Quantum dots (QDs) have emerged as powerful tools in biomedical imaging due to their unique optical properties, including high brightness, photostability, and tunable emission spectra. These semiconductor nanocrystals, typically ranging from 2 to 10 nanometers in diameter, offer significant advantages over traditional organic fluorophores, particularly for in vivo and in vitro fluorescence labeling applications. Their narrow emission peaks and broad absorption spectra enable multiplexed imaging, while their resistance to photobleaching allows for long-term tracking of biological processes. This article explores the use of quantum dots in biomedical imaging, focusing on biocompatibility, toxicity mitigation, targeting strategies, and advanced imaging techniques.

One of the most critical considerations for using quantum dots in biomedical applications is biocompatibility. Many QDs are composed of heavy metals such as cadmium (Cd), lead (Pb), or mercury (Hg), which pose potential toxicity risks. To address this, researchers have developed encapsulation strategies using biocompatible coatings like polyethylene glycol (PEG), silica, or zwitterionic ligands. These coatings not only reduce toxicity but also improve water solubility and stability in physiological environments. For example, PEGylation of CdSe/ZnS QDs has been shown to decrease cellular uptake by the reticuloendothelial system, prolonging circulation time in vivo. Additionally, cadmium-free alternatives, such as indium phosphide (InP) or carbon-based QDs, have been engineered to minimize toxicity while maintaining optical performance. Surface functionalization with biomolecules like peptides or proteins further enhances biocompatibility by mimicking natural biological interactions.

Toxicity mitigation extends beyond material composition to include size-dependent clearance pathways. Smaller QDs (less than 5.5 nm) are more readily excreted through renal clearance, while larger particles may accumulate in the liver or spleen. Studies have demonstrated that optimizing QD size and surface chemistry can significantly reduce long-term retention in tissues, addressing concerns about heavy metal leaching. For instance, ZnS shells around CdSe cores have been shown to prevent cadmium leakage, even under acidic conditions mimicking lysosomal environments. Moreover, the use of biodegradable coatings, such as poly(lactic-co-glycolic acid) (PLGA), enables gradual breakdown and clearance of QDs post-imaging.

Targeting strategies are essential for achieving specific labeling of cells or tissues. Quantum dots can be conjugated to antibodies, aptamers, or small molecules to direct them to biomarkers of interest. Antibody-conjugated QDs, for example, have been used to label HER2 receptors in breast cancer cells with high specificity. Similarly, folate-functionalized QDs target folate receptor-overexpressing tumors, enabling precise imaging of cancer margins. The high surface-to-volume ratio of QDs allows for multi-valent targeting, where multiple ligands are attached to a single dot, enhancing binding affinity. Additionally, bioorthogonal chemistry, such as click reactions, facilitates site-specific conjugation without interfering with biological processes.

Multiplexed imaging is a key advantage of quantum dots, as their narrow emission peaks enable simultaneous detection of multiple targets within the same sample. By tuning the size and composition of QDs, emissions can span the visible to near-infrared (NIR) spectrum. For example, a single excitation source can activate QDs emitting at 525 nm, 585 nm, and 655 nm, allowing for three-color imaging of distinct cellular markers. This capability is particularly valuable in pathology, where co-localization of biomarkers can provide insights into disease mechanisms. In vivo, NIR-emitting QDs (700–900 nm) are preferred due to reduced tissue autofluorescence and deeper penetration depth, enabling real-time tracking of tumor margins or immune cell migration.

Super-resolution techniques have further expanded the utility of quantum dots in biomedical imaging. Methods such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) leverage the blinking behavior of QDs to achieve resolutions below the diffraction limit (under 20 nm). Unlike organic dyes, which photobleach quickly under super-resolution conditions, QDs maintain their brightness over thousands of frames, enabling prolonged imaging of subcellular structures. For instance, QD-based STORM has been used to visualize synaptic proteins in neurons with nanometer precision, revealing dynamic changes during neurotransmission.

In vitro applications of quantum dots include high-throughput screening and single-molecule tracking. QDs conjugated to streptavidin, for example, are widely used in immunoassays to detect low-abundance proteins with high sensitivity. Their brightness outperforms enzymatic amplification methods, reducing background noise. In single-particle tracking, the photostability of QDs allows for millisecond-scale monitoring of receptor diffusion on cell membranes, providing insights into membrane dynamics and protein interactions.

In vivo imaging with quantum dots presents unique challenges, including tissue scattering and autofluorescence. To overcome these, researchers have developed QDs with emissions in the second NIR window (NIR-II, 1000–1700 nm), where tissue absorption is minimal. NIR-II QDs have been used to image vasculature and tumors in mice with millimeter-depth resolution, offering a non-invasive alternative to traditional histology. Furthermore, time-gated imaging techniques exploit the long fluorescence lifetime of QDs to separate their signal from short-lived autofluorescence, enhancing contrast in deep tissues.

Despite these advances, regulatory hurdles remain for clinical translation of quantum dots. Long-term toxicity studies and standardized protocols for surface functionalization are needed to ensure safety and reproducibility. However, the continued development of cadmium-free QDs, improved targeting strategies, and advanced imaging techniques positions quantum dots as indispensable tools in biomedical research. Their ability to enable multiplexed, super-resolution, and real-time imaging promises to unlock new understandings of biological systems at the molecular level.