells or tissues, cytotoxicity, and optimal conditions of bioimaging processes should be carefully studied to contribute significantly to biological and biomedical sciences.


### 3.2. Fluorescent Carbon Dots for the Bioimaging of Bacteria

Bacteria are abundant in the natural world and can lead to global bacterial infection, so it is essential to accurately specify them for efficient treatment. Nitrogen and sulfur codoped CDs (NSCDs) were obtained by a microwave-assisted hydrothermal method using thiourea as a precursor in tris-acetate-ethylenediamine (TAE) buffer, for use in the imaging of various bacteria. These NSCDs were well mono-dispersed with an average size of 3.62 nm and exhibited maximum fluorescence at 426 nm Em under irradiation of 340 nm with a quantum yield of 57%. With no toxicity to bacteria cells, NSCDs internalization or endocytosis occurred after incubation with bacteria, and emitted blue fluorescence that was observed inE. coli, Pseudomonas, and Staphylococcus, but not in Klebsiella. The untagging effect on Klebsiella may have resulted in the decimation or disorientation of NSCDs due to the catabolic characteristic of Klebsiella for different aromatic compounds. Additionally, the multicolor fluorescence of NSCDs as imaging nanoprobes was screened under different Ex wavelengths of (380, 450, and 505) nm in tagged E. coli cells, showing the bright colors of blue, green, and red at (430, 525, and 590) nm Ems, respectively [20].Taking advantage of the selective binding affinity of boronic acid to diols groups on the bacterial cell wall, phenylboronic acid-functionalized FL carbon dots (FCDs) with strong blue fluorescence were synthesized and conjugated to dye molecules, then successfully applied in the selective detection of bacteria via the imaging of diol-containing bacteria. Owing to the preferential affinity of phenylboronic acid toward diol-containing molecules through cyclic ester bond formation, glucose-containing bacteria were replaced with dye molecules that led to the release of dye from the FCD to recover the fluorescence, gradually increasing the FL intensity from (0 to 60) min under 405 nm Ex in the presence ofE. coli or S. aureus. These FCDs exhibited high sensitivity and chemical specificity for bacteria detection via fluorescence imaging (Figure 2(f)) [15].Accurate bacterial identification is required to characterize unknown bacteria, which is important for the fast diagnosis of bacterial infections. A one-pot solvothermal approach was exploited to obtain a quaternized CDs using glycerol and dimethyloctadecyl[3-(trimethoxysilyl)propy] ammonium chloride (Si-QAC) for the identification of gram-type bacteria through bacteria imaging. Si-QAC containing CDs interacted with the negatively charged bacterial cells via both electrostatic and hydrophobic interactions, selectively connecting with the gram-positive bacteria due to the different surfaces of gram-positive and gram-negative bacteria. CDs displayed Ex-dependent fluorescence from (320 to 500) nm with maximum intensity at 473 nm under 360 nm Ex, significantly enhancing the blue fluorescence once the CDs are adsorbed onto theS. aureus gram-positive bacteria, but not the E. coli cells, in the mixture of these bacteria. This CD-based Gram-type differentiation method was accurate, fast, and facile, suggesting that this CD FL staining method could act as an alternative to the conventional gram staining method for differentiating gram-type bacteria (Figure 2(g)) [14].CD-based bioimaging of bacteria is successfully utilized for FL imaging or differentiating gram-type bacteria. Nevertheless, these studies mostly focused on the imaging of several bacteria, such asS. aureus, and E. coli, other bacterial types that possess different influences should be further investigated. A CD with specific affinity towards each bacterium should be developed for efficient application. Cytotoxicity of CDs should be considered carefully for various types of bacteria for further utilization. The interference of culture media to FL of CDs and the real-time ability of monitoring bacteria are required to study deliberately.


### 3.3. Fluorescent Carbon Dots for the Bioimaging of Fungi

Along with bacteria, the bioimaging of fungi is also important to understand the fungal biology or their infection mechanisms more clearly. Carbon dots synthesized from methyl red (MR) as a precursor in the presence of ethylenediamine (EDA) by facile hydrothermal pyrolysis, namely as MR-EDA-CDs, were fractionated into four fractions with different sizes of (16.3, 12.6, 5.5, and 4.5) nm under column chromatography that emitted green, purple, blue, and yellow fluorescence at (398, 470, 511, and 530) nm, respectively. These biocompatible MR-EDA-CDs were investigated as imaging nanoprobes for the FL imaging of pathogenic fungal cells (C. gloeosporioides). MR-EDA-CDs internalized into the fungal cells upon 6 h of incubation at RT, brightly illuminating the multicolor fluorescence with Ex at 405 nm by confocal microscopy (Figure 2(h)) [99].Owing to their ease of availability and affordability, natural resources have been widely used as precursors for the preparation of CDs.Acacia concinna fruit seeds (shikakai) as a green precursor were used to fabricate CDs in the application as biocompatible nanoprobes for the multicolor imaging of fungal (Penicillium sp.) cells. Upon Ex of 390 nm, these CDs displayed a strong Em peak at 468 nm with a higher quantum yield (10.2%), with good biocompatibility with fungal cells. The ultra-small CDs (2.5 nm diameter) internalized into the cells via endocytosis, and located in the cytoplasm and especially the nucleus of cells, exhibiting bright blue, green, and red fluorescence colors under laser Ex wavelengths of (405, 488, and 561) nm [100]. Another multicolor emissive CD that generated blue, green, and yellow fluorescence was obtained from Manilkara zapota fruits in acidic solutions (sulphuric acid and phosphoric acids) at different temperatures of 100 and 80 °C for 60–15 min. These CDs exhibited the maximum Em peaks at 443, 515, and 563 nm (blue, green, and yellow) under excitation at 350, 420, and 440 nm, with the QYs of 5.7, 7.9, and 5.2 %, respectively. Due to the small size of these CDs of from 1.9–4.5 nm, they are easily internalized into Aspergillus aculeatus and Fomitopsis sp. cells via the cell membrane through endocytosis, located in the cell membrane and cytoplasm, resulting in blue, green, and yellow fluorescence at Ex wavelengths of 405, 488, and 561 nm with good intensity [101]. Overall, the FL CDs have proven to be potential agents for the multicolor imaging applications of fungal cells. However, there are many other types of fungi that are essential to be explored as well. These reported CDs are mainly for imaging of the whole bodies of fungi. Specific CDs for different organelles in fungi are thus highly required to develop for obvious understanding.


### 3.4. Fluorescent Carbon Dots for the Bioimaging of Animal Models

In vivo molecular imaging as a powerful tool for the analysis of the body significantly contributes to precise medicine through diagnosis and guiding therapy. N-doped carbon dots (NCDs) obtained by a hydrothermal method using pDA as precursor have embedded with Mn2+ onto the polydopamine (PDA) nanoparticle (NP) to fabricate the imaging nanoprobe, namely as PDA@NCDs(Mn). Using NCDs as FL agents, PDA as a photothermal agent, and Mn2+ as a T2 magnetic resonance contrast agent, these CD-based NPs were successfully applied as multimodal imaging nanoprobes for tumor diagnosis. These PDA@NCDs(Mn) NPs displayed maximum Em peak at 620 nm when Ex at 550, with strong red Em under the Ex wavelength of 500 nm. For in vivo fluorescence imaging investigation, PDA@NCDs(Mn) were injected into the left rear flank region of the mice model, then accumulated in the tumor site with good distribution, hence valid fluorescence was observed around the tumor. After 12 h post-injection, organs (heart, liver, spleen, lung, and kidney) and tumors were harvested to assess fluorescence intensities. These PDA@NCDs(Mn) NPs mainly accumulated in the liver, lung, and kidney tissues, as well as stronger fluorescence signal in tumor tissues than the spleen and heart, which was observed in a time-dependent fluorescence intensity manner, indicating red-emissive PDA@NCDs(Mn) NPs as promising fluorescence imaging agent for in vivo imaging (Figure 3(a)) [107]. Phosphonated compounds are widely applied for the imaging and treatment of severe bone turnover due to their high affinity to hydroxyapatite (HA) minerals. monophosphonated CDs obtained by a one-pot hydrothermal method using food-grade agave nectar and α-methoxy-ω-phosphate polyethylene glycol exhibited Ex-dependent Em spectra with maximum Ex/Em at 430/600 nm and high affinity towards calcium salt of the bone, indicating their potential as bone-seeking luminescent nanoprobes for the detection of bone microcracks via fluorescence imaging. The CDs-injected mice displayed a prominent difference in the luminescent emitted from the tibia when excited at 430 nm and imaged at 600 nm, suggesting that CDs can light up the tissue surrounding the bone. A statistically significant enhancement was detected in the treated mice, compared to the nontreated animal, by calculating the photon count after injection and the circulation time of about 30 min, indicating the feasibility of tissue imaging and sufficient signal sensitivity of these CDs nanoprobes as effective bone-seeking agent for the prediction of bone failure, considering the crack fracture mechanics (Figure 3(b)) [119].Figure 3
In/Ex vivo FL imaging of animals using CDs. (a) Real-time in vivo red FL images in nude mice at different time periods after (A) subcutaneous, and (B) intravenous injections of CDs via the tail vein revealed the accumulation of CDs in the tumor tissue. Adapted with permission from Ref. [107]. (b) In vivo FL of monophosphonated CDs in the tibia of (A) non-treated mice, (B) treated mice (Ex/Em = 430/600 nm); (C) treated the animal with the computed tomography-fused fluorescence image (Ex/Em = 465/700 nm), showing the feasibility of CDs for the detection of bone microcracks. (D) Significant difference of FL radiance between CDs-nontreated and CDs-treated mice (∗p value < 0.05). Adapted with permission from Ref. [119]. (c) (A) In vivo FL images, and (B) corresponding FL intensity of the tumors before and after the intratumoral injection of CDs for different time periods. (C) Ex vivo FL images, and (D) corresponding FL intensity of different organs of mice after CDs injection for different day periods (∗∗p value < 0.01, ∗∗∗p value < 0.001). (D) CLSM image, and (E) bright-field image of CDs-stained zebrafish. Adapted with permission from Ref. [108].Various metal ions doped into pDA during the hydrothermal treatment lead to the formation of FL CDs with Em wavelengths of up to 700 nm. The Ni-pDA-CDs were prepared using pDA and nickel ions (Ni2+) as raw materials that exhibited Ex-independent Em (at ∼605 nm) with good photostability. A549 live cells treated with Ni-pDA-CDs for 30 min emitted strong red fluorescence under laser Ex at 552 nm in nucleoli, indicating the nucleolus targeting mechanism of Ni-pDA-CDs. Further, the in vivo imaging performance of Ni-pDA-CDs in tumor-bearing mice and zebrafish was investigated. The tumor region emitted bright red fluorescence after the intratumoral injection of Ni-pDA-CDs with a high signal-to-noise ratio of the tumor fluorescence. These Ni-pDA-CDs mainly accumulated in the tumor, with negligible distribution in other organs (heart, liver, spleen, lung, and kidneys), suggesting their good biosafety. Additionally, the Ni-pDA-CDs-incubated zebrafish for 30 min were well stained with bright red fluorescence, confirming that they could pass through the mucus layer and interact with the cells in zebrafish due to their ultrasmall size, positively charged surfaces, and amphiphilicity. These experiments suggest the potential of these Ni-pDA-CDs as excellent red-emissive FL nanoprobes for in vivo imaging in both mice and zebrafish models (Figure 3(c)) [108].The above studies show that these FL CDs hold great promise in the bioimaging of animal models that can support efficient diagnosis and treatment in medicinal applications. Nevertheless, it is worth developing the FL CDs that exhibit excellent biocompatibility and efficient ability of clearance, providing excellent FL probes for tracking tumors, delivering drugs, monitoring the ongoing disease, and further biomedical applications. Surface modification of CDs is needed to study for the elimination of nonspecific absorption onto CDs, and specific imaging of targetsin vivo should be considered carefully when developing CDs.


## 3.1. Fluorescent Carbon Dots for the Bioimaging of Cells and Tissues

Various multicolor FL CDs have been widely fabricated and applied as excellent nanoprobes to effectively image cells and tissues. By simply mixing glucose as a carbon source, EDA as an N-dopant, and phosphoric acid as P-dopant, water-soluble FL nitrogen and phosphorus dual-doped carbon dots (NP-Cdots) of about 3.5 nm in diameter were rapidly synthesized by the acid-base neutralization spontaneous heat, and they exhibited multicolor fluorescence, with the optimal excitation and emission wavelengths at (403 and 502) nm, respectively. These biocompatible NP-Cdots were utilized as a multicolor FL agent for the intracellular imaging of human cervical carcinoma SiHa cells. The multicolor fluorescence of cells stained with NP-Cdots presented blue, green, yellow, and red Em under laser stimulation at 405, 488, 515, and 543 nm, respectively, showing internalization and good decentralization into the cytoplasm of SiHa cells, along with the entering of some NP-Cdots into the cell nucleolus. These results indicate the excellent ability of these NP-Cdots to permeate the cell membrane for the application inin vitro cellular imaging (Figure 2(a)) [25]. The double rare-earth-doped (gadolinium, ytterbium) carbon dots (Gd/Yb@CDs) of 5.26 nm in diameter were fabricated via a one-step hydrothermal process at 200°C for 10 h using Na2EDTA and L-arginine as raw materials, and GdCl3 and YbCl3 as dopants. These Gd/Yb@CDs exhibited Ex-dependent Em, with the strongest Em at 418 nm when excited at 340 nm, superhigh photostability without change of fluorescence intensity for 2 h, and superior biocompatibility without toxicity to HeLa cells up to 1 mg/mL of Gd/Yb@CDs. The in vitro HeLa cells and in vivo nude mice treated with Gd/Yb@CDs displayed significant blue fluorescence with very weak interference from the autofluorescence of cells [73].Figure 2
In vitro FL imaging of various targets using CDs. (a) CLSM images of CDs-treated SiHa cells. (A) Bright-field image, (B)–(E) multicolor FL images from blue, green, yellow, and red under excitation/emission wavelengths of ((405/422 ± 25), (488/500 ± 25), (515/570 ± 25), and (543/650 ± 25)) nm, respectively, adapted with permission from Ref. [25]. (b) Washfree selective nucleus imaging of Hela cells through hydrogen-bonding-induced emission, adapted with permission from Ref. [61]. (c) Selective lysosomal imaging of live Hela cells confirmed by well-overlapped image of CDs (collected at (420–580) nm) and Lyso-Tracker Red (commercial lysosomal probe, collected at (650–800) nm) in Hela cells under 405 nm excitation, adapted with permission from Ref. [70]. (d) Mitochondrial imaging of MCF-7 cells incubated with CDs (Ex: 488 nm), and then MitoTracker (Ex: 638 nm), ER-Tracker (Ex: 552 nm), Golgi-Tracker (Ex: 552 nm), or LysoTracker (Ex: 552 nm), confirmed by the coincident overlay between CDs green FL and MitoTracker red FL, but not ER-Tracker, Golgi-Tracker, or LysoTracker, adapted with permission from Ref. [63]. (e) Red-emissive FL imaging of different plant tissues through the selective cellulose-binding of CDs, adapted with permission from Ref. [81]. (f) CLSM images of the CDs FL monitoring of S. aureus and E. coli at various incubation periods, adapted with permission from Ref. [15]. (g) Differentiation of gram-positive S. aureus bacteria from gram-negative E. coli bacteria using CLSM images of a mixture of CDs-treated bacteria under 405 nm excitation, adapted with permission from Ref. [14]. (h) CLSM images of C. gloeosporioides fungus labeled by different color CDs, adapted with permission from Ref. [99].Various o-, m-, or p-phenylenediamines (pDA) with triethylenetetramine as precursors were used to synthesize the FL carbon dots with different colors for the imaging application of cellular ribonucleic acids (RNA). However, only m-phenylenediamines formed carbon dots (m-CDs) with a size of 2.75 nm, and maximum green Em at 510 nm under Ex at 360 nm demonstrated excellent ability to specifically bind to the cellular RNA of HEp-2 cells, allowing successful long-term real-time monitoring of RNA dynamics during cell apoptosis, mitosis, and proliferation. The RNA affinity of m-CDs is associated with the isoquinoline moieties and amines on the surface of m-CDs that bind to RNA throughπ–π stacking and electrostatic bonding, respectively. Furthermore, investigations of the in vivo zebrafish larvae body showed major accumulation in the lens, pronephros, intestine, and vessels, suggesting the efficient excretion of m-CDs after 48 h of incubation. Hence, m-CDs as smart nanoprobes for cellular RNA imaging could be valuable for the development of visualized screening of RNA [60]. Interestingly, hydrogen-bonding-induced Em CDs (HBIE-CDs) formed by a hydrothermal method using m-phenylenediamine (mDA) and folic acid (FA) as precursors displayed 460 nm of blue Em in nonhydrogen-bonding solution and 535 nm of green Em in hydrogen-bonding solution due to the inhibition of nonirradiation by a H-bond network. The HBIE-CDs possessed strong affinity toward nucleic acid as the donor of H-bond without interference, dramatically increasing 6-fold green fluorescence after binding with the DNA or RNA of Hela cells. After incubation of Hela cells with HBIE-CDs for 5 min, cells without washing exhibited strong green fluorescence in the nucleus after binding with DNA and RNA and almost no background fluorescence in the cytoplasm, which was almost similar to cells with PBS washing, allowing continuous monitoring of the cell nucleus without interruption (Figure 2(b)) [61]. Furthermore, monitoring the dynamics of DNA and RNA structures in live cells is essential for tracking cell behavior. However, the difficulty in fabricating a the nanoprobe that can distinguish between double-stranded DNA and single-stranded RNA, and cross multiple membrane barriers from the cell to the tissue level is still a challenge. Thus, a cationic carbon quantum dot of 2–3 nm in diameter and zeta potential at +20 mV synthesized from conductive carbon nanoparticles and p-phenylenediamine was successfully developed to address this challenge. The CD exhibited green fluorescence at 510 nm under 400 nm Ex, but significant fluorescence enhancement was obtained with the existence of DNA, up to 8-fold, due to the CD insertion into the grooves of dsDNA that restricted the rotation of the linkage bonds between the pDA and the CD core. In contrast to DNA, the presence of RNA induces the shift of Em fluorescence to red at around 620 nm under 540 nm Ex, because it leads to the formation of CD aggregates to make the strong fluorescent transit from green to red. Upon treatment of CD, the nuclei of a total of 12 cell types emitted strong green fluorescence under 488 nm Ex, while strong red fluorescence was observed in the cytoplasm under 542 nm Ex, due to the CD staining of DNA and RNA, respectively, indicating its ability to cross both the plasma and nuclear membranes to dye the target DNA and RNA. The CD’s ability to enter the intestinal pathway was confirmed through the greater concentration of CD in intestinal wall cells using the imaging of wireworms. Further, it was successfully applied to monitor DNA and RNA during cell division at different cell cycles, including prophase, metaphase, telophase, and interphase. CD’s selectivity towards DNA and RNA, and the excellent ability to penetrate through various biological barriers in cells and live organisms, make CD an efficient agent for basic cell biology, embryology, and clinical diagnosis [104].Lysosomes, as another subcellular organelle, also play critical roles in protein degradation, cell signal transduction, plasma membrane repair, homeostasis, and autophagy, making the development of a specific probe for the imaging of lysosomes extremely necessary to understand the dysfunction of lysosomes that is related to various diseases, such as Alzheimer’s disease, neurodegenerative diseases, and cancer. Through the conjugation of the morpholine group (ML) as lysosome targeting to CDs formed from polyethyleneimine (PEI) and citric acid (CA) as precursors, CDs-PEI-ML were successfully fabricated to act as a long-term tracking probe of lysosomes for cell imaging with excellent properties that include strong blue Em, high photostability, adequate biocompatibility, and high selectivity toward lysosomes. Imaging of the lysosomal localization of CDs-PEI-ML in live Hela cells was described that CDs-PEI-ML were translocated into lysosomes via an endocytic pathway, and accumulated in acidic lysosomes with less diffusion into the cytoplasm due to the presence of lysosome target group morpholine on the surface of CDs-PEI-ML. This CDs-PEI-ML exhibited high specificity in lysosomal imaging, presenting an alternative FL probe for lysosome labeling and imaging (Figure2(c)) [70].Mitochondrion as a vital energy-supplying organelle, plays an important role in cellular functions, such as the generation of ATP and reactive oxygen species (ROS), regulation of transmembrane potentials, and initiation of apoptosis and autophagy, making the dysfunction of the mitochondrial responsible for various diseases, such as Alzheimer’s disease, Parkinson’s disease, diabetes, and cancer. The green FL CDs synthesized using chitosan, ethylenediamine, and mercaptosuccinic acid as precursors, showed intrinsic mitochondrial targeting ability to visualize mitochondria for bioimaging. These CDs quickly entered cells and specifically targeted mitochondria through energy-dependent, caveolae-mediated endocytosis, exhibiting superior features that included multicolor fluorescence, good photostability, and long-term imaging capability for at least 24 h, compared to MitoTrackers (commercial mitochondrial probes), representing a good FL nanoprobe for mitochondrial imaging that could be an alternative to commercial probes (Figure2(d)) [63].As another targeting species in cellular bioimaging, hyaluronan (HA), a linear long-chain polymer, is intimately associated with human tumor progression by stimulating growth, survival, and angiogenesis within primary tumors or their metastases. About 3.2 nm N-doped CDs using starch as a carbon source and L-tryptophan as nitrogen dopants were coupled with molecularly imprinted polymer (MIP) and glucuronic acid (GlcA) as an epitope of hyaluronan to form CD-MIPGlcA that was employed to probe HA cancer biomarkers. CD-MIPGlcA with (3–10) nm diameter emitted the green maximum Em at 500 nm under 445 nm Ex with high selectivity to HA, showing about 4 times higher FL intensity on HeLa cells than that on HaCaT cells, suggesting the ability of CD-MIPGlcA to differentiate between cancer cells and normal cells. The possibility of CD-MIPGlcA in HA-targeting FL nanoprobes indicates how powerful these tools are for the imaging of tumor biomarkers [71].A desirable goal in bioimaging applications is to discriminate against interference from autofluorescence. However, it is still a challenge to develop room temperature (RT) phosphorescence (RTP) probes that feature ultralong Em lifetimes. CD-based silica composites (CDs@SiO2) with long Em lifetimes of 1.64 s were successfully fabricated for ultralong lifetime phosphorescence imaging of cells and tissue at RT using polyacrylic acid and EDA as CD precursors. CDs@SiO2 emitted blue fluorescence and the afterglow green Em maximum at 520 nm under an Ex of 356 nm. Although due to strong endogenous autofluorescence at 365 nm irradiation, it was difficult to distinguish between CDs@SiO2-untreated and treated onion bulb epidermal tissues, strong green phosphorescence of the CDs@SiO2-treated group in EM-6 mouse breast carcinoma cells and onion bulb epidermal tissue was observed after turning off the irradiation, highlighting the successful phosphorescence imaging of CDs@SiO2 in bioimaging in cells or plant and animal tissues by eliminating the autofluorescence interference. These CDs@SiO2 could serve as excellent nanoprobes for bioimaging with an efficient ability to discriminate against tissue autofluorescence, suggesting the potential for ultralong-lived RTP reporters [64]. In addition, a CD-grafted cellulose hybrid phosphor with a high affinity to bind cellulose was prepared for effective fluorescence mapping of cellulosic plant cell walls. These reddish-orange emissive CDs exhibited strong fluorescence at 633 nm with a 44% high quantum yield and high photostability. Various model plant tissues, including onion bulb epidermal tissue, the root of mung bean sprout, and the stem and leaf of Arabidopsis thaliana, were employed to demonstrate the bioimaging capacity of these CDs. CDs can readily have absorbed in plant tissues and bind to a cellulose-rich structure with deep penetration to 55.5 μm depth in the stem of Arabidopsis thaliana, 177.2 μm in the root of mung bean sprout, and 136.3 μm in onion bulb epidermal tissue, respectively, which could address the limitation of FL dyes in their difficulty in visualizing deeper tissue layers of ≥ (50 to 100) μm. These CDs could serve as useful probes for the study of cell wall structure in living plant cells (Figure 2(e)) [81].Undoubtedly, CDs exhibit excellent performance for bioimaging applications that can enhance the understanding of cells or tissues. CDs can also be applied as nanoprobes for the recognition of cancerous cells from normal cells. However, each specific mechanism of interaction between CDs and targets in cells or tissues, cytotoxicity, and optimal conditions of bioimaging processes should be carefully studied to contribute significantly to biological and biomedical sciences.


## 3.2. Fluorescent Carbon Dots for the Bioimaging of Bacteria

Bacteria are abundant in the natural world and can lead to global bacterial infection, so it is essential to accurately specify them for efficient treatment. Nitrogen and sulfur codoped CDs (NSCDs) were obtained by a microwave-assisted hydrothermal method using thiourea as a precursor in tris-acetate-ethylenediamine (TAE) buffer, for use in the imaging of various bacteria. These NSCDs were well mono-dispersed with an average size of 3.62 nm and exhibited maximum fluorescence at 426 nm Em under irradiation of 340 nm with a quantum yield of 57%. With no toxicity to bacteria cells, NSCDs internalization or endocytosis occurred after incubation with bacteria, and emitted blue fluorescence that was observed inE. coli, Pseudomonas, and Staphylococcus, but not in Klebsiella. The untagging effect on Klebsiella may have resulted in the decimation or disorientation of NSCDs due to the catabolic characteristic of Klebsiella for different aromatic compounds. Additionally, the multicolor fluorescence of NSCDs as imaging nanoprobes was screened under different Ex wavelengths of (380, 450, and 505) nm in tagged E. coli cells, showing the bright colors of blue, green, and red at (430, 525, and 590) nm Ems, respectively [20].Taking advantage of the selective binding affinity of boronic acid to diols groups on the bacterial cell wall, phenylboronic acid-functionalized FL carbon dots (FCDs) with strong blue fluorescence were synthesized and conjugated to dye molecules, then successfully applied in the selective detection of bacteria via the imaging of diol-containing bacteria. Owing to the preferential affinity of phenylboronic acid toward diol-containing molecules through cyclic ester bond formation, glucose-containing bacteria were replaced with dye molecules that led to the release of dye from the FCD to recover the fluorescence, gradually increasing the FL intensity from (0 to 60) min under 405 nm Ex in the presence ofE. coli or S. aureus. These FCDs exhibited high sensitivity and chemical specificity for bacteria detection via fluorescence imaging (Figure 2(f)) [15].Accurate bacterial identification is required to characterize unknown bacteria, which is important for the fast diagnosis of bacterial infections. A one-pot solvothermal approach was exploited to obtain a quaternized CDs using glycerol and dimethyloctadecyl[3-(trimethoxysilyl)propy] ammonium chloride (Si-QAC) for the identification of gram-type bacteria through bacteria imaging. Si-QAC containing CDs interacted with the negatively charged bacterial cells via both electrostatic and hydrophobic interactions, selectively connecting with the gram-positive bacteria due to the different surfaces of gram-positive and gram-negative bacteria. CDs displayed Ex-dependent fluorescence from (320 to 500) nm with maximum intensity at 473 nm under 360 nm Ex, significantly enhancing the blue fluorescence once the CDs are adsorbed onto theS. aureus gram-positive bacteria, but not the E. coli cells, in the mixture of these bacteria. This CD-based Gram-type differentiation method was accurate, fast, and facile, suggesting that this CD FL staining method could act as an alternative to the conventional gram staining method for differentiating gram-type bacteria (Figure 2(g)) [14].CD-based bioimaging of bacteria is successfully utilized for FL imaging or differentiating gram-type bacteria. Nevertheless, these studies mostly focused on the imaging of several bacteria, such asS. aureus, and E. coli, other bacterial types that possess different influences should be further investigated. A CD with specific affinity towards each bacterium should be developed for efficient application. Cytotoxicity of CDs should be considered carefully for various types of bacteria for further utilization. The interference of culture media to FL of CDs and the real-time ability of monitoring bacteria are required to study deliberately.


## 3.3. Fluorescent Carbon Dots for the Bioimaging of Fungi

Along with bacteria, the bioimaging of fungi is also important to understand the fungal biology or their infection mechanisms more clearly. Carbon dots synthesized from methyl red (MR) as a precursor in the presence of ethylenediamine (EDA) by facile hydrothermal pyrolysis, namely as MR-EDA-CDs, were fractionated into four fractions with different sizes of (16.3, 12.6, 5.5, and 4.5) nm under column chromatography that emitted green, purple, blue, and yellow fluorescence at (398, 470, 511, and 530) nm, respectively. These biocompatible MR-EDA-CDs were investigated as imaging nanoprobes for the FL imaging of pathogenic fungal cells (C. gloeosporioides). MR-EDA-CDs internalized into the fungal cells upon 6 h of incubation at RT, brightly illuminating the multicolor fluorescence with Ex at 405 nm by confocal microscopy (Figure 2(h)) [99].Owing to their ease of availability and affordability, natural resources have been widely used as precursors for the preparation of CDs.Acacia concinna fruit seeds (shikakai) as a green precursor were used to fabricate CDs in the application as biocompatible nanoprobes for the multicolor imaging of fungal (Penicillium sp.) cells. Upon Ex of 390 nm, these CDs displayed a strong Em peak at 468 nm with a higher quantum yield (10.2%), with good biocompatibility with fungal cells. The ultra-small CDs (2.5 nm diameter) internalized into the cells via endocytosis, and located in the cytoplasm and especially the nucleus of cells, exhibiting bright blue, green, and red fluorescence colors under laser Ex wavelengths of (405, 488, and 561) nm [100]. Another multicolor emissive CD that generated blue, green, and yellow fluorescence was obtained from Manilkara zapota fruits in acidic solutions (sulphuric acid and phosphoric acids) at different temperatures of 100 and 80 °C for 60–15 min. These CDs exhibited the maximum Em peaks at 443, 515, and 563 nm (blue, green, and yellow) under excitation at 350, 420, and 440 nm, with the QYs of 5.7, 7.9, and 5.2 %, respectively. Due to the small size of these CDs of from 1.9–4.5 nm, they are easily internalized into Aspergillus aculeatus and Fomitopsis sp. cells via the cell membrane through endocytosis, located in the cell membrane and cytoplasm, resulting in blue, green, and yellow fluorescence at Ex wavelengths of 405, 488, and 561 nm with good intensity [101]. Overall, the FL CDs have proven to be potential agents for the multicolor imaging applications of fungal cells. However, there are many other types of fungi that are essential to be explored as well. These reported CDs are mainly for imaging of the whole bodies of fungi. Specific CDs for different organelles in fungi are thus highly required to develop for obvious understanding.


## 3.4. Fluorescent Carbon Dots for the Bioimaging of Animal Models

In vivo molecular imaging as a powerful tool for the analysis of the body significantly contributes to precise medicine through diagnosis and guiding therapy. N-doped carbon dots (NCDs) obtained by a hydrothermal method using pDA as precursor have embedded with Mn2+ onto the polydopamine (PDA) nanoparticle (NP) to fabricate the imaging nanoprobe, namely as PDA@NCDs(Mn). Using NCDs as FL agents, PDA as a photothermal agent, and Mn2+ as a T2 magnetic resonance contrast agent, these CD-based NPs were successfully applied as multimodal imaging nanoprobes for tumor diagnosis. These PDA@NCDs(Mn) NPs displayed maximum Em peak at 620 nm when Ex at 550, with strong red Em under the Ex wavelength of 500 nm. For in vivo fluorescence imaging investigation, PDA@NCDs(Mn) were injected into the left rear flank region of the mice model, then accumulated in the tumor site with good distribution, hence valid fluorescence was observed around the tumor. After 12 h post-injection, organs (heart, liver, spleen, lung, and kidney) and tumors were harvested to assess fluorescence intensities. These PDA@NCDs(Mn) NPs mainly accumulated in the liver, lung, and kidney tissues, as well as stronger fluorescence signal in tumor tissues than the spleen and heart, which was observed in a time-dependent fluorescence intensity manner, indicating red-emissive PDA@NCDs(Mn) NPs as promising fluorescence imaging agent for in vivo imaging (Figure 3(a)) [107]. Phosphonated compounds are widely applied for the imaging and treatment of severe bone turnover due to their high affinity to hydroxyapatite (HA) minerals. monophosphonated CDs obtained by a one-pot hydrothermal method using food-grade agave nectar and α-methoxy-ω-phosphate polyethylene glycol exhibited Ex-dependent Em spectra with maximum Ex/Em at 430/600 nm and high affinity towards calcium salt of the bone, indicating their potential as bone-seeking luminescent nanoprobes for the detection of bone microcracks via fluorescence imaging. The CDs-injected mice displayed a prominent difference in the luminescent emitted from the tibia when excited at 430 nm and imaged at 600 nm, suggesting that CDs can light up the tissue surrounding the bone. A statistically significant enhancement was detected in the treated mice, compared to the nontreated animal, by calculating the photon count after injection and the circulation time of about 30 min, indicating the feasibility of tissue imaging and sufficient signal sensitivity of these CDs nanoprobes as effective bone-seeking agent for the prediction of bone failure, considering the crack fracture mechanics (Figure 3(b)) [119].Figure 3
In/Ex vivo FL imaging of animals using CDs. (a) Real-time in vivo red FL images in nude mice at different time periods after (A) subcutaneous, and (B) intravenous injections of CDs via the tail vein revealed the accumulation of CDs in the tumor tissue. Adapted with permission from Ref. [107]. (b) In vivo FL of monophosphonated CDs in the tibia of (A) non-treated mice, (B) treated mice (Ex/Em = 430/600 nm); (C) treated the animal with the computed tomography-fused fluorescence image (Ex/Em = 465/700 nm), showing the feasibility of CDs for the detection of bone microcracks. (D) Significant difference of FL radiance between CDs-nontreated and CDs-treated mice (∗p value < 0.05). Adapted with permission from Ref. [119]. (c) (A) In vivo FL images, and (B) corresponding FL intensity of the tumors before and after the intratumoral injection of CDs for different time periods. (C) Ex vivo FL images, and (D) corresponding FL intensity of different organs of mice after CDs injection for different day periods (∗∗p value < 0.01, ∗∗∗p value < 0.001). (D) CLSM image, and (E) bright-field image of CDs-stained zebrafish. Adapted with permission from Ref. [108].Various metal ions doped into pDA during the hydrothermal treatment lead to the formation of FL CDs with Em wavelengths of up to 700 nm. The Ni-pDA-CDs were prepared using pDA and nickel ions (Ni2+) as raw materials that exhibited Ex-independent Em (at ∼605 nm) with good photostability. A549 live cells treated with Ni-pDA-CDs for 30 min emitted strong red fluorescence under laser Ex at 552 nm in nucleoli, indicating the nucleolus targeting mechanism of Ni-pDA-CDs. Further, the in vivo imaging performance of Ni-pDA-CDs in tumor-bearing mice and zebrafish was investigated. The tumor region emitted bright red fluorescence after the intratumoral injection of Ni-pDA-CDs with a high signal-to-noise ratio of the tumor fluorescence. These Ni-pDA-CDs mainly accumulated in the tumor, with negligible distribution in other organs (heart, liver, spleen, lung, and kidneys), suggesting their good biosafety. Additionally, the Ni-pDA-CDs-incubated zebrafish for 30 min were well stained with bright red fluorescence, confirming that they could pass through the mucus layer and interact with the cells in zebrafish due to their ultrasmall size, positively charged surfaces, and amphiphilicity. These experiments suggest the potential of these Ni-pDA-CDs as excellent red-emissive FL nanoprobes for in vivo imaging in both mice and zebrafish models (Figure 3(c)) [108].The above studies show that these FL CDs hold great promise in the bioimaging of animal models that can support efficient diagnosis and treatment in medicinal applications. Nevertheless, it is worth developing the FL CDs that exhibit excellent biocompatibility and efficient ability of clearance, providing excellent FL probes for tracking tumors, delivering drugs, monitoring the ongoing disease, and further biomedical applications. Surface modification of CDs is needed to study for the elimination of nonspecific absorption onto CDs, and specific imaging of targetsin vivo should be considered carefully when developing CDs.


## 4. Fluorescent Carbon Dots for Bioimaging-Supported Sensing Applications

Table2 summarizes various CD-based imaging-supported sensing applications towards different targets such as metal ions, biomolecules, pH, temperature, and polarity. Case studies of their sensing performance are then described in this section based on their target.Table 2
Imaging-supported sensing applications using FL CDs.
Precursors/formsFL colorApplied Ex/Em (nm)MicroscopicApplicationBiotargetRef.Ascorbic acid, L-arginineGreen750/380–495Two-photon CLSMImaging of tissuesPigskin tissue[120]Red750/500–650Sensing of Fe3+Fibroblast skin cellsAbelmoschus manihotBlue330/410FMSensing of 2,4,6-trinitrophenol (TNT)Hela[121]CaseinBlue383/446CLSMSensing of Hg2+, biothiolsHeLa[122]Catechol, triethylene tetramineGreen415/515CLSMSensing of Fe3+, ascorbic acid (AA)MCF-7[123]p-phenylenediamine, ammoniaGreen361/515CLSMSensing of CrVI, TNT, and AASMMC7721[124]p-aminosalicylic acid, ethyleneglycol dimethacrylateGreen390/520FMSensing of Fe3+BT474[125]Glycerol, DAMOBlue350/442CLSMSensing of Fe3+Zebrafish, Hela,[126]Cancer/normal cell differentiationA549, Hep G2/ AT II, L02GlutaraldehydeBlue440/–CLSMSensing of microRNA-21MCF-7[127]nitro benzaldehyde360/–benzaldehyde360/–CDs-chitosanWhite pepper, H2N-PEG-NH2Green420/525–550CLSMSensing of coenzyme AHeLa[128]Red420/>560CA, PEIBlue350/450CLSMSensing of H2O2HeLa[129]CQD-AuNCRed550/620CA, melamineBlue360/440CLSMSensing of Hg2+, glutathione (GSH)BHK[130]o-phenylenediamineGreen488/–CLSMSensing of atrazineE. coli, S. aureus[131]2,3-diaminophenazineGreen495/530CLSMSensing of NAD+A375 cells[132]Fungus fibersBlue360/440FMSensing of tetracyclinesHepG2[133]CA, EDABlue345/453CLSMSensing of CA125OVCAR-3[134]CD-aptamerCA, L-cysteineBlue355/425FMSensing of testosterone293T[135]β-CD-CDsL-tartaric acid, triethylenetetramineBlue350/425CLSMSensing of metronidazoleMCF-7[136]CABlue380/450FMSensing of cathepsin BHeLa[137]AS1411-Ce6-CQDsm-aminophenolYellow–green450/520CLSMSensing of selenolL929[138]CD-DNSFood grade agave nectar,α-methoxy-ω-phosphate PEGRed430/600In vivo imaging systemImaging of hydroxyapatite-targeted boneAthymic nude mice[119]CA, EDABlue, yellow360/440, 565CLSMSensing of thioredoxin reductaseMCF-7, HeLa cells[139]Biotin-CD-NaphNeutral red, triethylamineYellow478/532, 618CLSMSensing of Ag+, GSHSMMC7721[140]Chestnut, onionBlue370/440CLSMSensing of coenzyme AT24[141]Aconitic acid, tryptophanBlue370/455CLSMSensing of cytochrome cHepG-2, zebrafish[142]FO-Pte, FO-PSeGreen800/400–600Two-photon CLSMSensing of superoxide anionHela, A549[143]Breast tumor tissueEDTA-2Na, CuCl2Blue370/480CLSMSensing of H2SH3122[144]CABlue405/470CLSMSensing of Fe3+, AAHepG2, zebrafish, liver ischemia tissue[145]CDs-DBRed405/450–550CA, phenosafranine,Orange525/600CLSMSensing of nitriteA549[146]HAuCl4, GSH, and glucoseGreen405/495–575FMSensing of SKBR3 exosomesHeLa[147]PEI-GCDs-AbCA, 2,3-PhenazinediamineYellow380/568FMSensing of Ag+, GSHH1299[148]CA, urea, and triethylenetetramineBlue380/470CLSMSensing of Cu2+, GSHYeast[116]CA, methionineBlue350/420FMSensing of Al3+HUVEC[149]Green350/502Dunaliella salinaGreen340/415FMImaging of cellsHEK-293[150]RedSensing of Hg2+, Cr6+Alkali lignin, EDA, and formaldehydeBlue405/–CLSMImaging of cellsRAW264.7[151]Green488/–Sensing of Fe3+Red558/–Ocimum sanctumBlue330–385/–FMImaging of cellsMDA-MB 468[152]Green450−480/–Sensing of Pb2+Red510–550/–Banana plantBlue408/–CLSMSensing of Fe3+Hela, MCF-7[153]Green488/–Red561/–Ganoderma lucidum sporesBlue405/–CLSMSensing of Fe3+MIHA[154]Green488/–Red559/–Phthalic acid, EDA, and H3PO4Blue405/422CLSMImaging of cellsSMMC-7721[155]Green488/500Sensing of Mn7+Red543/650PhthalocyanineGreen780/520Two-photon CLSMImaging of cellsMCF-7[118]Red850–890/600Sensing of Fe3+Magnolia liliifloraBlue405/–CLSMSensing of Fe3+Clone 9 hepatocytes[156]Green488/–Red555/–Sweet potatoBlue330–388/405CLSMImaging of cellsHela, HepG2[157]Green450–480/–Sensing of Fe3+Red510–550/–Glucose, octadecylamine, and octadeceneGreen425/485CLSMSensing of Cu2+HepG2, HL-7702[158]HCD-based micelleRed425/655Syringa obtata LindlBlue340/425CLSMSensing of Fe3+MCF-7[159]Green450/520Black soya beansBlue405/–CLSMImaging of cellsSMMC-7721[160]Green488/–Sensing of Fe3+Red543/–AspartameBlue405/420–480CLSMImaging of cellsSGC-7901[161]Green405/500–560Sensing of Fe3+Red555/600–700o-phenylenediamine, 4,5-difluoro-1,2-benzenediamine, and tartaric acidGreen480/600FMImaging of cellsHEK 293T, NIH 3T3,[162]Red540/550Sensing of Ag+COS-7, HepG2, and B16F10Aminosalicylic acidGreen405/–CLSMSensing of Fe3+H1299[163]Yellow488/–Red542/–CA, thioureaBlue405/450FMImaging of cellsMiaPaCa-2[164]Sensing of Fe3+L-glutamic acid, EDABlue360/459CLSMImaging of fungiC. gloeosporioides[165]Sensing of Fe3+m-phenylenediamine, diethylenetriaminepenta (methylenephosphonic acid)Green440/510CLSMImaging of cellsA549, KB[166]Sensing of Fe3+CA, diethylenetriamineBlue391/438FMSensing of Co2+HeLa[167]Osmanthus fragransBlue340/410FMSensing of Fe3+, AAA549[168]CA, methylamine hydrochloride, and lanthanumGreen420/510FMSensing of Fe3+HeLa[169]chloride heptahydrateTetraphenylporphyrin-Mn, CABlue330–385/–FMSensing of Fe3+HeLa, zebrafish[170]Green400–440/–Red510–550/–P-phenylenediamineOrange566/590CLSMSensing of intracellular pHSMMC7721[171]CA, 1,4-butanediamineBlue380/443CLSMSensing of intracellular pHPholiota adipose fungi[172]RhB-AB-CDsRed400/580p-phenylenediamineRed488/550–650CLSMImaging of cellsHepG2, HL-7702[173]Sensing of intracellular polarityAscorbic acidYellow405/415–550CLSMImaging of nucleolus293T[174]Sensing of temperature


### 4.1. Fluorescent Carbon Dots for the Bioimaging-Supported Sensing of Metal Ions

It is worth noting that effective probes with excellent biocompatibility and photostability that can sensitively detect metal ions in living cells andin vivo remain a highly essential and challenging goal. Diverse CDs have been developed for the sensing applications of various ions, such as Fe3+, Ag+, Cu2+, and Hg2+. Fluorine (F)-doped CDs were successfully synthesized by a solvothermal process using difluoro-1,2-benzenediamine as the F source, and tartaric acid to improve aqueous solubility, for the intracellular detection of Ag+ ions. The FCDs emitted Ex-dependent Em with yellow fluorescence (maximum Ex/Em at 360–500)/550 nm and red fluorescence (maximum Ex/Em at 600/540–580 nm). F-doped CDs possessed remarkable water solubility, high biocompatibility, and excellent photostability, and could act as nanoprobes for the selective detection of Ag+ in HEK 293 normal cell and B16F10 cancer cell, which was confirmed by the quenching FL image in the presence of Ag+ (Figure 4(a)) [162].Figure 4
Fluorescence imaging-supported sensing of various metal ions and biomolecules. (a) Intracellular silver ions detection in HEK 293T cells and B16F10 cells without (-), and with (+) Ag+, through obvious quenching phenomenon. Adapted with permission from Ref. [162]. (b) In vivo semiquantitative detection of Fe3+ through CLSM images of zebrafish treated with CDs and quenching FL effect after treatment with different concentrations of Fe3+ and corresponding FL intensity. Adapted with permission from Ref. [126]. (c) Action mechanism of AS1411–CQDs–Ce6 nanoprobe for the intracellular detection of endogenous CTSB through specific binding of AS1411 aptamer with nucleolins. Adapted with permission from Ref. [137]. (d) Strong FL imaging of living HeLa cells under different treatments with AS1411–CQDs–Ce6 and no FL signal of nucleolin-deficient HEK293 cells under treatment with AS1411–CQDs–Ce6 indicated the feasibility of nanoprobes for the detection of CTSB. Adapted with permission from Ref. [137]. (e) In vivo detection of Cyt (c) in zebrafish. CLSM images of zebrafish (A) untreated NCDs, (B) incubated with NCDs for 30 min; and subsequent treatment with (C) Cyt (c), or (D) etoposide for 20 min showed a significant FL quenching effect of Cyt (c). Adapted with permission from Ref. [142]. (f) Selective detection of superoxide anion in mice through the FL enhancement of O2−to CDs. FL imaging of O2−levels in the tumor tissues (A)–(C) and normal tissue (D)–(F) after treatment with Te-CDs, FO-PTe, and Se-CDs-treated mice, respectively, acquired using 800 nm two-photon excitation. (G) Corresponding FL intensity revealed the significant increase of O2−level between normal and tumor tissues. Adapted with permission from Ref. [143].An on/off/on FL CD-based nanosensor was successfully fabricated for in vitro and in/ex vivo Fe3+ detection. CDs prepared by a solvothermal treatment of glycerol and a silane molecule (N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) exhibited favorable biocompatibility and excellent fluorescence properties that included high QY blue fluorescence 45% at optimal Ex/Em of 350/442 nm, robust photostability in a wide range of ionic strengths and pH, selectively monitoring Fe3+ in Hela cells for 30 min and zebrafish for 2 h in the range 0–100 μM. Herein, FL imaging of Hela cells and zebrafish after treatment with Fe3+ was obtained and FL intensity measured, showing the gradual decrease of intensity with increasing concentration of exogenous Fe3+. These CDs could semiquantitatively detect Fe3+in vitro and in vivo. The feasibility of these CDs for the detection of Fe3+in vivo was further evaluated through the tumor-specific imaging of nude mice bearing subcutaneous U14 xenograft tumors. Normal and tumor regions of a mouse model were imaged under in vivo animal imaging systems after 1 h postinjection of CDs/Fe3+ dots. The fluorescence intensity of the tumor region was stronger than that of normal tissue, which was verified by ex vivo tissue imaging, suggesting their possibility of realizing tumor-site imaging (Figure 4(b)) [126].Amphiphilic FL carbon dots (A-CDs) were synthesized by cetyltrimethyl ammonium bromide (CTAB) modification of CDs that were prepared from CA and methionine. The fluorescence of these A-CDs was quenched by morin through electrostatic interaction between abundant amino groups of A-CDs and phenolic hydroxyl groups of morin, and then recovered in the presence of Al3+ by the release of A-CDs due to the strong complex of Al3+ and morin. These phenomena were used to detect Al3+ ions in HUVEC cells via cell imaging, showing fluorescence color that changed from light to deep green with increasing concentrations of Al3+ ions from 5–20 μM [149]. Green synthesis from Dunaliella Salina was used to obtain strong blue FL CDs with a maximum Ex/Em at 340/415 nm at a QY of 8% for the utilization of CDs as an FL turn-off sensor for Hg2+ and Cr6+ ions in living cells. These CDs exhibited good biocompatibility, high photostability, and negligible photobleaching, making them suitable for in vitro cell imaging. Upon treatment of exogenous Hg2+ and Cr6+ at 100 μM, the fluorescence of HEK-293 cells was strongly quenched in the red and green channels, indicating their possibility of detecting endogenous Hg2+ and Cr6+ contaminated live cells [150].These results indicate that CDs-based bioimaging-supported sensing platforms for metal ions have been successfully developedin vitro and in vivo. However, it is required to carefully study the specificity of these CDs in sensing ions using FL imaging. Autofluorescence is considered the major issue for bioimaging of cells and tissue. Therefore, it is necessary to develop FL CDs that will not be affected by the cell and tissue autofluorescence, leading to a better possibility to recognize the targets. These reported studies are qualitative analysis for metal ions through bioimaging, which is unable to explore the minimum detectable level of ions. Therefore, quantitative analysis is required to predict the possibility of prepared CDs for practical application.


### 4.2. Fluorescent Carbon Dots for the Bioimaging-Supported Sensing of Biomolecules

Detection of the presence of small biomolecules that may be toxic or play a role in physiological processes is critical for various biomedical purposes. Amine-rich CDs with blue fluorescence were synthesized from CA and PEI and were then covalently assembled with nucleolin-targeting recognition nucleic acid aptamer AS1411 and a CTSB-cleavable peptide substrate that tethered with near-infrared (NIR) fluorophore chlorin e6 (Ce6-Pep), forming the nanoprobe AS1411–Ce6–CDs for cancer targeting and the imaging of cathepsin B (CTSB, a biomarker for malignant tumors), in living cancer cells (Figure4(c)). Blue fluorescence of CDs at 450 nm was strongly quenched upon the conjugation with Ce6 via efficient fluorescence resonance energy transfer (FRET); otherwise, NIR fluorescence enhancement of Ce6 at ∼650 nm was observed. After being internalized into cancer cells via nucleolin-mediated endocytosis, the overexpressed CTSB in the lysosome could cleave Ce6–Pep from AS1411–Ce6–CDs to terminate the FRET process, recovering the efficient ratiometric fluorescence response toward endogenous CTSB from NIR to blue Em. Compared to no fluorescence in nucleolin-deficient HEK293 cells, significant fluorescence was observed in nucleolin-positive Hela cancer cells, confirming that AS1411 aptamer could bind to nucleolin-positive cancer cells with high specificity. Thus, these CD-based nanoprobes could be particularly suitable for monitoring the low abundance of CTBS biomarkers via precise cell imaging (Figure 4(d)) [137].Superoxide anion (O2−) is considered one of the primary reactive oxygen species (ROS) associated with major diseases. Se- and Te-containing CDs for the detection of O2− were successfully fabricated by the hydrothermal treatment of FO-PTe and FO-PSe as carbon sources, and their selectivity and sensitivity toward O2− was evaluated. These Se- and Te-containing CDs exhibited the optimal Ex/Em at 440/550 nm and 380/440 nm, respectively, showing enhanced fluorescence after the reaction with O2−, and quenching when reacting with glutathione, making it possible to monitor the reversible and dynamic response of O2˙−. The feasibility of these CDs for the analysis of O2− was investigated using two-photon fluorescence imagery of different cells (hepatocytes, HepG2 cells, macrophages, Hela cells, and lung cancer cells) and a mouse-bearing tumor model. Under 800 nm Ex, two-photon fluorescence Em of 400–500 nm for Te-CDs and 500–600 nm for Se-CDs were observed to be clearer in tumor cells than in normal cells, due to the high concentration of O2− in cancer cells, compared to in normal cells. Additionally, brighter fluorescence with deep tissue penetration and reduced background fluorescence was obtained in the breast tumor tissue of mice compared to normal abdomen tissue due to the significantly higher level of O2− in tumor tissue. Te-containing CDs exhibited the highest sensitivity toward O2−, suggesting that they could serve as nanoprobes for the real-time and dynamic imaging of O2− fluxes (Figure 4(f)) [143].Gold-carbon quantum dots (GCDs) synthesized by the modification of CDs and gold nanoclusters through a microwave-assisted method immobilized the tumor-specific antibodies to serve as FL nanoprobes for the intracellular imaging of cancer-derived exosomes. HER2, a common surface marker found on SKBR3 exosomes, was used as a target protein for the detection of exosomes in Hela cells. After being taken up by cells, the anti-HER antibody conjugated nanoprobes stuck to SKBR3 exosomes and showed bright green fluorescence in the cytoplasm, indicating the excellent intracellular fluorescence image of exosomes [147].Coenzyme A, an important coenzyme in many biochemical reactions of the human body, such as neurodegeneration, protein acetylation, autophagy, and signal transduction, might cause different types of damage due to the deficiency of CoA. Green synthesis was conducted under hydrothermal heating using two natural biomasses (water chestnut and onion) as precursors to obtain the S and N co-doped FL CDs (S, N/CDs) that were used to monitor the presence of coenzyme A in human bladder cancer T24 cells. These S, N/CDs possessed strong blue fluorescence with maximum Ex/Em at 370/475 nm that was dramatically quenched when bound to Cu2+ ions and then restored fluorescence in the presence of coenzyme A due to the stronger complex between thiol groups of coenzyme A and Cu2+. After the loading of S, N/CDs into T24 cells for 30 min, the stronger blue and green fluorescence was observed in the T24 cells excited at 405 and 488 nm in the presence of coenzyme A, indicating that S, N/CDs-Cu2+ probe penetrated cell membranes, and efficiently imaged intracellular Coenzyme A [141].Fluorescence imaging with effective monitoring of cytochrome c as an important biomarker for the early stage of apoptosis in cell apoptosis was developed using N-doped CDs that were prepared by the hydrothermal treatment of aconitic acid and tryptophan. These CDs showed strong blue fluorescence with an intensity maximum at 455 nm under 370 nm Ex and QY at 20%, which is obviously quenched by cytochrome c, facilitating thein-situ detection of cytochrome c for apoptosis signaling. After treatment of HepG-2 cells with N-doped CDs for 2 h, the bright blue Em was gradually decreased with increasing concentrations of etoposide, which is an apoptosis inducer to specifically trigger the release of cytosolic cytochrome c from mitochondria, indicating the quantitative imaging of etoposide-induced intracellular release during cell apoptosis. The ability to detect cytochrome c was further investigated in zebrafish imaging where brighter blue fluorescence in the abdomen of 3-day old CDs-treated zebrafish was observed, as compared to that when the zebrafish were consequently treated with cytochrome c, suggesting N-doped CDs as effective FL nanoprobes for the monitoring of cytochrome c mediated cell apoptosis pathway (Figure 4(e)) [142].Overall, there are several excellent CDs that have been used to detect biomoleculesin vitro and in vivo via bioimaging. Again, autofluorescence of cells and tissue, and the specificity of FL CDs must be carefully considered to enhance the sensing performance. Quantitative analysis should be studied for further practical application. The cytotoxicity of CDs is an urgent matter to investigate before the sensing process. The time and mechanism of CD penetration into the target should be clearly examined for further effective imaging-supported sensing application.


### 4.3. Fluorescent Carbon Dots for the Bioimaging-Supported Sensing of pH, Temperature, and Polarity

The direct measurement of some intracellular conditions in cells is essential but not easily achieved, making it difficult to explain their effects on bioreactions. The dual-Em CDs obtained by the hydrothermal pyrolysis of aniline blue (AB) and rhodamine B (RhB), and CDs made from CA and 1,4-butanediamine in the presence of PEG 400, denoted as RhB-AB-CDs, were exploited as intracellular pH nanoprobes in the mycelia of Pholiota adipose fungus. These RhB-AB-CDs emitted two distinct Em bands at the maximum Ex/Em of 380/443 nm and 400/580 nm, which were attributed to the Ex-dependent Em of CDs and the Ex-independent Em characteristic of RhB, respectively, making the signal sensitive to pH, corresponding to the ratio of Em intensity at 580 and 467 nm under Ex at 400 nm. They were then applied to monitor the intracellular pH of fungal mycelia using the ratio of FL intensity in the red/blue channels at 580/467 nm under continuous Ex at 405 nm, showing the increase of this ratio with an increase of pH value from pH = 5.52 to 7.50 with good linear regression of 0.997, suggesting the highly photostable fluorescence of these CDs for the reliable long-term monitoring of intracellular pH (Figure5(a)) [172].Figure 5
Fluorescence imaging-supported sensing of other effects (pH, thermal, polarity). (a) Response mechanism to pH of RhB-AB-CDs based on the inner filter effect of Aniline Blue on the emission of Rhodamine B and fluorescence color changes of CLSM images of RhB-AB-CDs-treated Pholiota adipose mycelia at pH of (5.5 to 7.5). Adapted with permission from Ref. [172]. (b) (A) Fluorescence linear relationship CDs during heating and cooling processes. (B) Thermal stability of FL lifetimes over seven cycles of heating and cooling between (30 and 70) °C ((I)0 and (I) are the FL intensity at 20°C and other temperatures, respectively). (C) CLSM images of CDs-treated 293T cells at (20, 30, and 40)°C; Em was collected at (415–550) nm at an excitation wavelength of 405 nm. Adapted with permission from Ref. [174]. (c) Intracellular polarity sensor through FL images of HepG2 cells incubated with (A) DMSO + CDs (obvious FL enhancement), (B) CDs, and (C) HBSS + CDs (quenching FL) at Ex/Em of 488/(550–650) nm. Adapted with permission from Ref. [173].Temperature is also an important factor in the life process, due to the role of thermal energy transfer in all biological reactions in organisms. Faint yellow fluorescence CDs were synthesized directly from ascorbic acid aqueous solution by an electrochemical method at RT and exhibited Ex-dependent Em with a maximum Ex at 400 nm. The fluorescence of these CDs with a high quantum yield at 35.2% was stable with different pH values, ionic strengths, and storage times, and sensitive to temperature, with the fluorescence intensity changing linearly with a temperature of 20–100 °C. Human normal HEK 293T cells emitted faint yellow fluorescence under 405 nm irradiation; moreover, as the temperature increased from 20 to 40 °C or decreased from 40 to 20°C, the fluorescence intensity gradually decreased or increased, respectively. Furthermore, these CDs can especially stain the nucleolus by entering the cell nucleolus and adsorbing on nucleic acids (DNA and RNA). Thus, the CDs could act as an effective nanothermometer to monitor in vitro and in vivo temperature by utilizing the yellow-emissive nanoprobes of nucleolus specific imaging (Figure5(b)) [174].Monitoring the change of intracellular polarity plays a great role in the understanding of its functions in live cells, due to its large impact on the activity of biomolecules or the function of domains. The FL carbon dots R-CDs with a 5 nm spherical shape and good monodispersity were successfully obtained through a hydrothermal method at 200°C for 12 h using p-phenylenediamine as a precursor, displaying significant red Em under 420 nm Ex, and were then utilized as FL nanoprobes for the real-time sensing of polarity in living cells. The R-CDs possessed a high quantum yield of 47.87%, high photostability, good biocompatibility up to 400μg/mL, and dual targetability for mitochondria and lysosomes in cells. Various solutions with increasing polarity from 10 to 99 % were obtained by mixing water and dioxane based on volume ratios and showed a decrease in FL intensity of up to 11-fold. For colocalization examination of these R-CDs, human liver cancer HepG2 cells costained with R-CDs, Mito-tracker green, and Lyso-Tracker Red presented partial overlaps of the regions between the red FL of R-CDs and the Mito-tracker green or Lyso-Tracker Red areas, suggesting the preferential accumulation of R-CDs in mitochondria and lysosomes. The ability of R-CDs to monitor the polarity change in HepG2 cells by the reduction of red-emissive fluorescence with increasing polarity was successfully studied. Interestingly, the imaging of HepG2 cells and normal human liver HL-7702 cells both treated with R-CDs showed the difference between the strong red fluorescence in HepG2 cells and weak fluorescence in HL-7702 cells, discriminating the lower polarity in cancer cells with high polarity in normal cells. These R-CDs nanoprobes could contribute to mitochondrial and lysosomal polarity-related studies (Figure 5(c)) [173].


## 4.1. Fluorescent Carbon Dots for the Bioimaging-Supported Sensing of Metal Ions

It is worth noting that effective probes with excellent biocompatibility and photostability that can sensitively detect metal ions in living cells andin vivo remain a highly essential and challenging goal. Diverse CDs have been developed for the sensing applications of various ions, such as Fe3+, Ag+, Cu2+, and Hg2+. Fluorine (F)-doped CDs were successfully synthesized by a solvothermal process using difluoro-1,2-benzenediamine as the F source, and tartaric acid to improve aqueous solubility, for the intracellular detection of Ag+ ions. The FCDs emitted Ex-dependent Em with yellow fluorescence (maximum Ex/Em at 360–500)/550 nm and red fluorescence (maximum Ex/Em at 600/540–580 nm). F-doped CDs possessed remarkable water solubility, high biocompatibility, and excellent photostability, and could act as nanoprobes for the selective detection of Ag+ in HEK 293 normal cell and B16F10 cancer cell, which was confirmed by the quenching FL image in the presence of Ag+ (Figure 4(a)) [162].Figure 4
Fluorescence imaging-supported sensing of various metal ions and biomolecules. (a) Intracellular silver ions detection in HEK 293T cells and B16F10 cells without (-), and with (+) Ag+, through obvious quenching phenomenon. Adapted with permission from Ref. [162]. (b) In vivo semiquantitative detection of Fe3+ through CLSM images of zebrafish treated with CDs and quenching FL effect after treatment with different concentrations of Fe3+ and corresponding FL intensity. Adapted with permission from Ref. [126]. (c) Action mechanism of AS1411–CQDs–Ce6 nanoprobe for the intracellular detection of endogenous CTSB through specific binding of AS1411 aptamer with nucleolins. Adapted with permission from Ref. [137]. (d) Strong FL imaging of living HeLa cells under different treatments with AS1411–CQDs–Ce6 and no FL signal of nucleolin-deficient HEK293 cells under treatment with AS1411–CQDs–Ce6 indicated the feasibility of nanoprobes for the detection of CTSB. Adapted with permission from Ref. [137]. (e) In vivo detection of Cyt (c) in zebrafish. CLSM images of zebrafish (A) untreated NCDs, (B) incubated with NCDs for 30 min; and subsequent treatment with (C) Cyt (c), or (D) etoposide for 20 min showed a significant FL quenching effect of Cyt (c). Adapted with permission from Ref. [142]. (f) Selective detection of superoxide anion in mice through the FL enhancement of O2−to CDs. FL imaging of O2−levels in the tumor tissues (A)–(C) and normal tissue (D)–(F) after treatment with Te-CDs, FO-PTe, and Se-CDs-treated mice, respectively, acquired using 800 nm two-photon excitation. (G) Corresponding FL intensity revealed the significant increase of O2−level between normal and tumor tissues. Adapted with permission from Ref. [143].An on/off/on FL CD-based nanosensor was successfully fabricated for in vitro and in/ex vivo Fe3+ detection. CDs prepared by a solvothermal treatment of glycerol and a silane molecule (N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) exhibited favorable biocompatibility and excellent fluorescence properties that included high QY blue fluorescence 45% at optimal Ex/Em of 350/442 nm, robust photostability in a wide range of ionic strengths and pH, selectively monitoring Fe3+ in Hela cells for 30 min and zebrafish for 2 h in the range 0–100 μM. Herein, FL imaging of Hela cells and zebrafish after treatment with Fe3+ was obtained and FL intensity measured, showing the gradual decrease of intensity with increasing concentration of exogenous Fe3+. These CDs could semiquantitatively detect Fe3+in vitro and in vivo. The feasibility of these CDs for the detection of Fe3+in vivo was further evaluated through the tumor-specific imaging of nude mice bearing subcutaneous U14 xenograft tumors. Normal and tumor regions of a mouse model were imaged under in vivo animal imaging systems after 1 h postinjection of CDs/Fe3+ dots. The fluorescence intensity of the tumor region was stronger than that of normal tissue, which was verified by ex vivo tissue imaging, suggesting their possibility of realizing tumor-site imaging (Figure 4(b)) [126].Amphiphilic FL carbon dots (A-CDs) were synthesized by cetyltrimethyl ammonium bromide (CTAB) modification of CDs that were prepared from CA and methionine. The fluorescence of these A-CDs was quenched by morin through electrostatic interaction between abundant amino groups of A-CDs and phenolic hydroxyl groups of morin, and then recovered in the presence of Al3+ by the release of A-CDs due to the strong complex of Al3+ and morin. These phenomena were used to detect Al3+ ions in HUVEC cells via cell imaging, showing fluorescence color that changed from light to deep green with increasing concentrations of Al3+ ions from 5–20 μM [149]. Green synthesis from Dunaliella Salina was used to obtain strong blue FL CDs with a maximum Ex/Em at 340/415 nm at a QY of 8% for the utilization of CDs as an FL turn-off sensor for Hg2+ and Cr6+ ions in living cells. These CDs exhibited good biocompatibility, high photostability, and negligible photobleaching, making them suitable for in vitro cell imaging. Upon treatment of exogenous Hg2+ and Cr6+ at 100 μM, the fluorescence of HEK-293 cells was strongly quenched in the red and green channels, indicating their possibility of detecting endogenous Hg2+ and Cr6+ contaminated live cells [150].These results indicate that CDs-based bioimaging-supported sensing platforms for metal ions have been successfully developedin vitro and in vivo. However, it is required to carefully study the specificity of these CDs in sensing ions using FL imaging. Autofluorescence is considered the major issue for bioimaging of cells and tissue. Therefore, it is necessary to develop FL CDs that will not be affected by the cell and tissue autofluorescence, leading to a better possibility to recognize the targets. These reported studies are qualitative analysis for metal ions through bioimaging, which is unable to explore the minimum detectable level of ions. Therefore, quantitative analysis is required to predict the possibility of prepared CDs for practical application.


## 4.2. Fluorescent Carbon Dots for the Bioimaging-Supported Sensing of Biomolecules

Detection of the presence of small biomolecules that may be toxic or play a role in physiological processes is critical for various biomedical purposes. Amine-rich CDs with blue fluorescence were synthesized from CA and PEI and were then covalently assembled with nucleolin-targeting recognition nucleic acid aptamer AS1411 and a CTSB-cleavable peptide substrate that tethered with near-infrared (NIR) fluorophore chlorin e6 (Ce6-Pep), forming the nanoprobe AS1411–Ce6–CDs for cancer targeting and the imaging of cathepsin B (CTSB, a biomarker for malignant tumors), in living cancer cells (Figure4(c)). Blue fluorescence of CDs at 450 nm was strongly quenched upon the conjugation with Ce6 via efficient fluorescence resonance energy transfer (FRET); otherwise, NIR fluorescence enhancement of Ce6 at ∼650 nm was observed. After being internalized into cancer cells via nucleolin-mediated endocytosis, the overexpressed CTSB in the lysosome could cleave Ce6–Pep from AS1411–Ce6–CDs to terminate the FRET process, recovering the efficient ratiometric fluorescence response toward endogenous CTSB from NIR to blue Em. Compared to no fluorescence in nucleolin-deficient HEK293 cells, significant fluorescence was observed in nucleolin-positive Hela cancer cells, confirming that AS1411 aptamer could bind to nucleolin-positive cancer cells with high specificity. Thus, these CD-based nanoprobes could be particularly suitable for monitoring the low abundance of CTBS biomarkers via precise cell imaging (Figure 4(d)) [137].Superoxide anion (O2−) is considered one of the primary reactive oxygen species (ROS) associated with major diseases. Se- and Te-containing CDs for the detection of O2− were successfully fabricated by the hydrothermal treatment of FO-PTe and FO-PSe as carbon sources, and their selectivity and sensitivity toward O2− was evaluated. These Se- and Te-containing CDs exhibited the optimal Ex/Em at 440/550 nm and 380/440 nm, respectively, showing enhanced fluorescence after the reaction with O2−, and quenching when reacting with glutathione, making it possible to monitor the reversible and dynamic response of O2˙−. The feasibility of these CDs for the analysis of O2− was investigated using two-photon fluorescence imagery of different cells (hepatocytes, HepG2 cells, macrophages, Hela cells, and lung cancer cells) and a mouse-bearing tumor model. Under 800 nm Ex, two-photon fluorescence Em of 400–500 nm for Te-CDs and 500–600 nm for Se-CDs were observed to be clearer in tumor cells than in normal cells, due to the high concentration of O2− in cancer cells, compared to in normal cells. Additionally, brighter fluorescence with deep tissue penetration and reduced background fluorescence was obtained in the breast tumor tissue of mice compared to normal abdomen tissue due to the significantly higher level of O2− in tumor tissue. Te-containing CDs exhibited the highest sensitivity toward O2−, suggesting that they could serve as nanoprobes for the real-time and dynamic imaging of O2− fluxes (Figure 4(f)) [143].Gold-carbon quantum dots (GCDs) synthesized by the modification of CDs and gold nanoclusters through a microwave-assisted method immobilized the tumor-specific antibodies to serve as FL nanoprobes for the intracellular imaging of cancer-derived exosomes. HER2, a common surface marker found on SKBR3 exosomes, was used as a target protein for the detection of exosomes in Hela cells. After being taken up by cells, the anti-HER antibody conjugated nanoprobes stuck to SKBR3 exosomes and showed bright green fluorescence in the cytoplasm, indicating the excellent intracellular fluorescence image of exosomes [147].Coenzyme A, an important coenzyme in many biochemical reactions of the human body, such as neurodegeneration, protein acetylation, autophagy, and signal transduction, might cause different types of damage due to the deficiency of CoA. Green synthesis was conducted under hydrothermal heating using two natural biomasses (water chestnut and onion) as precursors to obtain the S and N co-doped FL CDs (S, N/CDs) that were used to monitor the presence of coenzyme A in human bladder cancer T24 cells. These S, N/CDs possessed strong blue fluorescence with maximum Ex/Em at 370/475 nm that was dramatically quenched when bound to Cu2+ ions and then restored fluorescence in the presence of coenzyme A due to the stronger complex between thiol groups of coenzyme A and Cu2+. After the loading of S, N/CDs into T24 cells for 30 min, the stronger blue and green fluorescence was observed in the T24 cells excited at 405 and 488 nm in the presence of coenzyme A, indicating that S, N/CDs-Cu2+ probe penetrated cell membranes, and efficiently imaged intracellular Coenzyme A [141].Fluorescence imaging with effective monitoring of cytochrome c as an important biomarker for the early stage of apoptosis in cell apoptosis was developed using N-doped CDs that were prepared by the hydrothermal treatment of aconitic acid and tryptophan. These CDs showed strong blue fluorescence with an intensity maximum at 455 nm under 370 nm Ex and QY at 20%, which is obviously quenched by cytochrome c, facilitating thein-situ detection of cytochrome c for apoptosis signaling. After treatment of HepG-2 cells with N-doped CDs for 2 h, the bright blue Em was gradually decreased with increasing concentrations of etoposide, which is an apoptosis inducer to specifically trigger the release of cytosolic cytochrome c from mitochondria, indicating the quantitative imaging of etoposide-induced intracellular release during cell apoptosis. The ability to detect cytochrome c was further investigated in zebrafish imaging where brighter blue fluorescence in the abdomen of 3-day old CDs-treated zebrafish was observed, as compared to that when the zebrafish were consequently treated with cytochrome c, suggesting N-doped CDs as effective FL nanoprobes for the monitoring of cytochrome c mediated cell apoptosis pathway (Figure 4(e)) [142].Overall, there are several excellent CDs that have been used to detect biomoleculesin vitro and in vivo via bioimaging. Again, autofluorescence of cells and tissue, and the specificity of FL CDs must be carefully considered to enhance the sensing performance. Quantitative analysis should be studied for further practical application. The cytotoxicity of CDs is an urgent matter to investigate before the sensing process. The time and mechanism of CD penetration into the target should be clearly examined for further effective imaging-supported sensing application.


## 4.3. Fluorescent Carbon Dots for the Bioimaging-Supported Sensing of pH, Temperature, and Polarity

The direct measurement of some intracellular conditions in cells is essential but not easily achieved, making it difficult to explain their effects on bioreactions. The dual-Em CDs obtained by the hydrothermal pyrolysis of aniline blue (AB) and rhodamine B (RhB), and CDs made from CA and 1,4-butanediamine in the presence of PEG 400, denoted as RhB-AB-CDs, were exploited as intracellular pH nanoprobes in the mycelia of Pholiota adipose fungus. These RhB-AB-CDs emitted two distinct Em bands at the maximum Ex/Em of 380/443 nm and 400/580 nm, which were attributed to the Ex-dependent Em of CDs and the Ex-independent Em characteristic of RhB, respectively, making the signal sensitive to pH, corresponding to the ratio of Em intensity at 580 and 467 nm under Ex at 400 nm. They were then applied to monitor the intracellular pH of fungal mycelia using the ratio of FL intensity in the red/blue channels at 580/467 nm under continuous Ex at 405 nm, showing the increase of this ratio with an increase of pH value from pH = 5.52 to 7.50 with good linear regression of 0.997, suggesting the highly photostable fluorescence of these CDs for the reliable long-term monitoring of intracellular pH (Figure5(a)) [172].Figure 5
Fluorescence imaging-supported sensing of other effects (pH, thermal, polarity). (a) Response mechanism to pH of RhB-AB-CDs based on the inner filter effect of Aniline Blue on the emission of Rhodamine B and fluorescence color changes of CLSM images of RhB-AB-CDs-treated Pholiota adipose mycelia at pH of (5.5 to 7.5). Adapted with permission from Ref. [172]. (b) (A) Fluorescence linear relationship CDs during heating and cooling processes. (B) Thermal stability of FL lifetimes over seven cycles of heating and cooling between (30 and 70) °C ((I)0 and (I) are the FL intensity at 20°C and other temperatures, respectively). (C) CLSM images of CDs-treated 293T cells at (20, 30, and 40)°C; Em was collected at (415–550) nm at an excitation wavelength of 405 nm. Adapted with permission from Ref. [174]. (c) Intracellular polarity sensor through FL images of HepG2 cells incubated with (A) DMSO + CDs (obvious FL enhancement), (B) CDs, and (C) HBSS + CDs (quenching FL) at Ex/Em of 488/(550–650) nm. Adapted with permission from Ref. [173].Temperature is also an important factor in the life process, due to the role of thermal energy transfer in all biological reactions in organisms. Faint yellow fluorescence CDs were synthesized directly from ascorbic acid aqueous solution by an electrochemical method at RT and exhibited Ex-dependent Em with a maximum Ex at 400 nm. The fluorescence of these CDs with a high quantum yield at 35.2% was stable with different pH values, ionic strengths, and storage times, and sensitive to temperature, with the fluorescence intensity changing linearly with a temperature of 20–100 °C. Human normal HEK 293T cells emitted faint yellow fluorescence under 405 nm irradiation; moreover, as the temperature increased from 20 to 40 °C or decreased from 40 to 20°C, the fluorescence intensity gradually decreased or increased, respectively. Furthermore, these CDs can especially stain the nucleolus by entering the cell nucleolus and adsorbing on nucleic acids (DNA and RNA). Thus, the CDs could act as an effective nanothermometer to monitor in vitro and in vivo temperature by utilizing the yellow-emissive nanoprobes of nucleolus specific imaging (Figure5(b)) [174].Monitoring the change of intracellular polarity plays a great role in the understanding of its functions in live cells, due to its large impact on the activity of biomolecules or the function of domains. The FL carbon dots R-CDs with a 5 nm spherical shape and good monodispersity were successfully obtained through a hydrothermal method at 200°C for 12 h using p-phenylenediamine as a precursor, displaying significant red Em under 420 nm Ex, and were then utilized as FL nanoprobes for the real-time sensing of polarity in living cells. The R-CDs possessed a high quantum yield of 47.87%, high photostability, good biocompatibility up to 400μg/mL, and dual targetability for mitochondria and lysosomes in cells. Various solutions with increasing polarity from 10 to 99 % were obtained by mixing water and dioxane based on volume ratios and showed a decrease in FL intensity of up to 11-fold. For colocalization examination of these R-CDs, human liver cancer HepG2 cells costained with R-CDs, Mito-tracker green, and Lyso-Tracker Red presented partial overlaps of the regions between the red FL of R-CDs and the Mito-tracker green or Lyso-Tracker Red areas, suggesting the preferential accumulation of R-CDs in mitochondria and lysosomes. The ability of R-CDs to monitor the polarity change in HepG2 cells by the reduction of red-emissive fluorescence with increasing polarity was successfully studied. Interestingly, the imaging of HepG2 cells and normal human liver HL-7702 cells both treated with R-CDs showed the difference between the strong red fluorescence in HepG2 cells and weak fluorescence in HL-7702 cells, discriminating the lower polarity in cancer cells with high polarity in normal cells. These R-CDs nanoprobes could contribute to mitochondrial and lysosomal polarity-related studies (Figure 5(c)) [173].


## 5. Fluorescent Carbon Dots for Bioimaging-Guided Therapeutic Application

Table3 summarizes the imaging-guided therapeutic approaches using various CDs, including individual treatment (chemotherapy, photodynamic therapy, photothermal therapy) and combined treatment. Case studies of this CD-based imaging-guided therapy are then deliberated in this section based on their biotargets and performance.Table 3
: Imaging-guided therapeutic applications using FL CDs.
Precursors/FormsFL colorApplied Ex/Em (nm)MicroscopicApplicationBiotargetRef.Bamboo leaves CDs-CBBA-DoxGreen488/–CLSMImaging of cells Imaging-guided chemotherapyHeLa[86]Hypocrella bambusaeRed540/610CLSMImaging of cellsHela[175]In vivo imaging systemImaging-guided PDT/PTT4T1 tumor-bearing miceCA, hyaluronic acid, and PEIBlue341/461CLSMImaging of cells4T1[176]CD@p-CBA-DOXIn vivo imaging systemImaging-guided CD44-targeted chemotherapy4T1 tumor-bearing BALB/c miceDopamine hydrochloride, folic acidBlue425/485FMImaging-guided PTTLNCaP[177]4-amino salicylic acidYellow490/547FMImaging of cellsMCF-7/ADR[178]In vivo imaging systemImaging-guided chemotherapyMCF-7/ADR tumor-bearing miceCA, EDA, hyaluronic acidBlue405/–CLSMImaging-guided chemotherapyA549 tumor-bearing mice[179]MSN-SS-CDHA-DoxGreen488/–In/Ex vivo imaging systemRed561/–CA, Ru-AphenRed490/616CLSMIn vivo imagingZebrafish[180]Imaging-guided PDTHeLa1,3,6-trinitropyreneGreen457/570CLSMImaging of lysosome-targeted cellsHeLa[181]In vivo imaging systemImaging-guided PDT, PTT4T1-tumor-bearing nude miceCA, folic acid, DOXBlue405/488CLSMImaging of cellsHepG2[182]Imaging-guided chemotherapyLiver cancerL-cysteine, EDA, and Ru(II) complexRed405/660Two-photonImaging of lysosome-targeted cellsA549[117]810/660CLSMImaging-guided PDTZebrafishhydroxyapatite NPsGreen455/–In vivo imaging systemImaging-guided chemotherapyTumor-bearing mice[183]C-hMOS-DoxRed523/–CA, HPAPBlue405/–In vivo imaging systemImaging-guided gene therapyMCF-7 tumor-bearing BALB/c[184]PPD@HPAP-CDs/pDNAGreen488/–nude miceRed543/–Sodium citrate, ammoniumBlue350/435FMImaging of cellsA549[185]Imaging-guided chemotherapyBicarbonate CDs/Dox/ G5–RGD–TPGS6-O-((O,O’-Di-lauroyl)tartaryl)-d-glucose, WS2@CDsCyan405/445CLSMImaging of cellsHeLa[186]Green440/477Imaging-guided PTTRed568/6404-aminophenolRed540/620In vivo imaging systemImaging-guided chemotherapyHela tumor-bearing mice[187]CSCNP-R-CQDs/DoxWatermelon juiceBlue360/–In vivo imaging systemNIR-II FL imaging-guided PTTHela tumor-bearing BALB/C mice[188]NIR-II808/925F127-mPR, formalin, and phenolBlue405/–CLSMImaging of cellsU87[189]Green458/–In vivo imaging systemImaging-guided PTTGlioma-bearing miceYellow488/–Red514/–CA, PEIRed550/640CLSMImaging of cellsMCF-7, HeLa, and 4T1[190]CD-Ce6In vivo imaging systemImaging-guided PDT/PTT4T1 tumor-bearing BALB/c miceCA, GdCl3, poly-lysineBlue350/450CLSMImaging of cellsHela[191]FA-GdN@CQDs-MWCNTsIn vivo imaging systemImaging-guided chemo/PTTHeLa tumor-bearing miceManganese(II) phthalocyanineRed690/745CLSMImaging of cellsHela[192]In vivo imaging systemImaging-guided PDT4T1 tumor-bearing micePheophytin powderRed620/680CLSMImaging of cells4T1[193]In vivo imaging systemImaging-guided PDT4T1 tumor-bearing miceCA, formamideRed620/650CLSMImaging of cellsMCF-7[194]HFn(DOX)/CDsIn vivo imaging systemImaging-guided chemo/PDTS180 tumor-bearing miceCARed633/640/750CLSMImaging of cells4T1[195]CyOH–AgNP/CDIn vivo imaging systemImaging-guided PDT4T1 tumor-bearing micePolythiopheneRed635/690CLSMImaging of cellsHeLa[196]CDs/MnO2-PEGIn vivo imaging systemImaging-guided PDT4T1-luc tumor-bearing micePolythiophene, diphenyl diselenideRed500/730, 820Two-photonImaging of cellsHeLa[197]CLSMImaging-guided PTT4T1 tumor-bearing miceIn vivo imaging systemAs fluorescence nanoprobes, CDs have been widely investigated to enhance fluorescence imaging-guided therapeutics for the better intervention of tumors. Using folic acid (FA) that shows high receptors in hepatoma cells and DOX as an anticancer drug, FA-CDs-DOX with a high fluorescence quantum yield (97%) was synthesized to enhance the antitumor activity and imaging efficiency for imaging-guided chemotherapy of targeted liver cancer.In vivo images of tumor-bearing mice after the injection of FA-CDs-DOX displayed strong orange-red fluorescence under Ex at 480 nm light, while no FL signal was observed in tumor-bearing mice treated with CDs or DOX only, indicating their ability to penetrate tumor tissue and skin to achieve FL imaging effects. This FA-CDs-DOX also exhibited significantly higher tumor inhibition in vivo through the targeting ability of FA-CDs-DOX compared to that of free DOX, suggesting the effective improvement of FL image-guided chemotherapeutic effect in liver cancer therapy (Figure 6(a)) [182].Figure 6
Fluorescence imaging-guided therapy using CDs. (a) Schematic of FL imaging-guided therapeutics of liver cancer using FA-CDs-DOX. Adapted with permission from Ref. [182]. (b) Demonstration of ultrasound-enhanced theranostics of multidrug-resistant tumors using G5-TPGS@y-CDs)-DOX drug-loaded CD/dendrimer nanohybrids. Adapted with permission from Ref. [178]. (c) (A) Therapeutic schedule of (G5-TPGS@y-CDs)-DOX and ultrasound-targeted microbubble destruction (UTMD) treatment in vivo. (B) Tumor volumes, (C) body weights, and (D) survival rate of mice after intravenous injection periods of different groups (saline, free DOX, (G5-(m)PEG@y-CDs)-DOX, (G5-TPGS@y-CDs)-DOX, or (G5-TPGS@y-CDs)-DOX + UTMD) showed the effective chemotherapy of tumors. Adapted with permission from Ref. [178]. (d) In vivo FL images of glioma-bearing mice after intravenous injection of saline and HCCDs, and ex vivo images of major organs after administration under 465 nm excitation displayed the accumulation of HCCDs in the tumor region for the effective FL imaging-guided PPT of glioma. Adapted with permission from Ref. [189].Innovative nanomedicine for the theranostics of multidrug-resistant (MDR) tumors remains a challenge for scientists. Yellow FL CDs (y-CDs)/dendrimer nanohybrids have been developed as a platform for ultrasound-enhanced fluorescence imaging and chemotherapy of MDR tumors (Figure6(b)). These nanoprobes were prepared via electrostatic interaction between y-CDs and generation 5 (G5) poly(amidoamine) dendrimers and d-α-tocopheryl polyethylene glycol 1000 succinate (G5-TPGS), then physically loaded with anticancer drug doxorubicin (DOX) to form (G5-TPGS@y-CDs)-DOX complexes with high drug loading efficiency of 40.7%. The tumor fluorescence imaging by these complexes with a strong Em peak at 550 nm was further evaluated in the presence and absence of ultrasound-targeted microbubble destruction (UTMD). Images of in vivo mice-bearing MCF-7/ADR xenografts after the intravenous injection of y-CDs and (G5-TPGS@y-CDs)-DOX complexes (with or without UTMD) displayed stronger fluorescence signals in the tumor region, even at 6 h postinjection, compared to weaker fluorescence in the y-CDs control group, due to the quick metabolization of small free y-CDs. Significant enhancement of fluorescence was observed stably in the tumor region with the assistance of UTMD in vivo, as well as ex vivo, tumor and other organs, supporting the enhancing role of the UTMD-rendered sonoporation effect. There was no obvious inhibition effect on the tumor growth of UTMD, thus bioimaging-guided treatment with (G5-TPGS@y-CDs)-DOX with the support of UTMD was further investigated, demonstrating the effective therapeutic effect of this CD-based platform for MDR tumor chemotherapy (Figure 6(c)) [178].On the other hand, multicolor highly crystalline carbon nanodots (HCCDs) were successfully fabricated for the use in dual-modal imaging-guided PTT of glioma. HCCDs exhibited optimal Ex/Em 440/506 nm with Ex-dependent full-color fluorescence, where the Em was tuned from blue to green through increasing the Ex wavelength. With high water dispersity and good biocompatibility, U87 glioma-bearing mice were treated with HCCDs for the investigation of imaging-guide PTT performance, displaying that intravenously administrated HCCDs gradually accumulated in the tumor, emitted the brightest imaging at 30 min postinjection under 465 nm Ex, and achieved the effective PTT effect under an 808 nm laser. The capacity of HCCDs to permeate the glioma and specifically accumulate in glioma cells could provide imaging-guided PTT targeted to glioma in mice, without side effects to the normal tissues (Figure6(d)) [189].The therapeutic approach of combination phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has recently shown great promise for efficient cancer therapy by inducing reactive oxygen species (ROS) or heat into tumors to kill cancer cells. The yellow FL CDs with optimal Ex/Em 500/570 nm were easily obtained by the hydrothermal treatment of 1,3,6-trinitropyrene and possessed a strong ability to simultaneously generate singlet oxygen (1O2) (QY 5.7%), hydroxyl radical (OH.), and heat effect under a 635 nm laser irradiation (73.5% photothermal conversion efficiency). Interestingly, these CDs exhibited one-photon excited (OPE) and two-photon excited (TPE) fluorescence at a peak of 570 nm and displayed green fluorescence imaging in Hela cells under excitation by both 457 and 800 nm lasers, confirming the selective accumulation of CDs in lysosomes by the overlapping fluorescence of commercial lysosome targeting dye. With good biocompatibility, in vivo photoacoustic imaging and the photothermal performances of CDs on the 4T1-tumor-bearing nude mice were studied after the intravenous injection of CDs for 4 h, showing significant enhancement of the PA signal in the tumor region and an increase in temperature up to 70°C under an 800 nm laser, suggesting that these CDs could act as lysosome-targeting phototheranostic agents for simultaneous PA and fluorescence imaging-guided PDT/PTT [181].The magneto-fluorescent CDs prepared by the hydrothermal treatment of a mixture of poly lysine, CA, and GdCl3 were decorated on carbon nanotubes (CNTs)/doxorubicin (DOX) nanocomposites, then conjugated with FA to form FA-GdN@CDs-CNTs to serve as dual-modal fluorescence/magnetic resonance (MR) imaging. These CD-based complexes possess low toxicity, good biocompatibility, and drug release ability forced by pH and NIR light and are suitable for synergistic chemo-photothermal therapy (Chemo/PTT) in cancer excision. Fluorescence images of HeLa cells treated with FA-GdN@CDs-CNTs displayed green and red fluorescence under 488 and 543 nm illumination in cell membrane and cytoplasm, but not in nuclei, indicating that this complex could internalize in cell membranes, and enter HeLa cells via endocytosis. With the support of FL imaging, the combined chemo-PTT in HeLa tumor-bearing nude mice was effective in reducing the tumor with a great contribution to tumor inhibition, without damage to major organs [191]. Overall, owing to the high tumor-inhibition efficacy and low side effects, the capability of the CD imaging-guided therapeutic was proven as a smart approach for enhanced therapeutic effects, suggesting it offered an excellent strategy for potential clinical therapeutic application.This section indicates that CDs exhibit the promising potential for FL imaging-guided therapy due to their unique properties. Compared to other CDs, the red-emissive CDs possess a more efficient capability to serve as theragnostic nanoagentsin vivo. Surface modification of CDs is essential to precisely consider for reducing the non-specific protein absorption and improving the specifically tumor-targeted ability. The cytotoxicity and perfect ability of clearance must be attentively investigated for the best therapeutic efficacy without any side effects. It is becoming necessary to develop CDs with both high FL quantum yield and synergistic chemo/PDT/PTT, suggesting that CDs could be a promising theragnostic candidate for imaging-guided therapy.


## 6. Conclusions and Perspectives

Recently, fluorescent carbon dots have greatly accelerated the advancement of bioimaging-supported nanomedicine to supply efficient biomedical applications, including the efficacy of molecular imaging that increased the image properties over the conventional FL agents, accurate monitoring of the presence of ions and molecules via imaging for biological and medical studies, and efficient treatment of tumors via FL imaging guidance. As discussed above, various reports have successfully developed multicolor FL CDs with superior properties, such as high fluorescence, excellent photostability, ease of functionalization, and good biocompatibility, that have been utilized in multiple biomedical applications, from biology to medicine. Although the advancement of CD-supported imaging-based nanomedicine holds promising potential in further clinical medicine, some critical aspects require more attention before shifting to the clinical setting.Even though the affordability of CDs was confirmed, their exploitation in imaging-supported nanomedicine is difficult to offer globally, due to the limitation that the large-scale synthesis methods of functional CDs have not been standardized. Because fluorescence is the main property of CDs for imaging-supported medicine, the high quantum yield of CDs must be obtained for the best performance of bioimaging. For example, the high quantum yield of CDs can be achieved by heteroatom doping [198]. Although research on the toxicity of CDs has confirmed their biocompatibility in vitro and in vivo, the long-term accumulation of CDs in targeted tissue or organs might cause irritation, inflammation, renal toxicity, hepatotoxicity, and immune system problems in organisms. They remain challenges for scientists to evaluate the possibility of CDs in vivo, and the release capacity of CDs out of the body, without side effects. Sufficient photostability of CDs must be ensured to analyze the stability of the FL signal for further in vivo applications.In the field of bioimaging-supported sensing platforms for abnormal metal ions and molecules, almost all reports could only obtain qualitative detection or semiquantitative monitoring in the microgram range; hence, accurate quantitative detection should be acquired to provide significant information about biological processes or the diagnosis and prognosis of diseases. Specific organelle-targetable CDs possess the ability to image the selective targets, distinguish cancerous cells from normal cells, or detect further biomarkers in the targeted organelles. Thus, the selection of optimal CDs for specific imaging-supported applications is highly required for the improvement of sensing performance. Further, the high specificity of the bioimaging-supported sensing platforms should also be improved in the presence of various interferences in the organism’s body.For FL imaging-guided therapy, the assurance of tumor-targeted specificity that will be increased through image resolution and performance is required for the tracking of tumors during the therapeutic process. This tumor specificity is attributed to the loading optimization of specific agents towards each tumor target, the stability of conjugated nanoprobes in the body, and the improvement of FL microscopic techniques through the enhancement of contrast-to-noise ratio. Overcoming the nonspecific uptake in nontargeted organs for the best treatment of tumors should be concentrated on. CDs that can generate FL signals from deeply located tissue should continue to be developed to enhance the visualization capacity of CDs in deep tumor tissue. In addition, the CDs with highly biodegradable ability must be considerably focused on to develop facile utilization in clinical medicine without side effects. Based on the ease of functionalization of CDs, combined therapeutic approaches, such as chemo/PDT/PTT, which can achieve more efficacious treatment, should be integrated. For fighting bacterial infections, CDs-integrated biofilms were successfully utilized for imaging and selectively eliminating bacteria, suggesting that the integration of CDs along with antibiotics in biofilms could exhibit better control of bacterial infections than alone [199]. Hence, CDs-integrated materials should be intensively developed for their efficacy enhancement. Furthermore, technical professionals for accurate biomedicine are also important, so that medical scientists will have sufficient medicinal skills to perform the techniques and analyze the acquired data for thorough implementation.In summary, the applications of CDs in FL imaging-based biomedicine appear to hold promising innovation for further clinical use. Although these CD-supported imaging-based biomedicines have been reported in the initial stages, their substantial potential will be achieved in clinical medicine if their possibility is successfully evaluated in clinical trials. The toxicology, pharmacokinetic, and applicable studies of these CD-based imaging-supported biomedicine concepts in the human body should be intensively researched. The development of advanced CDs for these emerging biomedicine concepts is being completed day-by-day, leading to the realization of real medical accomplishment in the near future. Due to the superior properties of CDs in the fields of imaging-based biomedicine, their enormous applications could be proficiently handled globally for the best medicinal intervention.



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