Application of Quantum Dots in Biological Imaging

Quantum dots (QDs) are a group of semiconducting nanomaterials with unique optical and electronic properties. They have distinct advantages over traditional fluorescent organic dyes in chemical and biological studies in terms of tunable emission spectra, signal brightness, photostability, and so forth. Currently, the major type of QDs is the heavy metal-containing II-IV, IV-VI, or III-V QDs. Silicon QDs and conjugated polymer dots have also been developed in order to lower the potential toxicity of the fluorescent probes for biological applications. Aqueous solubility is the common problem for all types of QDs when they are employed in the biological researches, such as in vitro and in vivo imaging. To circumvent this problem, ligand exchange and polymer coating are proven to be effective, besides synthesizing QDs in aqueous solutions directly. However, toxicity is another big concern especially for in vivo studies. Ligand protection and core/shell structure can partly solve this problem. With the rapid development of QDs research, new elements and new morphologies have been introduced to this area to fabricate more safe and efficient QDs for biological applications.


Introduction
Semiconductor nanocrystals, or so-called quantum dots (QDs), show unique optical and electronic properties, including size-tunable light emission, simultaneous excitation of multiple fluorescence colors, high signal brightness, long-term photostability, and multiplex capabilities [1][2][3][4].Such QDs have significant advantages in chemical and biological researches in contrast to traditional fluorescent organic dyes and green fluorescent proteins on account of their photobleaching, low signal intensity, and spectral overlapping [5][6][7].These properties of QDs have attracted great interest in biology and medicine in recent years.At present QDs are considered to be potential candidates as luminescent probes and labels in biological applications, ranging from molecular histopathology, disease diagnosis, to biological imaging [8][9][10].Numerous studies have reported the use of QDs for in vitro or in vivo imaging of sentinel lymph nodes [11][12][13][14][15][16][17], tumor-specific receptors [18][19][20], malignant tumor detectors [21], and tumor immune responses [22].
However, the major concerns about potential toxicity of II-IV QDs (such as CdTe and CdSe) have cast doubts on their practical use in biology and medicine.Indeed, several studies have reported that size, charge, coating ligands, and oxidative, photolytic, and mechanical stability, each can contribute to the cytotoxicity of cadmium-containing QDs.Another critical factor that determines the cytotoxicity of QDs is the leakage of heavy metal ions from the core caused by photolysis and oxidation [23][24][25].The long-term effects of these ions enrichment and durations within the body raise concerns about the biocompatibility and safety of QDs [24,26].On the other hand, the rapid clearance of QDs from the circulation by the reticuloendothelial system (RES) or entrapment of QDs in the spleen and liver [27,28] can result in the degradation of the imaging quality of the objective and the enhancement of background noise.
One promising solution to these problems, including QDs sequestered in the liver/spleen and long-term retention in the body, is to evade the recognition and uptake by the reticuloendothelial system thus extending the circulation time of the particles in the body.In addition, for biological studies, QDs should be aqueous soluble in order to adapt the biological environment.Therefore, when QDs are employed in biological imaging, many factors should be considered and the system should be properly designed in order to meet a set of requirements [29][30][31].
In this report, we first briefly review different types of QDs that have been fabricated so far and their synthesis methods, including surface functionalization.Next, we focus on their applications in biological imaging.Finally, the new trends of QDs imaging are discussed.

Types and Characteristics of QDs
Traditionally, the typical QDs consist of a II-IV, IV-VI, or III-V semiconductor core (e.g., CdTe, CdSe, PbSe, GaAs, GaN, InP, and InAs; Figure 1) [3,32,33], which is surrounded by a covering of wide bandgap semiconductor shell such as ZnS in order to minimize the surface deficiency and enhance the quantum yield [5,34].The unique optical and electronic properties of QDs, including narrow light emission, wideband excitation, and photostability, provide them with significant advantages in the applications of multiplexed molecular targets detection [21] and as optical bioimaging probes [8][9][10].One major drawback which severely hinders the application of II-IV, IV-VI, or III-V QDs for in vivo biological research is the concern about the toxicity associated with the cadmium, lead, or arsenic containing QDs.The toxic heavy metal ions can be easily leaked out into biological systems if the surfaces are not properly covered by the shells or protected by ligands.
The limitation of heavy metal-containing QDs stimulates extensive research interests in exploring alternative strategies for the design of fluorescent nanocrystals with high biocompatibility.In this case, the practical strategy is to develop highly fluorescent nanoparticles based on nontoxic elements or explore luminescent π-conjugated polymer dots.
Silicon nanoparticle is another type of QDs which possess unique properties when their sizes are reduced to below 10 nm.Similar to their predecessors, silicon QDs also have many advantages over traditional fluorescent organic dyes [35], including resistance to photobleaching and wide emission range from visible to infrared region with relatively high quantum yields.Moreover, owing to silicon's nontoxic and environment-friendly nature, Si QDs are used as fluorescent probes for bioimaging [35].It has been reported that for in vivo applications, Si QDs mainly degrade to silicic acid which can be excreted through urine [36], and for in vitro use, Si QDs are considered 10-times safer than Cd-containing quantum dots (Figures 2 and 3) [37].However, the challenges present in employing Si QDs in bioimaging arise from their water solubility and biocompatibility.Usually, Si QDs are produced in nonpolar solvents with hydrophobic ligands (such as styrene and octene) on their surface in order to protect the Si cores.Therefore, it is a common problem that Si QDs show poor solubility and unstable photoluminescence (PL) in aqueous solutions [38][39][40].PL degradation can occur gradually after modified Si QDs are transferred from organic solvents to aqueous solutions.Thus, fabricating good water-disperse Si QDs with stable optical characters is vital to their applications in bioimaging studies.Recently, Tilley and Yamamoto reported that water-soluble Si QDs covered by allylamine [41,42] and poly(acrylic acid), respectively, had been successfully obtained and showed good performance in fixed-cell labeling [40].
The semiconducting properties of conjugate polymers derive from their π-electron delocalization along the polymer chain.Mobile charges are allowed to move between π and π * orbitals.Highly bright fluorescence can be stable under one-photon or two-photon excitation.Polymers with such structures are promising in the fields of LED and biosensors [43,44].Burroughes and coworkers first reported the luminescent conjugated polymers in 1990 [45].Later on, Friend and So successfully applied π-conjugated polymers to light-emitting devices [46,47].Recently, related works focused on fluorescent polymer encapsulation and functionalization to yield conjugated polymer dots (CP dots) for bioimaging and probes (Figure 4) [48,49].Compared with other fluorescent probes, CP dots are convenient for fluorescence microscopy and laser excitation because of their absorption ranging from 350 to 550 nm.Also, fluorescence quantum yields can be slightly variable around 40%.Moreover, CP dots can exhibit higher brightness than any other nanoparticles of the same size under certain conditions [50].

Synthesis Methods of QDs
3.1.Organometallic Method.Organometallic chemistry provides an efficient pathway to produce monodisperse QDs in nonpolar organic solvents, such as CdSe nanocrystals covered by tri-n-octylphosphine oxide (TOPO) ligand.In a typical preparation, Me 2 Cd and Bis(trimethylsilyl)selenium ((TMS) 2 Se) can be used as organometallic precursors.Monodisperse CdSe with uniform surface derivatization and regularity in core structure can be obtained based on the pyrolysis of organometallic reagents by injection into a hot coordinating solvent between 250 • C to 300 • C [51].The adsorption of ligands results in slow growth and annealing of the cores in the coordinating solvents.QDs with different sizes can be successfully obtained at different temperatures.The advantage of this method is obvious because this method can produce a variety of QDs with high quantum yields and the size distribution can be easily controlled by altering temperature or reaction time.Organometallic method is currently considered as the most important means to synthesize QDs.
In this process, ionic perchlorates such as Cd(ClO) 4 •6H 2 O and Al 2 Te 3 are used as the precursors.In the presence of ligands such as 3-mercaptopropionic acid (3-MPA), glutathione (GSH), or other hydrosulfyl-containing materials, CdTe QDs can be successfully obtained in aqueous medium.The advantage of this method lies in its environment-friendly synthesis procedure and relatively low cost.QDs produced by this method can be directly applied in biological researches without ligand exchange procedure.However, compared with the QDs produced by organometallic method, thiolcapped QDs show low quantum yields, and broad full width of half-maximum (FWHM), which can be attributed to its wide size distribution and poor stability in aqueous solution.

Hydrothermal Method/Microwave-Assisted Method.
In order to reduce QDs surface defects generated during the growth process, these two methods are put forward to synthesize QDs with narrow size distribution and high quantum yields.According to hydrothermal method, all reaction regents are loaded in hermetic container and then heated to supercritical temperature; high pressure produced by the temperature can efficiently reduce reaction time and surface defects of QDs [53].Moreover, because high reaction temperature can separate the processes of nucleus formation and crystal growth, this method has clear advantages over traditional aqueous synthesis, affording narrow size distribution QDs.
Microwave-assisted irradiation is another attractive method for QDs synthesis.This method was first introduced by Kotov group [54] and followed by Qian and coworkers to demonstrate a fast and simple synthesis of CdTe and CdSe-CdS QDs by microwave-assisted irradiation in 2006 [55].Microwave irradiation can be considered as heating source  to optimize synthesis conditions, and in this system, water is an excellent solvent for its tan δ is equal to 0.127.The whole solution can be quickly heated to above 100 • C to yield homogeneous QDs and the quantum yields can reach as high as 17%.

Toxicity, Solubilization, and Functionalization of Quantum Dots
In order to apply QDs to biological studies, safety, and biocompatibility are the issues that must be concerned.Toxicological studies have suggested that certain potential adverse human health effects may be resulted from their exposure to some novel nanomaterials, such as gold nanoparticles and QDs, but the fundamental cause-effect relationships are relatively obscure [23].Thus, it is necessary to figure out the interaction of QDs with biological systems and their potentially harmful side effects in cells.Several studies reveal that the toxicity of QDs depends on many factors which can summarized as inherent physicochemical properties and environmental conditions [23,24,26].Not all QDs are alike, each type of QDs should be characterized individually as to its potential toxicity.QD sizes, charges, concentrations, outer coatings materials and functional groups, oxidation, and mechanical stability all have been implicated as contributing factors to QD toxicity [24].Derfus et al. studied the cytotoxicity of CdSe QDs and pointed that without coating, the cytotoxicity of CdSe cores correlated with the liberation of free Cd 2+ ions due to deterioration of QDs lattice.And in vivo, the liver is the primary site of acute injury caused by cadmium ions even though the concentration of Cd 2+ is reduced to 100-400 μM [23].In order to eliminate QD toxicity resulted from heavy metal ions releasing, surface coating and core/shell structure are considered to be efficient solutions to this problem.Many new types of QDs such as core/shell (CdSe/ZnS) and core/shell/shell (CdSe/ZnS/TEOS) QDs with high luminescence are developed [34,35].Some researchers also found that the quantum size effect which resulted from the minute size of nanomaterials could lead to cytotoxicity.Since nanoparticles within certain diameters (this value may be different according to biological environment) are of similar size to certain cellular components and proteins, they may bypass natural mechanical barriers, thus leading to adverse tissue reactions [26].
The application of QDs in biological imaging also requires QDs with good water solubility.Several strategies have been developed to circumvent this problem, which are summarized as follows.
The first one is to synthesize QDs in aqueous solution directly, which has been depicted in this review (vide supra).The second is to obtain QDs in organic solvents, and then transfer them to aqueous solutions (Scheme 1).Nie and Weiss groups pioneered the preparation of water-dispersed QDs by ligand exchange method (Figure 4) [5,56].In addition, polymer and lipid coating is another efficient way to render QDs with good water solubility [57,58].
To improve in vitro and in vivo imaging quality and detection sensitivity in biological fields, multiplex capabilities QDs have been developed.Recent efforts mainly focus on the functionalization of QDs to accommodate the demands of imaging sensitivity and specificity [59].Nie reported multicolor QD-antibody conjugates for the rapid detection of rare Hodgkin's and Reed-Sternberg (HRS) cells from infiltrating immune cells such as T and B lymphocytes [60]; Shi combined Fe 3 O 4 with amine-terminated QDs to yield fluorescent superparamagnetic QDs for in vivo and in vitro imaging (Figure 5) [61].

QDs in Biological Imaging
In order to apply QDs in biological imaging, recent studies have focused on developing near-IR luminescent QDs which exhibit an emission wavelength ranging from 700 to 900 nm.Light within this range has its maximum depth of penetration in tissue and the interference of tissue autofluorescence (emission between 400 nm and 600 nm) is minimal [62,63].In addition, for the purpose of gathering more information during specific cellular processes in real time, QDs must be conjugated with molecules which have the capabilities of recognizing the target.These surface modifications can also help prevent aggregation, reduce the nonspecific binding, and are critical to achieving specific target imaging in biological studies.Further functionalization of QDs can be achieved by the modification of protecting ligands [64][65][66], introducing targeting groups such as apolipoproteins and peptides (Scheme 1) [64].In this part, we mainly introduce the recent development of conjugated QDs for in vitro and in vivo imaging; some new QDs related to bioimaging are also included.

In Vitro Imaging. Early studies of in vitro imaging used
QDs to label cells.For instance, PbS and PbSe capped with carboxylic groups had been obtained in aqueous solution by ligands exchange to label cells [67]; CdTe capped with 3mercaptopropionic acid (3-MPA) was used as imaging tool to label Salmonella typhimurium cells [68].Jaiswal et al. reported labelling HeLa cells with acid-capped CdSe/ZnS QDs [69].It is found that the cells can store QDs in vesicles through endocytosis after washing away the excess QDs.Further, they also demonstrated the potential application of QDs in cell tracking by using avidin-conjugated QDs to label cells.With the development of biomarkers in cell biology, the tracking of some specific cells (such as cancer cells) becomes possible.Gac et al. have successfully detected apoptotic cells by conjugating QDs with biotinylated Annexin V, which enables the functionalized QDs to bind to phosphatidylserine (PS) moieties present on the membrane of apoptotic cells but not on healthy or necrotic cells (Figure 6) [70].The detection and imaging of apoptotic cells makes it possible to monitor specific photostable apoptosis.Wolcott reported silicacoated CdTe QDs decorated with functionalized groups not only for labelling proteins, but also for preventing toxic Cd 2+ leaking from the core [71].Nie developed cellpenetrating QDs coated with polyethylene glycol (PEG) grafted polyethylenimine (PEI), which were capable of penetrating cell membranes and disrupting endosomal organelles in cells [72].Recently, in the demand of using biocompatible and nontoxic QDs as nanoprobes, rare earth (RE) elements are used to fabricate a new type of QDs, such as Gd-doped ZnO QDs.RE-doped QDs have distinct advantages over heavy metal-containing QDs, not only because of avoiding the increase of particle size by polymer or silica coating in synthesis procedure, but also providing a simple, green synthesis method.Liu et al. reported the development of Gd-doped ZnO QDs with enhanced yellow fluorescence, and these QDs can be used as nanoprobes for quick cell detection with very low toxicity [73].Other progresses include CdSe/ZnS with PEG coating and CdSe/ZnS-peptide conjugation for cytosol localization and nucleus targeting [74][75][76][77], CdS passivated by DNA to yield nontoxic QDs as new biological imaging agent [78], CdSe/L-cysteine QDs designed to label serum albumin (BSA) and cells [79], polymer QDs (PS-PEG-COOH) with conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG) to label cell surface receptors and subcellular structures in fixed cells [21,80], and other non-Cd-containing QDs such as Ge-QDs and near-IR InAs for imaging of specific cellular proteins [81,82].
In order to broaden QDs applications, MRI detectable QDs were demanded.Many studies have set out to explore paramagnetic QDs.Sun reported a new kind of magnetic fluorescent multifunctional nanocomposite which contained silicacoated Fe 3 O 4 and TGA-capped CdTe QDs.This nanocomposite was successfully employed for HeLa cell labelling and imaging, as well as in magnetic separation [83].Mulder et al. have successfully modified CdSe/ZnS with paramagnetic coating and arginine-glycine-aspartic acid (RGD) conjugation in order to combine the functions of specific protein binding and contrast agent for molecular imaging [84].The challenges encountered in cancer therapy stimulate comprehensive studies.Many efforts focus on the tracking and diagnosis of cancer cells.Cao et al. used near-IR QDs with an emission wavelength of 800 nm (QD800) to label squamous cell carcinoma cell line U14 (U14/QD800).The fluorescent images of U14 cells can be clearly obtained after 6 h by cell endocytosis [62].In 2007, Bagalkot et al. reported a more complex QDs-aptamer-(Apt-) doxorubicin (Dox) conjugate system [QD-Apt(Dox)] to endow QDs with the capability of targeting, imaging, therapy, and sensing the prostate cancer cells that express the prostate-specific membrane antigen (PSMA) protein (Figure 7) [85].Other types of QDs that have been applied in pancreatic cancer cells   imaging include CdSe/CdS/ZnS using transferrin and anti-Claudin-4 as targeting ligands, InP QDs conjugated with cancer antibodies, and water-soluble Si-QDs micelles encapsulated with polymer chains [86][87][88].Another important role played by QDs in biological application is transfection.Many studies reported fluorescence imaging and nucleus targeting of living cells by transfection and RNA delivery.Biju identified an insect neuropeptide, also-called allatostatin, which can transfects living NIH 3T3 and A431 human epidermoid carcinoma cells and transports quantum dots (QDs) inside the cytoplasm and even the nucleus of the cells [89].This method showed the conjugation of QDs with allatostatin could achieve high transection efficiency, which was promising for DNA gene delivery and cell labelling.Also, with specific conjugation with allatostatin, the QDs had another potential application in cancer, called photodynamic therapy [90].

In Vivo Imaging.
In addition to their usage as nanoprobes and labels for in vitro imaging, QDs have also been widely used as in vivo imaging agents (Figure 8) [91][92][93][94].Cancer-specific antibody, coupled to near-IR QDs with polymer coatings is the most popular QDs agent for tumortargeted imaging.Progress in this field has been reviewed in one literature [94,95].However, the depth of targets makes the QDs in vivo imaging very challenging.This special application of QDs requires them with low toxicity, high contrast, high sensitivity, and photostability.Dubertret et al. fabricated one type of CdSe/ZnS QDs which were encapsulated individually in phospholipid block-copolymer micelles.After further modification by DNA, this multifunctionalized DNA-QD-micelles can be developed to obtain Xenopus embryo fluorescent images in PBS.Moreover, after injection into embryos, the QD micelles showed high stability and nontoxicity (<5 × 10 9 nanocrystals per cell) [96].Another study reported by Michalet showed that CdSe/ZnS QDs can exhibit high contrast and imaging depth in two-photon excitation confocal microscopy by visualizing blood vessels in live mice [3,97].They confirmed that coatings such as PEG on the surfaces of QDs could further reduce their toxicity and accumulation in liver.Some studies also pointed out that the increase of the length of PEG coating polymer on the surface of QDs was able to slow down their extraction toward the liver, so coating polymer on the surfaces was another factor worthy of considering when QDs are employed for in vivo imaging [98].Other reports that used low-toxicity QDs include the development of non-Cd-containing QDs such as CuInS 2 /ZnS core/shell nanocrystals for in vivo imaging [95].In addition, sensitivity is another important factor that must be considered in QDs-related in vivo imaging.One study used nude mice for in vivo imaging after near-IR QD800-labeled BcaCD885 cells (BcaCD885/QD800) being implanted [99].Fluorescence signals of QDs accumulated in the tumor could be detected after 16 days of incubation at certain concentrations.It suggested that, compared with CT and MRI, QD800-based imaging could efficiently increase the sensitivity of early diagnosis of cancer cells.
With the development of QDs in recent years, many studies tried to explore the potential of QDs in wider fields.Kobayashi reported fluorescence lymphangiography by injecting five QDs with different emission spectra [100].Through simultaneous injection of five QDs into different sites in the middle of phalanges, the upper extremity, the ears, and the chin, different parts of the mouse body can be identified by certain fluorescence color.This is the first demonstration of simultaneous imaging of trafficking lymph nodes with QDs having different emission spectra.As biological fluorescent markers, no matter in vitro or in vivo, much attention has been concentrated on developing spherical QDs conjugations, but there are few reports on how other morphologies of QDs will interact with the biological systems.Yong et al. reported CdSe/CdS/ZnS quantum rods (QRs) coated with PEGylated phospholipids and RGD peptide for tumor targeting [101].This conjugation was demonstrated to be a bright, photostable, and biocompatible luminescent probe for the early diagnosis of cancer and was believed to offer new opportunities for imaging early tumor growth.

Prospects
Owing to the initial success of QDs-biomolecule conjugates employed for in vitro and in vivo imaging, it is conceivable that future researches will continue to focus on the development of QDs conjugations for cell labelling and cancer diagnoses.However, for bioapplications, biocompatibility of QDs remains the major challenge which must be carefully dealt with.Many studies have showed the short-term stability and long-term breakdown of Cd-containing QDs in in vivo imaging, which raise concerns about their chronic toxicity.Some studies showed that even robust coatings on the surfaces of QDs cannot prevent their releasing of Cd 2+ ions.The emerging picture of Cd-containing QDs toxicity not only suggests that QDs are required to improve safety before their wide biological applications as fluorescent nanoprobes, but also a variety of noncadmium alternatives are in urgent demand.At present, many efforts have focused on exploring new types of fluorescent nanomaterials such as upconversion materials NaYF 4 and NaGaF 4 .In addition, with the development of new techniques and detection methods, QDs are shown to have applications in wider fields.Zhang et al. successfully introduced Kelvin force microscopy (KFM) as a method to examine the binding of QDs with DNA in vitro and in vivo [102]; Huang et al. demonstrated a QDs sensor for quantitative visualization of surface charges on single living cell [103].We believe that with the development of new QDs with low toxicity and multiplex functionalities, QDs will find more versatile applications in biological fields other than bioimaging.

Figure 1 :
Figure 1: Emission maxima and sizes of quantum dots of different composition (with permission from [3]).

Figure 6 :
Figure6: Comparison of the staining efficiency of Annexin Vfunctionalized Qdots (staining achieved in one step using 2 nM of Qdots and 4 nM of biotinylated Annexin V, red), FITC-Annexin V (0.5% v/v, green) and Alexa Fluor 647-Annexin V (0.5% v/v, pink).(a) Light microscope pictures and pictures taken at t = 0, 500, 1000, 1500 s.(b) Intensity variation as a function of time for the three staining agents conjugated to Annexin V, FITC, Alexa Fluor 647 and Qdots.Intensity values expressed as relative values I/I 0 , where I 0 is the intensity measured at the first irradiation of the sample (with permission from[70]).

Figure 7 :
Figure 7: (a) Schematic illustration of QD-Apt(Dox) Bi-FRET system.(b) Schematic illustration of specific uptake of QD-Apt(Dox) conjugates into target cancer cell through PSMA mediate endocytosis.The release of Dox from the QD-Apt(Dox) conjugates induces the recovery of fluorescence from both QD and Dox ("ON" state), thereby sensing the intracellular delivery of Dox and enabling the synchronous fluorescent localization and killing of cancer cells (with permission from [85]).
mouse, colored NIR on top of field (a)

Figure 8 :
Figure 8: Targeting of lung melanoma tumor by injecting 2C5 QD-Mic in two mice.(a) Composite images (white field image superimposed with the fluorescence intensity), and cumulative histograms for the tumor region and the whole body of two mice; (b) composite ex vivo images (white field image superimposed with the fluorescence intensity) of lungs from mice bearing metastatic B16F-10 lung melanoma tumor (with permission from [94]).