Advances of gold nanoclusters for bioimaging

Summary Gold nanoclusters (AuNCs) have become a promising material for bioimaging detection because of their tunable photoluminescence, large Stokes shift, low photobleaching, and good biocompatibility. Last decade, great efforts have been made to develop AuNCs for enhanced imaging contrast and multimodal imaging. Herein, an updated overview of recent advances in AuNCs was present for visible fluorescence (FL) imaging, near-infrared fluorescence (NIR-FL) imaging, two-photon near-infrared fluorescence (TP-NIR-FL) imaging, computed tomography (CT) imaging, positron emission tomography (PET) imaging, magnetic resonance imaging (MRI), and photoacoustic (PA) imaging. The justification of AuNCs applied in bioimaging mentioned above applications was discussed, the performance location of different AuNCs were summarized and highlighted in an unified parameter coordinate system of corresponding bioimaging, and the current challenges, research frontiers, and prospects of AuNCs in bioimaging were discussed. This review will bring new insights into the future development of AuNCs in bio-diagnostic imaging.


INTRODUCTION
Bioimaging, including visible light fluorescence (FL) imaging, near-infrared fluorescence (NIR-FL) imaging, two-photon near-infrared fluorescence (TP-NIR-FL) imaging, computed tomography (CT) imaging, positron emission tomography (PET) imaging, magnetic resonance imaging (MRI), and photoacoustic (PA) imaging, is an indispensable method in biological research and medical diagnosis. They can play important roles in understanding the structure of biological tissues and elucidating the various physiological functions of life. Advances in optical technology, especially the introduction of digital imaging and computer vision techniques, the development of contrast agents with multiple imaging modalities, and the combination of different imaging modalities have improved the efficiency and accuracy of medical diagnosis.
In 1969, the photoluminescence of gold caused by the electronic interband transition was first discovered (Mooradian, 1969). Subsequently, the investigation of FL within the other Au-based materials has been studied. However, the weak FL of Au-based materials was almost undetectable to the naked eye, and their low quantum yields limited further applications in biomedical fields. It was not until 1994 that Brust et al. (1994) proposed a chemical reduction method using alkanethiols as surface ligands and successfully synthesized gold nanoclusters (AuNCs). With the continuous improvement and innovation of the synthesis methods and ligand modifiers of AuNCs, the quantum yield of AuNCs is constantly improving, and some even reach 70% (Wang et al., 2005;Lee et al., 2004). Inspired by these findings, a series of AuNCs protected by various ligands were prepared, and the investigations on the structures, properties and related applications of AuNCs have increased rapidly. As a bridge between Au atoms and gold nanoparticles (AuNPs), AuNCs are ultra-small nanoparticles (NPs) (typically less than 3 nm in diameter) consisting of Au(I)-organic ligand shells with different numbers of Au atoms Goswami et al. (2016a). Because of their size comparable to the Fermi wavelength of electrons, AuNCs exhibit molecular-like properties, including intense photoluminescence (PL), molecular chirality, magnetism, and catalysis (Maity et al., 2019;Xu et al., 2019). AuNCs also possess unique optical and electrical properties, including discrete electronic transitions and highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy level transitions (Chan and Nie, 1998;Guo and Wang, 2011). The FL of AuNCs can be influenced by various factors, including the size of the Au nucleus, the type of surface ligands, and the surrounding environment (Zheng et al., 2004;Zhu et al., 2020a). Because of their excellent photostability and biocompatibility, AuNCs have potential uses in biomedical imaging (Du et al., 2018;Xu et al., 2019). As shown in Figure 1, existing common imaging modalities include FL, magnetic resonance (MR), X-ray computed characterized as ligand-to-metal charge transfer, ligand-to-metal charge transfer, and electron leap within the Au nuclei (Abbas et al., 2018). All three Au 24 (SR) 20 reported by Gan et al. (2016) exhibited robust red FL, which increased with the intensity of the ligand-electron-donor. The quantum yields of the two Au 28 (SR) 20 modified with different-R groups showed prominent differences, with Au 28 (CHT) 20 (CHT: cyclohexanethiolate) having a more than 15-fold higher PL quantum yield ratio than Au 28 (TBBT) 20 (TBBT: p-tert-butylbenzenethiolate). This gap originates from the different structural arrangements of the staple motifs in the shell .

Phosphorescence
Phosphorescence is a slow luminescence phenomenon originating from radiative transitions between different states. Unlike FL, the luminescence phenomena of phosphorescence remain when the incoming light is turned off. In the aggregated state of [Au 8 ] 4+ , Sugiuchi et al. (2017) discovered the transition of PL emission from FL to phosphorescence. The monomeric cluster emits visible FL at 596 nm, but on aggregation, it emits a second near-infrared peak at 708 nm witha phosphorescent character. Excitation spectroscopy studies revealed that phosphorescent emission originates from exciton-coupled cluster assemblies, which have higher emission activity than monomeric clusters. In addition, significant phosphorescence emission was seen in solid-state clusters, with a quantum efficiency two orders of magnitude higher than that of monomeric clusters in solution. Similarly, the monomeric and solid-state clusters of GSH-AuNCs emitted for 4.3 and 7.9 s, respectively, confirming phosphorescent emission (Pan et al., 2019). Under UV light, the aqueous solution of GSH-AuNCs displayed relatively mild red emission; however, when it was dissolved in an acidic solution or an ethanol solvent, its GSH-AuNCs aggregation was activated, resulting in enhanced red emission.
The self-assembly of AuNCs can be induced and the optical characteristics of AuNCs can be modified by introducing different chemicals. For example, electrostatic and hydrophobic interactions between hyaluronic acid, protamine, and fluorescent AuNCs impair the non-radiative transition of AuNCs, boosting phosphorescence emission (Xia et al., 2019). Shen et al. (2021) prepared L-/D-cysteine modified AuNCs, which self-assemble into highly ordered chiral microflower structures upon Cd 2+ /H + induction, and this compact structure promotes Au(I)-Au(I) interactions and ligand-metal charge transfer (LMCT), resulting in fluorescence-phosphorescence conversion and emission lifetime extension up to 3.34 ms. Furthermore, dual FL and phosphorescence emission was observed in both NCs of Au 11 Cu 1 NCs and Au 10 Ag 2 NCs (Lei et al., 2016;Wu et al., 2021). At present, researchers focus on the study of the phosphorescence properties of AuNCs, and the development of AuNCs phosphorescence imaging probes is still in the development stage.

Two-photon (TP) excitation
Compared to single-photon excitation, nanomaterials with two-photon (TP) excitation can emit high-energy FL by absorbing two relatively low-energy photons, whereas the energy level jump requires only half the energy difference between these two energy levels. Thus, TP excitation is of great benefit in bioimaging. Ramakrishna et al. (2008) produced hexane thiolate-stabilized Au 25 NCs with good TP absorption (TPA) properties in hexane. According to their results, the TPA cross-section of this cluster was up to 17080 GM, which is close to the theoretical limit. Captopril-or GSH-protected AuNCs also show high TPA absorption (Olesiak-Banska et al., 2016;Polavarapu et al., 2011). Recently, dihydrolipoic acid (DHLA)-coated AuNCs were found to have an even higher TPA cross-section, with a cross-section of 10 6 GM for the 800 nm emitter . The extremely high TP cross-section of AuNCs can be used as an excellent bioimaging agent for TP imaging.

Tunable emission
Although the source of AuNCs PL is unknown, various factors affecting the luminescence of AuNCs have been reported. The ability to tune the brightness of AuNCs is an essential feature for their widespread use in bioimaging. Therefore, many strategies for tuning the emission wavelength of AuNCs and increasing their FL intensity have been developed. A list of them is given below.
(1) Limiting the number of Au atoms in the system. Because AuNCs are the primary source of cluster luminescence, varying the size of AuNCs can control the emission wavelengths. Zheng et al. (2004) synthesized AuNCs with different numbers of Au atoms by varying the concentration of Au 3+ in the tree-like macromolecule, producing Au 5 , Au 8 , Au 13 , Au 23 and Au 31 , resulting in emission spectra from the visible to the NIR region.
(2) Using different ligands, such as proteins, peptides, deoxyribonucleic acids (DNAs), and polymers (El-Sayed and Schneider, 2020). Studies have suggested that the FL of AuNCs could be enhanced by increasing the ability of the ligand to provide charge and increasing the oxidation state of the metal nuclei (Li et al., 2019b;Wu and Jin, 2010). Groups with electron-rich molecules were also found to promote FL. Kundu et al. (2020) used four different peptide chain lengths of thiol ligands to modify AuNCs, resulting in FL emissions ranging from 510 to 612 nm.
(3) Rigidifying the nuclear shells of AuNCs. Mixing tetraoctylammonium bromide (TOA) with GSH-AuNCs and pairing the TOA cation with the carboxylic acid heel anion of GSH, the shell layer of AuNCs was rigidified, leading to a 9-fold increase in FL intensity (Pyo et al., 2015(Pyo et al., , 2016. (4) Aggregation-induced emission (AIE). Luo et al. (2012) synthesized Au(I)-SR oligomers with GSH, which did not fluoresce in an aqueous solution. However, after adding enormous amounts of ethanol to the aqueous solution, the Au(I)-SR oligomers fluoresced with different intensities due to aggregation. The link between the length of the Au(I)-SR motifs and the AIE was further investigated, allowing to modify of the emission wavelength of AuNCs by changing the length of the Au(I)-SR motifs (Goswami et al., 2016b;Wu et al., 2020;Yu et al., 2014).
(5) Doping with other metal ions. For example, using Ag ion doping, Ag/AuNCs with different FL emission and FL intensity can be obtained by controlling the molar ratio of Ag: Au. (Jia et al., 2020;Zhai et al., 2018). Because of the synergistic effect of Ag and AuNCs, doping Ag can improve the FL intensity, and its intensity usually increases first and then decreases with the increase of Ag.

High X-Ray attenuation coefficient
When an X-ray penetrates an item, it interacts with the atoms of the material through absorption and scattering, causing the intensity of the X-ray to be attenuated during transmission. The attenuation coefficient is generally proportional to the atomic number and density of the substance. Compared to the commonly used I, Au has a higher atomic number (Au, 79; I, 53) and X-ray absorption coefficient (at 100 keV: Au, 5.16cm 2 g À1 ; I, 1.94cm 2 g À1 ). The contrast effect of Au is also 2.7-times higher than that of I per unit mass (Hainfeld et al., 2006). Several studies have shown that the K-edge (the minimum energy required to excite the elements in a sample to produce FL X-rays) of Au is 80.7 keV. When Au atoms are irradiated with X-rays of energy 80.7 keV or slightly more significant, their absorption coefficient increases exponentially, resulting in a significant increase in their corresponding attenuation coefficients (Hainfeld et al., 2006). They also pointed out that AuNPs should be prepared at a specific mass concentration when used as a contrast agent. X-ray attenuation increases with the mass concentration of AuNPs. At the same mass concentration, the size of AuNPs significantly affects the X-ray attenuation ability. Smaller AuNCs with larger specific surface areas exhibit more excellent apparent X-ray attenuation, showing their great potential as CT contrast agents (Tsvirkun et al., 2018;Xu et al., 2008).

Magnetism
Au is a diamagnetic element, but it exhibits magnetic characteristics at the nanoscale scale, as revealed by AuNPs (Trudel, 2011). According to research, the magnetism of AuNPs is influenced by their electronic structure, which is connected to their size and surface ligands (Krishna et al., 2014;Trudel, 2011;Ulloa et al., 2021 (Agrachev et al., 2017). Li and Jin (2021) recently hypothesized that the magnetic behavior of AuNCs can be predicted using their specific chemical formula (Li and Jin, 2021). They also propose that after adding or withdrawing one electron, AuNCs with an icosahedral core transfer magnetic. Window and Ackerson (2020) discovered chargedependent paramagnetism in Au 102 (SPh) 44 , as well as paramagnetism when two electrons were removed from Au 102 (SPh) 44 (Window and Ackerson, 2020). Zhu et al. (2009) have hypothesized that the magnetism of Au 25 (SR) 18 nanoparticles can be reversed by accurately manipulating their charge state. Although several investigations have been conducted on the formation of magnetism in AuNCs, its origin remains unknown.

Easy surface functionalization
The rational design of AuNCs surface modification ligands can further expand the biological applications of AuNCs. Surface functionalization of AuNCs can be divided into in-synthesis functionalization and postsynthesis functionalization (Song et al., 2016). In-synthesis functionalization, namely, the addition of pre-designed ligands during the synthesis process. Many studies have used different types of molecules with good biocompatibility, such as small organic molecules, amino acids, proteins, peptides, DNAs, and polymers . These ligands not only act as stabilizers but also greatly improve the properties of AuNCs by increasing their PL wavelengths, conferring targeting properties and treating diseases such as cancer (Gao et al., 2019;Li et al., 2019a). In addition, increasing the targeting of AuNCs can greatly improve the contrast and accuracy of the imaging site. Because tumor cells highly express receptors on their surface, modification of AuNCs using targeting molecules such as folic acid (FA) and various antibodies enable tumor cell-specific imaging (Hada et al., 2021;Zhou et al., 2013).
Functionalization after synthesis is usually based on ligand exchange, bioconjugation and non-covalent interactions . Ligand exchange is one of the most commonly used strategies for functionalizing AuNCs. Through ligand exchange, the optical and electronic properties of AuNCs can be modified. A typical example is the conversion of non-FL AuNPs (AuNPs@DDAB, DDAB: didodecyldimethylammonium bromide) into red-emitting AuNCs (AuNCs@DHLA) through ligand exchange (Lin et al., 2009). Fernando and Aikens (2015) studied the reaction kinetics of ligand exchange of AuNCs (Fernando and Aikens, 2015). They found that ligand exchange occurred between-SH and embedded Au atoms. In addition, the structure of AuNCs may change during ligand exchange. The ligand exchange reaction of [Au 11 (PPh 3 ) 8 Cl 2 ]Cl (PPh: triphenylphosphine) with water-soluble arylphosphines synthesized water-soluble [Au 9 (PPh 3 ) 8 ] 3+ , and the phosphine-stabilized Au 11 reacted with GSH in water under a nitrogen atmosphere to give Au 25 (SG) 18 (Ndugire and Yan, 2021;Shichibu et al., 2005). A series of thiol ligands of different chain lengths were exchanged with GSH-AuNCs ligands to prepare AuNCs of different sizes, which exhibited a tunable emission (Kundu et al., 2020).
Bioconjugation is usually achieved by further modifying other molecules on AuNCs with the modified ligands, mainly through functional group interactions between the two ligands. The standard coupling method is between amine and carboxylate groups activated by N-(3-dimethyl aminopropyl)-N-ethyl carbodiimide (EDC). In the synthesis of AuNCs, stabilizers with exemplary terminal functional groups, such as alcohols, carboxylic acids, primary amines and thiols, are usually selected to prevent the aggregation of AuNCs. Then superior biomolecules are further selected to couple with them to achieve multi-functionalization of AuNCs (Song et al., 2016;Wang et al., 2017;Zheng et al., 2017). For example, FA is conjugated to GSH capped AuNCs to achieve targeted recognition of cancer cells . Anti-human immunoglobulin E (IgE) is coupled to lipoic acid-modified fluorescent AuNCs to measure IgE in human serum (Alonso et al., 2016). Horseradish peroxidase (HRP)-coupled AuNCs are internalized by breast cancer cells, generating free radicals and inducing apoptosis (Vivo-Llorca et al., 2022).
Non-covalent interactions are also an effective strategy for the functionalization of AuNCs. For example, through electrostatic interactions, lysozyme (Lys)-AuNCs-curcumin (Cur) couples can bind to bacteria for real-time imaging of bacteria with excellent antibacterial effects . In addition, the quantum yield of AuNCs was significantly improved by hardening the Au(I)-thiol salt shells with TOA cations (Pyo et al., 2015).

Easy to doping
Doping with other metals can significantly improve the physicochemical properties of homogeneous AuNCs. As previously stated, FL emission can be tuned and FL intensity can be increased. Doping other metals in AuNCs is thus a useful method of tailoring the structure and properties of nanoclusters (Kang et al., 2020). The position of the doping of the doped metal is related to its kind by investigating the detailed structure of the doped clusters (Jin et al., 2016). Alkan et al. (2019) investigated the doping theory of clusters such as Au 24 and Au 37 and discovered that the X group elements (Ni, Pd, and Pt) prefer the center position, and the radius of the doping metal can greatly impact the geometry of the alloy clusters and relative stability. For the Au 25 À1 cluster, Pd and Pt tend to be in the center position, Ag tends to the Au core surface, and Cd and Hg tend to the core surface and the ligand shell part (Tian et al., 2016;Yao et al., 2015). iScience Review Wang et al., 2015b). Kang et al. (2020) provide a detailed description of the structure and behavior of atomic-scale alloy nanoclusters.
Doping allows AuNCs to be used for multimodal imaging. The use of radionuclides (e.g., 64 Cu, 68 Ga, 124 I, etc.) to label AuNCs enables PET and PET/CT imaging and improves the sensitivity of detection (Han et al., 2020a;Sultan et al., 2021;Zheng et al., 2021a). Modification of AuNCs using Gd ions or Gd complexes also enables MRI (Liang and Xiao, 2017;Liang et al., 2013;Sun et al., 2013). Therefore, AuNCs are expected to be ideal multimodal imaging probes ( Figure 2C).

Good biocompatibility
After injection, safe and valuable imaging probes must be properly eliminated from the body within a certain period. According to previous studies, AuNCs can enter the body either by injection or oral administration. Once in the body, AuNCs enter many organs through the bloodstream, including the heart, liver, spleen, lungs, kidneys, and brain Zhang et al., 2012b). AuNCs are usually absorbed by the liver, secreted with bile, and finally excreted in the urine and feces. Previous studies have shown that specific NPs with hydrodynamic diameters less than 5.5 nm can be efficiently and rapidly cleared by the kidneys (Choi et al., 2007;Du et al., 2018). Chen et al. (2016) observed the renal clearance of 64 Cu-labeled AuNCs by dynamic PET imaging of these AuNCs. It was shown that the kidney rapidly cleared synthetic AuNCs with an elimination half-life of <6 min, much shorter than previously reported AuNCs. Because the glomerulus more readily captured smaller AuNCs, a reduction in the size of several atoms also decreased renal clearance efficiency, causing an inverse size-dependent renal clearance of AuNCs in the subnanometer region (Du et al., 2017). The 24-h urinary excretion of kidney-clearable AuNCs around 2 nm was typical> 50% ID, comparable to the renal clearance efficiency of clinically used small molecule probes (Alric et al., 2013;Simpson et al., 2013;Yu and Zheng, 2015;Zhang et al., 2015a;Zhou et al., 2011Zhou et al., , 2012. In addition, the modification of different ligands also affects the renal clearance of AuNCs, with GSH-modified AuNCs showing much higher renal clearance over 24 h than BSA-modified AuNCs (Zhang et al., 2012b).
AuNPs have an inert gold nucleus, and therefore AuNPs are usually considered to have good biocompatibility. However, it has been shown that the size of AuNPs affects the cytotoxicity of AuNPs (Schrand et al., 2010). Pan et al. (2007) conducted a systematic study on the cytotoxicity of triphenylphosphine derivativemodified AuNPs with sizes ranging from 0.8 to 15 nm. They found that the cytotoxicity depended on the particle size of AuNPs, and the cytotoxicity of AuNCs was more significant than that of the larger 15 nm AuNPs. They suggested that AuNCs when endocytosed by cells, may interact with cell membranes and thus trigger apoptosis or necrosis of cells. As the particle size decreased, its specific surface area increased, and it was more bioactive, putting it at increased risk of toxicity (Schrand et al., 2010;Yang et al., 2022). This size dependence is also observed in silver thiolated nanoparticles . In another study, AuNPs of 3-100 nm were injected into mice separately, where AuNPs of 3, 5, 50 and 100 nm showed no deleterious effects. However, AuNPs of 8-37 nm caused severe disease and death in mice within 21 days (Chen et al., 2009). They concluded that AuNPs smaller than 5 nm could be associated with enhanced clearance activity of the immune system, thus reducing the biotoxicity of AuNPs. Also, the ligands on the surface of AuNPs also affected cytotoxicity, and the selection of biocompatible ligands could significantly reduce the cytotoxicity of AuNPs (Peng and Liang, 2019). A large number of experiments have confirmed that the ligand-modified AuNCs have the advantage of low toxicity, and injection into mice will not affect their vital organs. Therefore, AuNCs have excellent biomedical prospects.

ADVANCES OF AUNCS APPLIED IN BIOIMAGING
Visible light fluorescence (FL) imaging FL imaging has been widely used in the biomedical field to diagnose and guide disease treatment. It is known that fluorescent substances release fluorescent signals when activated by an external light source, and their intensity is proportional to the number of fluorophores in a particular range. AuNCs are widely used for FL imaging because of their robust photostability, photobleaching ability, and tunable luminescence.
Using suitable biomolecules and ligands with different activities, the FL emission wavelength of AuNCs can be modified, and the imaging categories of AuNCs in cells or animals can be identified. Cong  iScience Review nucleus of MCF-7 cells, allowing precise differentiation between the cytoplasm and nucleus within the cell. Liu et al. (2020) prepared red-and blue-light-emitting AuNCs that could target the nucleus for imaging by adjusting the order of modulating pH using the same peptide, as shown in Figure 3A. Yang et al. (2021) prepared GSH-AuNCs, which emitted red FL at a high quantum yield (8.9%) emitting red FL, which was used for brain neuroimaging by current displacement. Endocytosis allows the clusters to enter the cell, where they accumulate in the cytoplasm and undergo some AIE augmentation. They remained stable in mice for up to 21 days and red FL was still visible near the sciatic nerve. AuNCs can monitor the development of amyloid (Kundu et al., 2020;Lai et al., 2016). 16-polymer-nucleoprotein-protected AuNCs (7A-AuNCs) prepared by Lv et al. (2020) were also able to image hazardous substances (methylmercury) in the mouse brain in an acute toxicity model. In addition, persistent FL-specific labeling of tumor cells can be rapidly achieved by in situ synthesis of AuNCs in cancer cells . Zhu et al. (2019) prepared core-shell AuNCs with rigid host-guest components (Prot/MTU-AuNCs) by modifying the antimicrobial agent protamine (Prot) to 6-methyl-2-thiouracil (MTU)-stabilized AuNCs, which greatly enhanced their FL and allowed easy imaging of bacteria.
To enable tumor cell-targeted imaging and recognition of specific receptors on the surface of tumor cells, receptors targeting peptides and antibodies (such as FA) are usually modified on the surface of AuNCs. Feng et al. (2020) used DNA Mucin 1(MUC1) inducers to target 4T1 cancer cells that recognize mucin overexpression. Zhang et al. (2020) used herceptin, a monoclonal antibody against the human epidermal growth factor 2 (HER2) receptor monoclonal antibody, to stabilize synthetic fluorescent Her-AuNCs. During long-term observations, the synthetic Her-AuNCs showed good specific target recognition imaging of HER2-positive tumors, as shown in Figure 3B. Because cancer cells over-express L-type amino acid transporter proteins, which detect methionine (Met) ligands on the surface of Met-AuNCs, Met-modified AuNCs BSA is the most commonly used protein in biochemical experiments due to its high stability and biocompatibility. BSA is commonly used as a stabilizer and reducing agent in preparing AuNCs Zhang and Han, 2018). In situ sequestered BSA-AuNCs synthesized from BSA showed higher FL and quantum yields (about 6%) than NaBH 4 as a reducing agent (Xie et al., 2009). Subsequent studies also confirmed the targeted recognition of tumors by BSA-AuNCs . Compared to BSA-AuNCs, FAmodified BSA-AuNCs showed better-targeted imaging of human ovarian cancer cells and improved cellular uptake due to overexpression of FA receptors in ovarian tumor cells (Hada et al., 2021). Similarly, mannose-modified BSA-AuNCs specifically identified human breast cancer cells (Sha et al., 2020).
In bioimaging, FL in the visible band will be significantly affected because biological tissues have autofluorescence, usually generated by near-UV and blue light excitation (Ballou et al., 2005). Although the FL signal is much more potent than the bioluminescence signal, the signal-to-noise ratio of non-specific FL is significantly lower than that of bioluminescence. Because of this background noise, the sensitivity of FL imaging is limited and challenging to reduce, limiting the application of AuNCs. In addition, visible light is scattered and absorbed when passing through tissues, which limits penetration depth and spatial resolution (Zelmer and Ward, 2013). As a result, FL imaging can only image superficial tissues, resulting in a loss of physiological and pathological information (Leblond et al., 2010).

Near-infrared (NIR) FL imaging
NIR imaging can penetrate deep tissues well, which compensates for the shortcomings of imaging in the visible wavelengths. In the NIR region, including the NIR I region (700-900 nm) and the NIR II region (1000-1700 nm), the human body exhibits low absorption and no interference from biological tissues, showing its great potential for bioimaging. Compared with other NIR materials, AuNCs have the advantages of simple preparation, high biocompatibility, easy surface modification, significant Stokes shifts and rapid elimination (Gong et al., 2021a). Therefore, modulation of the FL emission wavelength of AuNCs has been a hot research topic, and selecting suitable ligands for modification is one of the most commonly used strategies, such as proteins, polymers, and peptides ( Figure 4)   This efficient binding is attributed to the specific binding of Au 25 (GSH) 18 to Ca in HA, which is caused by the carboxyl group at the end of the GSH ligand on the Au 25 (GSH) 18 surface. Within 8.5 min after Au 25 (GSH) 18 injection, the whole mouse body exhibited a vital NIR-II FL. As the time was prolonged, Au 25 (GSH) 18 was drastically reduced. Its signal was attenuated in other body parts but was enhanced in skeletal sites. After 8.3 h of injection, most mouse bones showed clear FL of the skeleton under the skin. When the skin was pulled away, almost all skeletal structures were photographed in high quality. This nanomaterial for direct observation of bone has great potential for guiding skeletal surgery and is an excellent NIR-II bone imaging probe ( Figure 5A). Liu et al. (2019) created an Au 25 (GSH) 18 cage structure with a red-shifted FL emission center at 1120 nm. Its surface ligand is altered by doping with metal atoms. The quantum yield of Au 25 (GSH) 18 was increased by altering the electron transfer between the surface ligand and the metal nucleus by doping with metal atoms, which allowed penetration into the skull for NIR-II cerebrovascular imaging. Using principal component analysis techniques, primary tumors can also be clearly distinguished from tumor metastases ( Figure 5B). When AuNCs are combined with Pt-based therapeutic agents, Pt transport can be observed by NIR-II imaging, allowing visualization of the tumor treatment process . These AuNCs can also remove intracellular GSH, thus improving the therapeutic effect.
The diversity of ligands modified on the AuNCs surface offers several possibilities for NIR-II FL imaging. pHsensitive AuNCs protected by a-lipoic acid (LA) and g-cyclodextrin (g-CD) were designed by Das et al. (2016).
In an acidic environment, LA on the surface of AuNCs was converted to DHLA, leading to structural disruption. This eventually leads to efflux of-CD, which allows selective imaging of MCF7 human breast cancer cells. Compared to rare earth and silver-based NIR-II emitters, RNase-A@AuNCs were more sensitive to the gastrointestinal tract, allowing precise examination of intestinal tumor nodules as small as 2.5 mm. NIR-II imaging with RNase-A@AuNCs was provided four times with a higher resolution of the gastrointestinal tract than the barium swallow method. Using AuNCs as a model, Er 3+ -doped rare earth (Ln) nanoparticles were further assembled, which significantly enhanced the PL intensity of AuNCs and could emit double the NIR-II luminescence under a single laser excitation, located at 1100 and 1550 nm, respectively . The ratio of H 2 S generation in NIR-II-targeted monitoring. AuNCs modified with short dithiol peptide chains (AuMHA/ TDT, MHA: 6-mercaptohexanoic acid, TDT: terminal deoxynucleotidyl transferase) have a detection wavelength of 1250 nm and a high quantum yield (approximately 6%), allowing high-resolution imaging of whole-body blood arteries in mice (Yu et al., 2020). AuNCs with various functional components stabilized by peptides has also been created (Xia et al., 2018).
To provide an excellent combinatorial strategy for the multi-functionalization of AuNCs, researchers have used polyethylene glycol (PEG) to increase blood circulation time, matrix metalloproteinase (MMP)-2 peptides (CPLGVRGDs) to induce tumor targeting ability, cis-nicotinic anhydride modified doxorubicin to control drug release, and the photosensitizer e6 chloride (Ce6) to provide photodynamic therapy for photodynamic therapy. The AuNCs-LHRHa nanosystems were synthesized by combining luteinizing hormone releasing hormone analogs (LHRHa) with AuNCs, and LHRHa was used to enhance the uptake of nanosystems by tumor cells and improve the targeted imaging and photothermal therapy of prostate cancer . Using Keratin-modified AuNCs (AuNCs@Keratin) as a template, Li et al. (2020b) further synthesized silver ion-modified AuNCs@Keratin (AuNCsAg@Keratin) and gadolinium-induced AuNCs-Ag@Keratin (AuNCs-Ag@Keratin-Gd). Among them, the NIR FL intensity of AuNCs-Ag@Keratin-Gd was enhanced by 6.5 times compared with that of AuNCs@Keratin because silver modification promoted the formation of dense aggregates after gadolinium ion-induced assembly. In addition, because of gadolinium ion induction, AuNCs-Ag@Keratin-Gd can also achieve MR imaging, and it can be used as a drug carrier loaded with doxorubicin hydrochloride (DOX), which significantly inhibits tumor growth. Yang et al. (2020a) reported three a-lactalbumin-stabilized AuNCs, under the same exciter, had different emission spectra of 450, 520, and 705 nm (named AuNCs 450 , AuNCs 520 , and AuNCs 705 , respectively). The emission wavelengths of the three clusters were pH dependent and intensity dependent on a-LA concentration, with AuNCs 705 having superior in vivo imaging signal-to-noise ratio and radiation efficiency for multimodal imaging and guiding surgical resection of breast tumors. These research results are expected to promote the multi-functionalization of AuNCs nanoprobes, stimulate the rational design of nanodrugs, and promote the development of in vivo bioimaging for multiple applications, laying the foundation for the integration of diagnosis and treatment.

Two-photon near-infrared fluorescence (TP-NIR-FL) imaging
Two-photon excitation of AuNCs is typically in the red to the near-infrared range, allowing them to penetrate deeply into the tissue. In addition, TPA occurs only in the focal plane, improving the spatial resolution ( Figure 6). Jiang et al. (2019) used etching to create zwitterionic ligands (ZW) and 11-mercaptodecanoic acid (MUA)-stabilized AuNCs with excellent stability, resistance to photobleaching, and a large TPA cross-section (2.27 3 10 5 GM). By binding streptavidin and biotin alkyl ligands to AuNCs, the two clusters bioassembled intracellularly, amplified the firing signal, and successfully resisted quenching caused by AuNCs aggregation. Gong et al. (2021b) developed rhodamine B derivative-modified AuNCs that can be used as TP ratio fluorescent probes for real-time sensing and imaging of NO in neurons in brain regions. He et al. (2021) synthesized transferrin-modified AuNCs, which can emit TP near-infrared FL. They combined AuNCs with MnO 2 nanosheets to prepare a novel TP nanoprobe. The probe has a tissue penetration depth of 300 mm, much larger than previously reported TP probes (<200 mm). The probe can monitor the dynamic changes of endogenous GSH in living cells and tissues and enable dual TP-NIR FL and MIR imaging. Han et al. (2020b) prepared DHLA-modified AuNCs by etching, which has an ultra-high TPA cross section ($10 6 GM) for TP imaging and can efficiently generate ROS, which, in combination with photodynamic therapy, exhibits superior antitumor effects.
Time-gated imaging with background and signal management based on TP imaging can improve the signal-to-noise ratio while reducing the impact of auto-fluorescence on image quality. Tian et al. (2021)  iScience Review detecting liver cancer cells. The clusters have a long FL lifetime (>300 ns) and significant TPA (TPA cross section up to 1.59 3 10 5 GM). Using an 800 nm laser for TP and TP time-gated imaging, the cluster could significantly remove the auto-fluorescence signal from the tissue, allowing real-time imaging of the liver and tumor.

Computed tomography (CT) imaging
CT is currently the most widely used medical diagnostic method. In clinical practice, high atomic number I-based compounds are often utilized as contrast agents to increase the contrast of CT scan sites (Hallouard et al., 2010). On the other hand, I-based contrast agents can produce allergic reactions, kidney damage, and short imaging times. These side effects severely limit their use in medical imaging. According to the X-ray absorption coefficient law, Au has a good X-ray absorption capacity because of its high density and atomic number. Au-based nanoparticles have attracted much attention as CT contrast agents due to their biocompatibility and modifiability (Figure 7). Because of their superior X-ray attenuation qualities, Hainfeld et al. (2006) pioneered the use of AuNPs as CT contrast agents with less interference to tissue imaging and longer imaging times. The results demonstrated the great potential of AuNPs as CT contrast agents, as they showed significantly higher CT attenuation than type I contrast agents of the same molar concentrations. Similarly, AuNCs are considered promising CT contrast agents. BSA-stabilized AuNCs have been shown to have higher CT values (Hounsfield units, HU) compared to type I contrast agents of the same concentration, with low BSA-AuNCs having HU values comparable to those of high concentrations of iopromide (Wang et al., 2015c;Zhang et al., 2012a). Since BSA-AuNCs are mainly excreted in the kidney, details of the renal tissue can be observed, and renal lesions can be diagnosed ( Figure 8A). In addition to dual-mode imaging by combining FL and CT, the FL properties of AuNCs can enhance tumor accumulation and enable tumor radiotherapy Zheng et al., 2021b).
The ability of AuNCs to perform CT imaging can be improved by adding different functional auxiliary complexes to their surfaces. Guo et al. (2010) created dendrimer-encapsulated AuNCs (AuDENPs) using a fifthgeneration polyamidoamine (G5 PAMAM) dendrimer as a template. They altered AuDENPs by surface PE-Gylation and acetylation to circumvent the toxicity and non-specificity of surface amino groups (Peng et al., 2011(Peng et al., , 2012. All modified AuDENPs outperformed type I contrast agents in imaging contrast, circulation time, and local enrichment because of the effects of electron paramagnetic resonance (EPR) and lack of comparable cytotoxicity. AS1411 aptamer-modified AuNPs (AS1411-DA-AuNPs) targeted ditrizoic acid

OPEN ACCESS
and nucleolin to localize CL1-5 tumor sites accurately . Both I and Au contribute to high X-ray absorption coefficients and strong CT signals Clark et al., 2013). They allow simultaneous FL and CT imaging to guide tumor resection. NPs composed of Au and I can also diagnose thyroid cancer and image tumor vasculature. Zhou et al. (2013) designed a silica-coated FA-modified AuNCs targeting FR (+) MGC803 and FR (À) GES-1 cells for dual-mode imaging of FL and CT in gastric cancer ( Figure 8B). Chu et al. (2019) in BSA-AuNCs introduced Ag + and found that a molar ratio of Au: Ag of 3:2 showcased the best X-ray attenuation coefficient, approximately twice that of iohexol (80 mM concentration). This compound can be used as a potential CT contrast agent. Similarly, the complex of Fe 2+ with AuNCs improves CT imaging, and the presence of Fe 2+ allows MRI, thus laying the foundation for multimodal imaging (Zhao et al., 2016a). AuroVist TM is a commercially available nano Au X-ray contrast agent with a size of 1.9 nm (Clark et al., 2013). It exhibits the following characteristics: high imaging contrast, which is three times higher than that of type I contrast agents; low toxicity, which is more than four times the concentration of type I contrast agents; and long retention time, which the kidneys can clear.

Positron emission tomography (PET) imaging
PET imaging allows imaging at the cellular and molecular levels, making it one of the most advanced diagnostic medical imaging techniques available. Living metabolites are labeled with short-lived radionuclides, and their aggregation in human metabolism can reflect the living metabolism (Ziegler, 2005). Currently, PET is widely used for diagnosing and treating cancer, coronary heart disease, and brain diseases (Burgess et al., 2001;Delbeke and Martin, 2001;Hagge et al., 2001;Machac, 2005). PET imaging agents are typically glucose analogues, such as 18 F-FDG, which are based on glucose metabolism and cause massive 18 F-FDG aggregation at tumor locations (Duysinx et al., 2004). This non-specific tracer makes diagnosis and prognosis more difficult because it cannot target the tumor locations. In addition, the poor stability of commonly used radionuclides hinders PET imaging studies. Directly doping radionuclides into AuNCs can significantly improve their stability and sensitivity detection. The commonly used radionuclides are 64 Cu, 68 Ga, and 124 I (Ballegeer et al., 2013;Cho et al., 2015;Petersen et al., 2011;Wu et al., 2019). Zhao et al. (2014a) prepared 64 CuAuNPs with diameters of 9.4 G 1.2 nm by directly doping 64 Cu into the lattice of AuNPs in an alloyed manner. The NPs showed good radionuclide labeling stability with no degradation in mouse serum for 48 h. The NP retention was high in liver and kidney sites. Therefore, they further reduced the size of 64 CuAuNPs to 2.5 G 0.8 nm and PEGylated the surface of 64 CuAuNCs, improving the biosafety and enhancing the renal clearance of NCs (Zhao et al., 2014b). However, the AuNCs made this way were non-specific, and their distribution was not uniform in targeting and identifying specific iScience Review tumors. Zhao et al. (2016b) modified 64 CuAuNCs using AMD3100 to achieve high sensitivity and accuracy in detecting C-X-C chemokine receptor 4 (CXCR4) expression ( Figure 9A). In a mouse model of breast cancer, these clusters can target and identify tumor sites and show better PET imaging of tumor metastases, especially lung metastases. 64 Cu-AuNCs-ECL1i (ECL1i: extracellular loop 1 inverso peptide) was synthesized by conjugating chemokine (C-C Motif) receptor 2 (CCR2) to 64 CuAuNCs to specifically identify triple-negative breast cancer and improve tumor and contrast of background PET imaging ( Figure 9B) . This receptor is also highly expressed in cells with atherosclerotic lesions. In an ApoE À/À mouse model with progressive atherosclerosis, 64 Cu-AuNCs-ECL1i is sensitive to early lesions and shows dynamic changes in lesions, indicating its great potential for detecting and imaging atherosclerotic disease .
Several researchers labeled AuNCs with 68 Ga, 124 I for PET-CT imaging. Han et al. (2020a) labeled human serum albumin and luteinizing hormone-releasing hormone-modified AuNCs with 124 I and achieved a 73% labeling rate for targeted identification of tumor tissue in the lungs. Zheng et al. (2021a) prepared two types of 68 Galabeled AuNCs, namely GSH-AuNCs ( 68 Ga-GSH@AuNCs) and GSH and arginine-glycine-aspartate tripeptide (RGD) co-modified AuNCs ( 68 Ga-RGD-GSH@AuNCs). 68 Ga-RGD-GSH@AuNCs have a shorter distribution half-life and longer elimination half-life, allowing them to rapidly enter the tumor site and provide a longer blood circulation time. Compared with 68 Ga-GSH@AuNCs, 68 Ga-RGD-GSH@AuNCs can specifically identify tumor sites, exhibit more pronounced aggregation at tumor sites and play a more significant role in early tumor PET-CT imaging and detection due to the presence of RGD. However, due to the high affinity of RGD for renal tubules, 68 Ga-RGD-GSH@AuNCs also aggregated mainly in the kidney.

Magnetic resonance imaging (MRI)
MRI uses the resonance of atomic nuclei in a magnetic field to reconstruct images. This method determines the spatial distribution of the H + density and its relaxation time in human tissues (Huk and Gademann, 1984). Since it utilizes the hydrogen nucleus of water in the body, there is no danger of ionizing radiation. MRI provides images with high spatial resolution and excellent tissue information without radiation, thus offering advantages in diagnosing diseases. Although MRI has long been used for clinical diagnosis, its low sensitivity limits its usefulness. Thus, MRI contrast agents play a crucial role in enhancing image contrast by significantly altering the differences in physicochemical quality of nearby tissues (Aime et al., 2006;Na et al., 2009).
Gd 3+ complexes, such as Gd-DTPA (DTPA: Diethylenetriaminepentaacetic acid) and Gd-EOB-DTPA (EOB: gadolinium ethoxybenzyl), are currently used as MRI contrast agents (Tamada et al., 2009;Werner et al., 2008). However, the low tumor targeting, high renal clearance and toxic side effects of Gd 3+ complexes severely limit their use. Coupling of AuNCs with Gd 3+ complexes is a novel approach to developing MRI contrast agents (Figure 10). The FL properties of AuNCs enable AuNCs probes to be optically imaged, and the combination of multiple imaging modalities can guide subsequent treatment more effectively. MR and FL imaging probes were prepared by covalently grafting Gd-DTPA on BSA-stabilized AuNCs. These probes showed significantly higher Gd 3+ relaxation rates, five times higher than clinical Gd-DTPA while maintaining the FL intensity of AuNCs . Han et al. (2017) prepared nanocrystals of Gd 2 O 3 based on a BSA biomineralization method and constructed a new therapeutic platform by combining them with AuNCs. The platform was loaded with indocyanine green (ICG) for NIRF/MR/CT triple-mode imaging, which allowed simultaneous photothermal and photodynamic therapy due to the absorption of ICG in the NIR range. The relaxation rates of Gd-AuNCs generated in one step by biomineralization were slightly higher than those of the probes as mentioned above, implying that the properties of the clusters were modified by the synthesis process (Le et al., 2016;Liang and Xiao, 2017). Liang and Xiao (2017) investigated the effect of the production process on the optical and chiral characteristics of nanoprobes using BSA and Lys as protein models (Liang and Xiao, 2017). The results showed that AuNCs templated by proteins were first constructed to bind directly to DTPA with higher purity and iScience Review relaxation rates. Compared to BSA-AuNCs, Lys-AuNCs are smaller, exhibit a larger Gd: AuNCs ratio, and show better MR images when using commercial Gd-DTPA probes ( Figure 11A) (Hou et al., 2017). Gd 3+ and the negatively charged carboxyl groups of AuNCs can also self-assemble AuNCs into monodisperse spherical particles (GNCNs) through electrostatic interactions, enhancing the PL of AuNCs. Because of the EPR effect, GNCNs aggregated at the tumor site and showed a high MR signal within 60 min, resulting in a significant increase in the brightness of the tumor site. Wang et al. (2019) covalently linked the scavenger receptor (SRAI)-specific peptide pp1 to Gd-AuNCs to target atherosclerotic plaques and enhance plaque contrast ( Figure 11B). Bonnet et al. (2009) combined a cyclic dodecapeptide (CP) with Gd 3+ , which showed excellent relaxation times. Following this work, altered the complex by replacing three amino acid residues on the CP backbone with tyrosine and two cysteine residues, producing a novel CP that was used to create CP-AuNCs (Liang et al., 2013). When GdCl 3 was added to CP-AuNCs, Gd 3+ adsorbed on the surface of AuNCs via CP to generate Gd-AuNCs, dramatically increasing the chirality of the Gd 3+ complex by 10-fold compared to Gd-DTPA and prolonged the intravascular circulation of Gd 3+ . The addition of Gd 3+ did not affect the size of AuNCs, and their tiny size distribution allowed them to be excreted via urine.

Photoacoustic (PA) imaging
Imaging is a new biomedical imaging approach based on laser ultrasound that has been developed in recent years. High imaging resolution, low acoustic attenuation, and significant tissue penetration depth (about 10 cm) are all advantages of PA imaging (Wang and Hu, 2012;Wang and Yao, 2016). In addition, the non-invasive and iScience Review non-ionizing nature of PA imaging ensures its safety and reproducibility. Endogenous chromophores (oxyhemoglobin, deoxyhemoglobin, melanin, lipids, and collagen) are used as imaging molecules in PA imaging and exogenous contrast agents with solid light absorption properties (fluorescent moieties, precious metal NPs, carbon-based nanomaterials, polymers, and other inorganic materials) (Upputuri and Pramanik, 2020). Exogenous contrast agents are used to improving the resolution and signal-to-noise ratio of PA imaging and to achieve directional imaging. AuNPs exhibiting surface plasmon resonance effects and NIR absorption have attracted much attention as exogenous contrast agents . AuNPs commonly used for PA imaging include Au nanorods, Au nanoflowers, Au nanocages, vesicles, and oversized spherical AuNPs (>5 nm in diameter) Huang et al., 2013;Santos et al., 2021;Wang et al., 2021;Yan et al., 2021).
AuNCs have also been used for PA imaging.  prepared BSA-AuNCs with a particle size of approximately 3 nm as a contrast agent for dual-mode imaging of FL and PA. The imaging capability of the clusters was verified in an adult zebrafish model. AuNCs with ICG were more effective in PA imaging (Higbee-Dempsey et al., 2019). In addition, AuNCs-ICG nanoprobes (AuNCs-INPs), synthesized with AuNCs components conjugated to ICG, can be used not only for NIRF and PA imaging but also for real-time monitoring of tumor therapy (Figure 12) . Compared to ICG, The PA intensity of AuNCs are more intense and significantly increase in intensity with increasing ICG concentration. This probe enhances the accumulation of ICG at the tumor site and prolongs the residence time of ICG, allowing it to be used for targeted tumor iScience Review therapy. Photoacoustic signals were better in AuNCs stabilized by zwitterionic ligands (Shen et al., 2017). Ning et al. (2017) produced AuNPs of various sizes (2.5-6 nm) and investigated the effect of AuNP size and ligand ratio on FL and PA signals. The number of ligands decreases with increasing Au: Zw ratio, which increases the size of Au nuclei. As a result, more heat and more powerful vibrations are generated under light irradiation, increasing the intensity of the PA signal. At the same time, non-radiative losses rise, FL increases and intensity decreases. This finding suggests that the intensity of the PA signal is closely related to the size of the Au nucleus and the number of ligands. Therefore, future studies should focus on modulating the size of the Au nucleus and the coverage of ligands to provide optimal PA and FL imaging results.

SUMMARY AND OUTLOOK
This article presents the scientific progress of AuNCs in bioimaging applications in detail. By adjusting the synthesis method and selecting different ligands for modification, the prepared AuNCs can show tunable FL from the visible to near-infrared region, allowing them to be employed as fluorescent probes for visible and nearinfrared wavelengths. Because of their high atomic number and electron density, AuNCs can also be used as contrast agents for CT and PA imaging. The FL properties of AuNCs can be modified by doping the backbone with metals (such as 64 Cu, 68 Ga, 124 I, Ga 3+ ) and can be used for PET and MR imaging. Because the modification is easy, AuNCs with various ligand alterations can be produced, allowing the creation of imaging probes with many advantages.
Medical research relied on early and precise disease diagnosis and targeted therapy, and the superior physicochemical qualities and biocompatibility of AuNCs make them a particularly intriguing material choice. Despite the tremendous progress made in recent years, many difficulties still need to be addressed before AuNCs may be widely used.
First, most AuNCs currently produced have a low quantum yield, which reduces the detection sensitivity and also affects the detection limit. Second, despite the remarkable biocompatibility of AuNCs, studies on AuNCs imaging have so far included only cellular and animal tests, with no application in humans. Extensive studies on the metabolism, tissue distribution and toxicity of AuNCs are needed for in vivo imaging in humans.
Finally, each imaging modality has its own set of restrictions. Developing nanomaterials that combine multiple imaging modalities to enable nanoplatforms with high sensitivity, real-time, and precise localization of anatomical components could improve disease detection and treatment. Although AuNCs probes have been used in certain multimodal imaging studies, these studies are still in their early stages and need to be further explored.