Introduction

In vitro live-cell imaging is the study of the cellular structure and function in living cells, requiring advanced technologies to record the high-resolution and spatiotemporal images of cellular events such as migration, cellular development, and intracellular trafficking. The progress of this field depends on the efforts to synthesize new fluorescent reagents, develop new microscopy techniques, and finally increase the understanding of the complex nature of cellular structures and functions [1, 2]. Fluorescent reagents used in live-cell imaging need to have specialized optical, chemical, and biological properties, such as brightness, photostability, fluorogenicity, biocompatibility, chemical stability, and up-conversion. The fluorescent probes used commonly in live-cell imaging are composed of organic dyes, quantum dots (QDs), fluorescent proteins, and CDs [3,4,5,6,7].

Currently, great progress has been made in the development of green fluorescent proteins, but the development of other colors of fluorescent proteins is in the early stages [8,9,10,11,12,13,14]. In addition, their short fluorescence lifetime and high requirements of the operation process are factors that limit their development [6]. Organic dyes are the most common markers that show high brightness in biological environments and undergo an appropriate number of switching cycles, but the photobleaching and the buffer-dependent blinking limit their applications [15,16,17,18,19,20,21,22,23]. QDs as a new nano-size material represents attractive optical properties, including very low photobleaching, high absorption coefficient, and remarkable brightness, but the defect of fast blinking is not resolved yet in these nano-particles. Additionally, high on–off duty cycle and high toxicity have reduced the QD applications [24,25,26,27,28].

CDs are carbon-based structures and belong to the fluorescent emissive nano-materials [29]. They have applied in various new technologies and medical science such as optical devices, sensors, catalysis, drug delivery, and bioimaging [30]. These nano-particles have zero-dimensional size (typically ∼5 nm), unique multicolor emission, appropriate solubility, and low toxicity, but their brilliant properties are simple and low-cost synthesis procedures. Another exciting feature of CDs is their graphitic-like core structure with the possibility of bonding to the various chemical groups, while nitrogen, sulfur, and phosphorus doping into its structure make them more attractive [31,32,33,34,35,36]. CDs are easily obtained from simple reactants and expose remarkable photostability [37, 38]. CDs can emit durable multicolor fluorescence light [39, 40] and enable the imaging of live cells via their photostability and biocompatibility [41, 42]. This nano-particle easily interacts with the cellular membranes and penetrates to the cytoplasm via the endocytosis process [43]. Dependence of fluorescence emission to the excitation wavelength is responsible for multicolor imaging of cells or organelles by the CDs [44, 45]. Furthermore, the pH of the cellular environment can affect the fluorescent emission of CDs that provide the advantage of cell pH detection by these nano-particles [46]. Hydrophilicity, chemical functional groups, and the electrical charges of the CD surface are key factors in their penetration into the cells and targeting of organelles [45,46,47,48]. All of these properties can be modulated rationally during the synthesis process and post modification of CDs [36, 49].

All these features have led the CDs to various fluorescence imaging applications. The aspects of this topic and the obtained results have been compiled and categorized in some review articles during the last years, covering the common uses of CDs in fluorescence in vivo imaging, high-resolution imaging, and intercellular organelle targeting [3, 50,51,52,53,54,55,56,57,58]. Meanwhile, the importance of in vitro live-cell imaging technologies in pharmaceuticals and the necessity of the continuous introduction of the new nano-based probes with various critical characteristics are still attractive and challenging issues in this area. Hence, this review has focused on these new possibilities and provides the latest progress in live-cell imaging using novel modulated CDs. To the best of the authors’ knowledge, no review article has been published on this topic. A list of the CD advantages and their applications in live-cell imagining are presented in Scheme 1.

Scheme 1
scheme 1

Important features, advantages, and applications of CDs in live-cell imaging

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CD properties for in vitro live-cell imaging

The properties of CDs vary according to the precursors and the synthesis method, which is very explained in various review articles [51, 57,58,59,60,61,62,63,64,65]. Thus, among the features of CDs, only important features related to in vitro live-cell imaging are briefly summarized in this section.

Red and near infrared (NIR) fluorescence

The CD fluorescent emission wavelength has a broad range from deep UV to NIR region [41, 42], with large Stokes shifts originating from the different electronic transition pathways [42]. NIR fluorescence emission is generally generated by the large rigid conjugated structure of CDs and their quantum confinement effect. In this range, due to the reduction of tissue absorption and inherent fluorescence of the biological environment, the depth of light penetration is greater. Therefore, extensive research has recently been conducted on the synthesis and preparation of long-wavelength CDs that have lowered phototoxicity in the biological transparency window [36]. For example, D-π-A-conjugated fluorine and nitrogen-doped CDs were prepared from uric acid and NH4F as doping agents via the solvothermal method. Because of the D-π-A-conjugated formation, prepared CDs show absorption peaks in the full UV–Vis–NIR spectrum. In addition to the excellent cytocompatibility and appropriate cellular uptake, the brilliant property of these CDs is their very sharp deep-red emission that confirms their competence for cell imaging applications (Fig. 1A) [66].

Fig. 1
figure 1

A Confocal laser scanning microscopy (CLSM) images of HepG2 cells treated with CDs, Reproduced with permission from ref [66]. Copyright 2020 Wiley–VCH. (B) The emission spectrum of prepared CDs excited by a 700-nm two-photon laser and two-photon image of HeLa cells incubated with CDs. Reproduced with permission from ref [68]. Copyright 2021 Elsevier. (C) CLSM images of HUVEC cells incubated with CDs taken at different excitation wavelengths: (a) bright field, (b) 403, (c) 488, and (d) 640 nm. Reproduced with permission from ref [72]. Copyright 2020 American Chemical Society. (D) CDs show only 23% decay in the period of 2 h at 365-nm irradiation [75]. (E) On–off fluorescence imaging of CDs. Reproduced with permission from ref [42]. Copyright 2018 American Chemical Society. (F) CLSM of PC-12 cells treated with CDs: (a) bright-field image and (b) confocal images taken by excited at 401. Reproduced with permission from ref [77]. Copyright 2017 American Chemical Society. (G) (a) Spectral overlap between the fluorescence spectrum of CDs and the absorption spectrum of dopamine-quinone during the FRET process, the image of MDA-MB 468 cells incubated with (b) the 10 µg/mL of CDs, and (c) after adding dopamine solution at the excitation wavelength (450–480 nm). Reproduced with permission from ref [79]. Copyright 2020 Elsevier. (H) Comparative staining with CDs and SYTO RNASelect. Reproduced with permission from ref [81]. Copyright 2019 American Chemical Society. (I) The cytotoxic effects of CDs against HeLa cells with 94% cell viability even at 500 μg/mL. Reproduced with permission from ref [87]. Copyright 2020 American Chemical Society

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Up-conversion fluorescence

Up-conversion fluorescence is often cited as an important feature in CDs. It can be detected by measuring the excitation intensity dependence of the fluorescence [67]. Multiphoton excitation provides the capability of deep-tissue penetration by using lower energy wavelength during the bioimaging process. Recently, a new class of CDs was obtained using p-aminoazobenzene as a nitrogen doping precursor with remarkable properties, including high affinity to the RNA, red fluorescence emission, two-photon excitation, and photodynamic activity. Owing to these characteristics, CDs enable real-time fluorescence monitoring of dynamic nucleolar changes during photodynamic therapy. Because of the overlapping between CD and RNA adsorption, a two-photon laser was used to stimulate CD at 700 nm (λem = 625) to prevent adsorption overlap (Fig. 1B) [68].

Multicolor imaging

In contrast with organic dyes and inorganic QDs, the emission peak of some CDs exhibits excitation-dependent behavior. In other words, the emission wavelength of CDs changes in the different excitation wavelengths that are considerable for multicolor and high-resolution imaging applications [69, 70]. This phenomenon is related to the changes in the size distribution and the number of surface defects in the CDs [71]. According to a novel report, in vitro imaging of human umbilical vein endothelial cells were done by the VEGF-conjugated CDs. The CDs were firstly obtained via the hydrothermal method and then conjugated with VEGF by carbodiimide coupling. As shown in Fig. 1C, multicolor emission was observed due to the excitation wavelength-dependent emission of CDs [72].

Photostability

Photobleaching is the biggest problem of fluorescent dyes that strongly restricts their imaging efficiency [73]. In addition, photobleaching makes storing and preparing samples difficult because it also occurs under natural light irradiation. By resistance to the photobleaching, the CDs provide longer imaging time and overcome all mentioned limitations [74]. For example, the green synthesis of CDs was prepared by using sugarcane as the sole carbon precursor. Based on this study, doping of nitrogen and phosphorus is responsible for better solubility, high quantum yield (QY), and photostability of synthesized CDs. In order to investigate the possibility of photobleaching CDs, the effect of continuous ultraviolet light was studied using a 365-nm lamp for 2 h. As shown in Fig. 1D, CDs display only 23% decay [75].

Photoblinking

Spontaneous photoblinking is the other attractive feature of some CDs that is necessary for super-resolution imaging. Usually, to prevent emission overlapping from neighboring fluorophores, a small section of the sample is isolated and selected for the imaging process. Therefore, a kind of photoswitchable fluorescent probes or devices such as interference grids is required for this purpose. Interference grid role is to excite a select population of fluorophores at the same time within one sample. Some modulated CDs are capable of switching from an “on” emission state to an “off” state under continuous illumination, so there is no need for additional adjustment [76]. Malic acid-CDs are a clear example of a photoswitchable probe that exhibited an acceptable signal-to-noise ratio in different imaging buffer solutions. Because of their photoblinking properties, this kind of CD is able to significantly enhance spatial resolution in imaging experiments. Figure 1E shows the bright spots of malic acid-CDs in the on-state were distributed throughout the imaging session [42].

Brightness

The fluorescence output per fluorophore molecule, known as brightness, is proportional to the extinction coefficient and the fluorescence QY. Lower laser intensity is needed to excite fluorophores with higher brightness, providing higher resolution imaging and lower phototoxicity for live cells. Many different approaches exist to enhance the brightness of semiconductor QDs or metal nano-particles, but controlling CD synthesis in order to achieve more brightness is significantly easy and more accessible. For example, a simple one-step approach was utilized to prepare bright multicolor CDs with QY up to 58%. These CDs were used for bioimaging of HeLa cell that showed bright confocal images (Fig. 1F). Also, CDs show a high dopamine neurotransmitter fluorometric detection ability in the blood plasma samples without any surface functionalization [77].

Stimulus-responsive fluorescence

CDs are able to change their optical properties in response to stimuli such as pH, light radiation, and analytes (gases, ions, biologically active molecules, etc.). By responding to stimuli, such materials have been used as Forster resonance energy transfer (FRET) fluorophores. The ratiometric fluorescence sensing in FRET requires two or more fluorophores with different emission bands: one of them is a reference, and others act as a response molecule [78]. By considering CD advantages in comparison with common fluorophores, CD-based FRET systems are introduced to the biosensing and bioimaging application. A recent examination has used the pyrolysis method to prepare CDs from porcine pancreatic lipase (PPL). The synthesized CDs are used as a fluorescence probe for the detection of dopamine. The tendency to oxidization into the dopamine-quinone triggered the adsorption of dopamine on the CD surface and effectively quenched the CD emission. As shown in Fig. 1G(a), the absorption spectrum of dopamine-quinone has an overlap with the emission spectrum of CDs, and this overlapping shows the occurrence of FRET. As a result, quenching of CD fluorescent emission is a sensitive sensing way of dopamine detection (Fig. 1G(b, c)) [79].

Fluorogenicity

Fluorogenicity explains the enhancement of a fluorophore emission during the interaction with its target molecule. This dramatically removes background fluorescence from the unspecific localization of probes in live cells and other biological environments [80]. For example, CDs exhibit fluorescence emission, which is able to photostable, wash-free, high-resolution, and high-quality nucleolar imaging. Most RNAs accumulate in the nucleoli that could be stained clearly by CDs. With a high affinity to the nucleolus and excellent imaging performance, CDs have exposed more advantages to the commercial nucleolar dye (Fig. 1H) [81].

Biocompatibility

Carbon is the basic element of life and constitutes the body of living organisms. Therefore, carbon-based nano-materials such as CDs are biocompatible with living organisms and different cell lines [42, 82,83,84,85]. In vivo examination confirms the nontoxic nature of CDs in zebrafish as an animal model [86]. Water-soluble fluorescent-nitrogen-doped CDs were synthesized from the lemon juice and ethylenediamine via hydrothermal methods. The fluorescence emission intensity of reported CDs depends on the pH value and shows appropriate photostability under different salt conditions. Biocompatibility and nontoxicity of these CDs were demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of the MCF7 cell lines (Fig. 1I) [87].

Applications of CDs for in vitro live-cell imaging

The obvious understanding and tracking of biochemical and physiological streams in all biological levels (biomacromolecules, cellular level, tissues, and body organs) are easily and quickly available via various cell imaging methods. The existing organic dyes and semiconductor QDs are challenged with related photobleaching, poor water solubility, and intrinsic toxicity. Due to their stable photoluminescence, multicolor emission, low cytotoxicity, and good biocompatibility, CDs are a powerful alternative for common bioimaging dyes. In the following, the last advances in CD-based in vitro live-cell imaging, including whole cell, cell organelles, pH, ion, molecule, and protein imaging, are discussed in detail (Scheme 2).

Scheme 2
scheme 2

Application potential of fluorescent CDs as a live-cell imaging probe

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Whole-cell imaging (cytoplasm imaging)

A wide variety of CDs with different optical properties are applied to cell imaging without any requirement for external transfection and specific ligand [75, 88,89,90,91,92]. The penetration mechanism of CDs into the cells are mainly consists of endocytosis, aquaporins aided entrance, and diffusion through the ion channels [93].

In an advanced report, Citrus limon was selected as a low-cost carbon source for synthesizing nitrogen-doped CDs via a hydrothermal approach. The MCF7 cells are treated with 0.025 mg/mL of obtained CDs to examine their bioimaging potency. All blue, green, and red colors luminescence are recorded separately in the cytoplasm region, revealing sufficient CD entrance to the sample cells. The brightness of stained cells with reported CDs proved their competence as a multicolor probe for whole-cell imaging [89]. Other high biocompatible nitrogen-doped CDs were prepared by the hydrothermal procedure. Figure 2 shows the blue, green, and red fluorescence images of the HeLa cells after incubation with 100 μg mL−1 of CDs under different times and excitation wavelengths. Increasing the incubation time provides more opportunities for accumulation of the CDs into the cytoplasm and causes higher fluorescent emission. This indicates the appropriate permeability of CDs across the HeLa membrane cells [87]. Nitrogen and sulfur-doped CDs were synthesized from scallion and applied as imaging agents of the A549 cells. After incubation in prepared CDs, the A549 cells were excited at 405 nm by fluorescence microscopy. According to the observation of the resulting images, the main fluorescent light comes from perinuclear regions of the cytosol. As a result, surface functional groups and the small size of reported CDs are responsible for their excellent cell permeability into the living cells [91].

Fig. 2
figure 2

Confocal fluorescence images of HeLa cells treated with prepared CDs for 2, 4, and 6 h. The 1–4 columns showed different excitation wavelengths at 405, 488, and 561 nm, Reproduced with permission from ref [87]. Copyright 2020 American Chemical Society

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Because of sufficient penetration and accumulation into various cells, the emission light of CDs is appropriately detected and pictured by fluorescent microscopy. One of the entrance ways is related to the basic molecule derivatives such as arginine. The guanidyl group in arginine has a positive charge, which can electrostatically bind with the negative components of the plasma membrane and trigger the endocytosis process [94]. In spite of some reports, there is no strong explanation about the exact mechanism of CD uptake by the cells.

Organelle imaging

The mechanism of diseases, growth, repair, and many other molecular pathways is detectable and understandable via subcellular imaging. A wide variety of organelles are targeted in this imaging field, including lysosome, nucleus, nucleolus, mitochondria, endoplasmic reticulum (ER), and lipid droplet. The main difficulties of organelles imaging are the entrance of the probe into the cell and its specific attachment to the target. Typically, the colocalization experiment with proven commercial probes such as LysoTracker Deep Red and NucRed Live 647 is performed to track the precise probe location. The common targets of subcellular imaging by using CDs are presented in the following.

Lysosome

The lysosome is a digestive organelle with a spherical shape and acidic environment in eukaryotic cells [95]. Lysosome participate in macromolecule digestion, energy balance, and angiogenesis [96, 97]. Lysosome defections are the cause of cardiovascular disease, neurodegenerative diseases, cancer, and Alzheimer’s disease [29, 98, 99]. In recent years, researchers have been continuously designing lysosome-targeting CDs [100,101,102,103,104,105].

The CDs are a fast targeting agent for lysosome imaging through clathrin-mediated endocytosis. Detecting lysosomal polarity can help to better understand the physiological and pathological processes of lysosomes. Moreover, the CDs show that an obvious fluorescence intensity decreases with the increase of polarity with the addition of dithiothreitol (DTT) that stimulates polarity changes in living cells (Fig. 3A). Therefore, the mentioned CDs were used successfully in monitoring the polarity of lysosomes [106]. New finding indicates that the morpholine group in the chemical structure of CDs serves as a targeting agent for lysosome imaging. These CDs particularly accumulate in the lysosome through the endocytic pathway and are able to long-time tracking of lysosomes in live cells. Figure 3B exposes bright orange fluorescence in the live cells when the green emission of the CDs was merged with the red emission of the LysoTracker Deep dye. In addition, the CDs show location conformity with the LysoTracker Deep Red during lysosome staining [45]. New CDs prepared from the rose bengal and polyethyleneimine via one-step hydrothermal procedure amazingly possess an intrinsic affinity to the lysosome. The image (Fig. 3C) shows the rapid entrance of prepared CDs into the cells within 10 min. Moreover, they accumulated in the lysosomes organelle and emitted strong localized fluorescence after the following 30 min. Long-term imaging, lack of leakage, ultrahigh photoluminescence efficiency, and low cytotoxicity are excellent properties of these CDs as a proven alternative for common commercial probes [107]. The CDs were prepared from selenocysteine as a precursor using the hydrothermal method and then modified with morpholine. Obtained CDs prevent lysosomes from the high amount of •OH and are introduced for treating •OH-related inflammation [108].

Fig. 3
figure 3

A CLSM images of HeLa cells treated with CDs and CDs + DTT, Reproduced with permission from ref [106]. Copyright 2020 American Chemical Society. (B) The comparative images of CDs and Lyso-Tracker Red probe in live HeLa cells. Reproduced with permission from ref [45]. Copyright 2017 American Chemical Society. (C) Lysosomal imaging of the CDs in the HL-7702 cell line. Reproduced with permission from ref [107]. Copyright 2020 American Chemical Society

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Briefly, CD modification with morpholine and amine groups is utilized for specific lysosome targeting. However, amino groups show affinity to the ER that faced its specificity to the lysosome with unresolved challenges. The cells uptake CDs through energy-dependent, micropinocytosis, caveolae-mediated, and clathrin-mediated pathways. CDs with various functional groups can be transiently localized in lysosomes.

Nucleus

The nucleus is the center of cellular metabolism, activity, and many disorders such as cancer. Detail imaging and staining of the nucleus are one of the most important biological and clinical issues because our knowledge about the nucleus is incomplete for its desired manipulation. Specific imaging of the nucleus is necessary for targeted gene therapy and drug delivery, especially cancer therapy. In addition to its potential for drug loading and drug delivery, the CDs have been found to be applicable in nucleus imaging as a specific and stable fluorescent probe [109,110,111,112].

The high water-soluble and nontoxic CDs are synthesized by a hydrothermal approach for bioimaging applications. The proposed mechanism for the nucleus targeting potential of reported CDs (Fig. 4A) is related to its positive surface charge, which creates electrostatic interactions with DNA macromolecules existing in the nucleus [109]. In some cases, the physicochemical environment of the cell (L929 cells) causes a blue shift in the fluorescent emission of CDs. The lowest and highest intensity of CDs was observed in cytosol and nucleus (green and red color), respectively (Fig. 4B) [110]. For the first time, an electrostatic assembly between positive CDs and negative graphene oxide (GO) provides a new biocompatible probe of the nucleus without significant affinity to the cytoplasm. GO nano-sheets do not quench CD fluorescent emission after assembly. These electrostatic interactions of CD and GO moderate the electrical charge of the final nano-structure, which results in selective cell nucleus labeling [111].

Fig. 4
figure 4

A Fluorescence images of A549, HEK293, and MDA-MB-231 cells treated with CDs, fluorescence signal accumulated in nuclear substance, especially in A549 and MDA-MB-231 cells, Reproduced with permission from ref [109]. Copyright 2021 Elsevier. (B) The nuclei of NIH/3T3 cells filled with CDs (red color). False colored maps of intensity. Reproduced with permission from ref [110]. Published under a CC BY license

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There are no particular and proven methods for selective labeling of the nucleus by using CDs. According to the previous report, the zwitterionic surfaces of CDs can specify their attachment to the nucleus. In a more complex way, surface functionalization of CDs is a certain approach of nucleus targeting, but this mechanism is also not precisely characterized and understood. It is obvious that the utility of CDs in nucleus imaging is in its beginning steps and is a very attractive field for interested researchers.

Nucleolus

Ribosomes synthesize all proteins needed by the cell that the nucleolus is responsible for the production of ribosomes [113]. Proteins are the functional form of cellular molecules. Therefore, the imaging of the nucleolus provides an overview of the cellular status and metabolism [114]. Immunohistochemistry (IHC) [115], fluorescence in situ hybridization (FISH) [116], and silver staining [117] are the most common methods for morphological evaluation of nucleolus that are served for fixed cell lines. Although organic fluorophores such as SYTO RNA [118] are utilized in both fixed and living cell imaging [119], they suffer from photobleaching during the nucleolus imaging process. Recent reports confirm the competence of CDs as wash-free, high-resolution, and high-quality nucleolar imaging probe with a high affinity to the nucleolus (Fig. 5A) [81]. Doping of fluorine element into the CD structure creates a new nucleolus and tunneling nano-tube (TNT) staining agent with the high photoluminescence quantum yield (56%), low toxicity, anti-photobleaching, and good water solubility (Fig. 5B) [120].

Fig. 5
figure 5

A The confocal image of the nucleolus of A549 (I, II, and III) cells targeted by CDs, Reproduced with permission from ref [81]. Copyright 2019 American Chemical Society. (B) CLSM images of living 4T1 cells treated with CDs, the nucleolus (left), and tunneling nano-tubes (right) were visualized with sharp fluorescent signals. Reproduced with permission from ref [120]. Copyright 2019 Springer

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In summary, the affinity of CDs to interaction with nucleolus RNAs and ribosomes is the main reason for their application in nucleolus labeling [68, 120], which is related to noncovalent interactions. Although the subcellular labeling, the use of CDs in critical nucleolus diagnostic such as immune activation processes, transformation processes, and chemo-drug treatment are not reported, which are very attractive to examination.

Mitochondrion

The mitochondrion is the factory of energy production in mammalian cells by the creation of adenosine triphosphate (ATP) as a common form of energy in live cells [121]. Therefore any defect in mitochondrion causes significant disorders such as cardiac dysfunction, Alzheimer’s, and Parkinson’s diseases [122]. The mitochondrion has an internal negative charge, positive surface charge, and an internal alkaline environment (pH = 8) [123, 124]. Hence, two strategies are possible for specific imaging of mitochondrion: one is a modification with triphenylphosphonium (TPP) or targeting peptides, and the other way is to synthesize lipophilic labels containing positive surface groups such as ammonium, pyridine, and rhodamine [51].

The TPP ligands are practically utilized for the improvement of CD affinity to the mitochondria [125]. In spite of appropriate targeted imaging of mitochondria using these CDs, TPP shows high toxicity against various cell lines. On the other side, some works are in progress to obtain CDs with intrinsic mitochondrial targeting property [42, 123, 126]. Long-term photostable, water-soluble, mono-sized, and biocompatible CDs were synthesized with intrinsic mitochondrial affinity and tunable long-wavelength fluorescence emission from green to red colors. A high-resolution mitochondrial image of HeLa cells was obtained using as-synthesized CDs (Fig. 6A) [127]. The fast and simple microwave-assisted method was applied as a synthesis method of new excellent CDs with shuttling label features between the mitochondria and the nucleolus. These positive CDs prefer to accumulate in mitochondria with negative membrane within the healthy cells. However, in the case of damaged cells, such as inhibition of the cell metabolism by reactive oxygen species (ROS) agents such as H2O2, the negative charge of mitochondria decreases, and CDs move out from mitochondria and localize into the nucleolus. Amazingly, if the damaged cell succeeds in recovering itself in the presence of powerful antioxidants such as ascorbic acid (AA), the negative charge of mitochondria will recover too, which triggers the return of CDs to the mitochondria (Fig. 6B). In this way, the viability of a particular cell can be easily visualized [128].

Fig. 6
figure 6

A Confocal images of HeLa cells incubated with green, yellow, orange, and red CDs, Reproduced with permission from ref [127]. Copyright 20,193 Wiley–VCH. (B) Shuttling of CDs between mitochondria and the nucleolus, (top) schematic and (bottom) fluorescent images of HeLa cells. Reproduced with permission from ref [128]. Copyright 2020 American Chemical Society

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Endoplasmic reticulum

The main functionality of ER in the cell is the formation of proteins, transporting of protein, synthesis and storage of lipids, storage and regulation of calcium, and glucose metabolism. Therefore, ER plays a vital role in cell metabolism and regulation [129]. Polarity is one of the most important properties of healthy cells, and it is responsible for lots of cellular functions and metabolism. ER can affect cell polarity and consequently cell functionality via protein post translational modification, the transformation of particular proteins, and the synthesis of secretory proteins. As a result, any disorder in ER polarity can be a significant symptom of human diseases such as diabetes and Alzheimer’s [130]. Accordingly, clear imaging of ER provides proven information about cell functionality and human diseases.

Dual emissive CDs for ER labeling are obtained from o-phenylenediamine (OPD) and lysine. Adding lysine into the flask of OPD carbonization reaction strongly suppressed the reaction and caused emission shifting from green to blue. The synthesized CDs have entered the ER lumen via lipid raft-mediated endocytosis pathway because of their lipophilicity and electrophilicity. Introduced CDs are responsive to the changes of ER polarity in living cells that indicate their potential to the imaging of the ER stresses [131].

Briefly, CDs can electively visualize ER and provide sufficient morphological information. More information about the activity of ER and other organelles can be obtained via the aggregation and fluorescence responses of the CDs inside ER to environmental differences, including pH values within these organelles.

Lipid droplet

Lipid droplets (LDs) are a hydrophobic core made from lipid covered by a monolayer of phospholipid membrane [132]. LDs roles in the cell are lipid storage, membrane synthesis, and transportation, so it is supposed to be related to lipid metabolic diseases such as obesity, atherosclerosis, and cancer [133]. Consequently, imaging lipid droplets with CDs is an advantageous method of tracking cellular lipid metabolism and related diseases.

The biocompatible and amphiphilic CDs were synthesized via the hydrothermal method for LD imaging. The high cell viability, photostability, intracellular retention ability, and long-term ability to LD tracking are the highlight properties of prepared CDs. After six passages, selective fluorescence CDs can still be observed in LDs in the cytoplasm of the cells (Fig. 7A). The imaging of autophagy in the catabolic process and the effect of atorvastatin (a typical drug for lowering lipid levels) on hepatocyte cells are accomplished by reported CD [134]. The CDs with the ability of intrinsic LD targeting are obtained from 4-piperidinoaniline (PA) as a lipophilic reactant. By using these CDs, the dynamic behavior of LDs and also fatty liver disease-like changes in LD are visualized (Fig. 7B) [135].

Fig. 7
figure 7

A Confocal images of LoVo cells treated with CDs at different passages. CDs: excitation at 405 nm, Reproduced with permission from ref [134]. Copyright 2021 American Chemical Society. (B) Monitoring of the LD movement by PA CDs: the movement of LDs at 0, 30, 60, and 90 s is illustrated by four different colors, and the merged of all images. Reproduced with permission from ref [135]. Copyright 2021 American Chemical Society

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Detecting and imaging of intracellular pH

Similar to all chemical reactions, intracellular pH values play a central role in cellular functions, including metabolism pathways, growth, proliferation, apoptosis, and disease. A little variation in the cellular pH can be a signal of particular metabolic reactions, disorders, and diseases such as Alzheimer’s, cancer, and stroke [136, 137].

Application of common nano-size pH probes such as organic dyes and QDs [138,139,140] are restricted by their disability to cell entrance and high toxicity. The CDs are new alternatives for overcoming all previous obstacles [141, 142]. A biocompatible, tunable, and multicolor CD was prepared from ammonium persulfate, glucose, and ethylenediamine via the hydrothermal method. Recently, designed CDs could be used as a fluorescence platform to monitor pH fluctuations in living cells. The HepG2 cells labeled with these CDs emitted multi-color fluorescence at pH 5.0. As the pH increases from 5 to 9, the fluorescent brightness labeled cells decreases. Therefore, the changes in the brightness of CDs in the living cells can be translated to the intracellular pH variation. The prepared CDs are able to easily penetrate into the living cells and simultaneously visualize the pH statements of the cells (Fig. 8A) [136]. Also, the pH-responsive nitrogen- and chloride-doped CDs were synthesized by choline chloride and glycerin as an intracellular pH sensing probe (Fig. 8B) [143]. Another hydrothermal synthesis of nitrogen-rich CDs as pH sensor from melamine and triethanolamine was recently reported. A wide range of sensing, low cell cytotoxicity, and excellent biocompatibility were the remarkable properties of prepared CDs that confirm via imaging of pH sensing in live T24 cells (Fig. 8C) [144].

Fig. 8
figure 8

A Confocal images of HePG2 cells treated with CDs at pH (5.0, 7.0, 9.0), Reproduced with permission from ref [136]. Copyright 2020 Elsevier. (B) Confocal images of HeLa cell incubation with CDs at pH (5.37, 6.38, 7.38, 8.34, and 9.57). Reproduced with permission from ref [143]. Copyright 2018 American Chemical Society. (C) Confocal images of T24 cells treated with CDs at pH (5.0, 7.0, 9.0). Reproduced with permission from ref [144]. Copyright 2016 Elsevier

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In spite of the proposed mechanism of pH imaging by CDs, including protonation or deprotonation, energy level variation, particle aggregation, protective shell, and proton transfer, there is a priority to exact explanation about this phenomenon [145].

Detecting and imaging of intracellular ion

Heavy metal ions are toxic to life in nature, but the main concern about heavy metals relates to their unknown and intricate effects on living organisms. So, any method of detecting this heavy metal ion has its own priority for researchers [146, 147]. Functionalized CDs are one of the recent tendencies to metal ion detection [49, 148]. The attractive CDs with high sensitivity to Fe3+ were obtained via microwave pyrolysis within just 4 min. As an intrinsic fluorescence probe, CDs practically visualize intracellular Fe3+ in live HeLa cells (Fig. 9A) [149]. Also, the CDs prepared from microalgal biomass are a candidate as potent endogenous imaging labels of Hg (II)- and Cr (VI)-contaminated live cells (Fig. 9B) [150].

Fig. 9
figure 9

A Bright-field transmission images and their corresponding fluorescent microscopy images of HeLa cells that were incubated with CDs and then Fe3 + , Reproduced with permission from ref [149]. Copyright 2018 Springer. (B) Cell imaging of HEK-293 cell line after treatment of 50 µg/mL of CDs, 100 µM Hg (II), and 100 µM Cr (VI). Reproduced with permission from ref [150]. Copyright 2018 Elsevier

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The reported mechanisms of heavy metal detection by CDs are coordination/complexation, inner filter effect (IFE), light-induced electron transfer (LET), and ion aggregation [151]. CDs have a great potential for visualization of other heavy metal ions such as Cr and As.

Detecting and imaging of intracellular small molecule

High-resolution imaging of small cellular molecules such as glutathione and dopamine always provides more details about biochemical pathways, the mechanism of cellular activity, and the pathophysiology of disease progression [152, 153]. The new NIR fluorescence CDs can be directly used for the quantitative evaluation of H2S. More surprising, this kind of CD could specifically be used for imaging of the cell membrane H2S transporting. As shown in Fig. 10A, the MCF-7 cells incubated only with CDs emit a bright red fluorescence from the cell membrane, while the fluorescence intensity of the NIR channel decreases obviously in the presence of the H2S. In addition to the labeling of the cell membrane by CDs, more detail about the signal pathways and cell–cell communications are accessible by visualization of gaseous signal molecule transportation across the cell membrane. The mechanism of fluorescent quenching and affinity to the H2S by CDs are not illustrated yet [154]. Simple and fast preparation of CDs from L-tartaric acid and triethylenetetramine was examined via the microwave-assisted method. Fluorescent quenching is the mechanism of metronidazole (MNZ) detection by these biocompatible CDs. Accordingly, CDs are a potent tool for cell imaging and MNZ detection (Fig. 10B) [155]. Also, CD-based fluorescent nano-probes for intracellular biothiols (cysteine, homocysteine, and glutathione) imaging in living cells were introduced due to their appropriate biocompatibility [156, 157].

Fig. 10
figure 10

A Red channel images (λex = 640 nm) of MCF-7 cells incubated with CDs (top) and CDs + NaHS (sodium hydrosulfide) (bottom), Reproduced with permission from ref [154]. Copyright 2021 Elsevier. (B) CLSM images of MCF-7 cells treated with CDs in with or no MNZ. Reproduced with permission from ref [155]. Copyright 2020 Elsevier

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Detecting and imaging of intracellular protein

The imaging of protein distribution and tracking of their cellular movement is necessary for a complete understanding of biological pathways and disorders [158]. Conjugation of Au nano-rods (AuNR) and red-emissive CDs (RCDs) provides a specific fluorescent label for caspase-1 activity assay (Fig. 11A). The emission of fluorescent energy occurs by CD and adsorbs by the Au nano-rods via surface energy transfer (SET) phenomena. The obtained fluorescent probe shows high sensitivity, specificity, and stability for in situ monitoring and imaging of the caspase-1 activity level in the living cells (Fig. 11B) [159]. As an imaging agent, CDs show selective emission response to cytochrome C (cyt-c) via an unclear mechanism. The HeLa cells were incubated with synthesized CDs and dexamethasone for various times. The dexamethasone triggers cyt-c releasing from the mitochondria and simultaneously the beginning of the apoptosis process, which could be monitored by confocal fluorescence imaging (Fig. 11C) [143].

Fig. 11
figure 11

A Schematic synthesis procedure of the caspase-1 and its response mechanism of the AuNR-Pep-RCD nano-probe. (B) Confocal images of MCF-7 and RAW264.7 cells treated with AuNR-Pep-RCD. The red color is related to the nano-probe response to active caspase-1, and the blue color comes from Hoechst 33,342 for cellular nucleus staining, Reproduced with permission from ref [159]. Copyright 2021 Elsevier. (C) Confocal fluorescence images of HeLa cells incubated with CDs for 5 h followed by further incubation for 0, 8, 16, and 24 min in the presence of dexamethasone. Reproduced with permission from ref [143]. Copyright 2018 American Chemical Society

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Table 1 shows precursors, emission color, size, biocompatibility, and important features and targets of CDs for live-cell imaging reported in the literature. In this table, we have tried to summarize the latest published articles in this field during the last few years.

Table 1 An overview of the use of different CDs for in vitro live-cell imaging
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Conclusion and future perspective

Cellular imaging is intricate and expensive, but it is one of the most valid techniques for understanding metabolic pathways and the mechanism of disease. The unlimited amounts of biological molecules are synthesized, modified, and transformed per second in the cells, interfering in targeted chemical labels. CDs can answer the highly variable and streaming nature of cells by the possibility of preparation by unlimited and cheap precursors from pure citric acid to contaminant waste water. Therefore, a wide variety of surface chemical groups and surface modifications are available in the designation of CDs as a selective probe for cellular organelles and bioactive molecules. High-resolution imaging, stable fluorescent emitting, ability to cell penetration, surface multifunctional capabilities, biocompatibility, low-cost synthetic, and low toxicity are the additional advantages of CDs against the common dyes and nano-probes. The newest progress in in vitro live-cell imaging based on CDs, including the whole cells, cell organelles, pH, ions, molecules, and proteins imaging, are elaborately presented in this review. However, the fundamental mechanism of selective behavior of CDs against a particular organelle or biomolecule is not clearly reported. Undoubtedly, the future progress in cell imaging depends on the illustration of the mechanism of cellular and subcellular specific imaging by various chemically CDs. In spite of attempts to improve the targeting ability of CDs via post surface modification such as chemical conjugation with Au nano-rods, researchers are tending to achieve this goal by altering simple and low-cost reactions to synthesize CDs with intrinsic affinity to a particular target.