Ultrasmall fluorescent gold nanoclusters represent a significant advancement in bioimaging due to their atomically precise structure, tunable emission in the red to near-infrared (NIR) range, and excellent biocompatibility. Unlike larger plasmonic gold nanoparticles, which rely on surface plasmon resonance for optical properties, these nanoclusters exhibit molecule-like fluorescence due to quantum confinement effects. Their ultrasmall size, typically below 2 nm, allows for efficient renal clearance and reduced nonspecific interactions, making them ideal for high-contrast imaging in biological systems.

The atomically precise structure of gold nanoclusters is a defining feature. These clusters consist of a few to several dozen gold atoms, often stabilized by ligands such as thiolates, phosphines, or biomolecules. The exact atomic arrangement determines their electronic transitions and emission properties. For example, Au25(SR)18, where SR represents a thiolate ligand, exhibits a well-defined structure with a central Au13 icosahedron protected by six Au2(SR)3 units. This precise configuration leads to discrete energy levels, enabling fluorescence with large Stokes shifts and minimal photobleaching compared to organic dyes.

Synthesis methods play a crucial role in determining the properties of gold nanoclusters. Protein-templated synthesis has emerged as a particularly effective approach for producing biocompatible clusters. Proteins such as bovine serum albumin (BSA), lysozyme, and transferrin act as scaffolds, stabilizing the clusters while also providing functional groups for further modifications. In BSA-templated synthesis, gold ions are reduced in the presence of the protein, forming clusters within its cysteine-rich pockets. This method yields nanoclusters with emission peaks tunable between 600 and 800 nm, depending on the protein and reaction conditions. The resulting clusters inherit the protein’s biocompatibility and can be further functionalized for targeting specific cellular structures.

Stability is a critical factor for bioimaging applications. Gold nanoclusters must maintain their fluorescence and structural integrity in physiological conditions. Ligand choice significantly influences stability; thiolate-protected clusters exhibit robust resistance to oxidation and aggregation, while protein-templated clusters benefit from the natural shielding provided by the protein matrix. Studies have shown that BSA-stabilized gold nanoclusters retain their fluorescence for weeks in buffer solutions and under laser irradiation, outperforming many organic fluorophores. However, challenges remain in high-salt environments or in the presence of competing thiols, which can lead to ligand displacement and fluorescence quenching.

The red to NIR emission of gold nanoclusters is highly advantageous for bioimaging. This spectral range minimizes interference from tissue autofluorescence and allows deeper penetration due to reduced scattering and absorption by hemoglobin and water. For instance, clusters emitting at 650–700 nm have been used for in vivo imaging with high signal-to-noise ratios. Their long fluorescence lifetimes further enable time-gated imaging, effectively suppressing background signals. Unlike larger gold nanoparticles, which are limited to scattering-based contrast, nanoclusters provide both luminescence and the ability to penetrate tissues more effectively.

Applications in organelle-specific imaging highlight the versatility of gold nanoclusters. By conjugating targeting moieties such as peptides or small molecules, these probes can localize to mitochondria, nuclei, or lysosomes with high specificity. Mitochondria-targeting clusters, for example, utilize triphenylphosphonium derivatives that exploit the organelle’s membrane potential for accumulation. Nuclear localization sequences derived from viral proteins enable clusters to traverse the nuclear pore complex, allowing for real-time monitoring of nuclear dynamics. The ultrasmall size ensures minimal disruption to cellular processes, a significant advantage over larger nanoparticles that may induce steric hindrance or stress responses.

Gold nanoclusters offer several advantages over traditional plasmonic gold nanoparticles. Plasmonic particles, typically larger than 10 nm, lack intrinsic fluorescence and rely on surface-enhanced Raman scattering or two-photon luminescence for imaging, which often requires complex instrumentation. In contrast, nanoclusters provide direct, tunable fluorescence without the need for additional enhancement techniques. Their small size also facilitates faster diffusion and better access to subcellular compartments, whereas larger particles may be restricted to extracellular or endosomal spaces. Additionally, nanoclusters do not exhibit the photothermal effects associated with plasmonic particles, reducing the risk of localized heating during prolonged imaging.

Despite these advantages, challenges remain in optimizing gold nanoclusters for widespread use. One major limitation is their relatively low quantum yield, typically below 10%, which can restrict sensitivity in low-abundance target imaging. Strategies to improve quantum yield include ligand engineering to reduce nonradiative decay pathways and doping with other metals such as silver or copper to enhance emission intensity. Another challenge is batch-to-batch variability in synthesis, particularly for protein-templated methods, where slight changes in pH, temperature, or protein conformation can alter cluster properties. Standardization of protocols and rigorous characterization are essential to ensure reproducibility.

Future directions for gold nanoclusters in bioimaging include multimodal applications and theranostics. Their ability to serve as both imaging agents and drug carriers has been demonstrated in proof-of-concept studies, where clusters conjugated with therapeutic compounds enabled simultaneous imaging and treatment. The integration of radioactive isotopes or magnetic resonance contrast agents could further expand their utility in hybrid imaging techniques such as PET-fluorescence or MRI-fluorescence. Advances in ligand design and surface chemistry will continue to refine their targeting efficiency and stability in complex biological environments.

In summary, ultrasmall fluorescent gold nanoclusters represent a promising class of bioimaging probes with unique advantages stemming from their atomic precision, red/NIR emission, and biocompatibility. Their synthesis, particularly through protein-templated methods, offers a pathway to highly functionalized and stable probes capable of organelle-specific imaging. While challenges such as low quantum yield persist, ongoing research into ligand engineering and hybrid nanostructures holds significant potential for overcoming these limitations and expanding their applications in biomedical imaging.