Small-Molecule NIR-II Probes for Biological Imaging: A Four-Dimensional Design Framework

The second near-infrared (NIR-II, 1000-2000 nm) window, also known as the short-wave infrared (SWIR), represents a transformative frontier for optical bioimaging, offering unprecedented penetration depth and minimal autofluorescence in living tissues. Since 2015, small-molecule fluorophores have been recognized as promising platforms for translating these optical advantages into biological discovery. Yet converting NIR-II fluorophores into reliable, application-ready probes remains challenging because multiple performance requirements must be balanced simultaneously, often under conflicting constraints. Over the past decade, feedback from practical biological applications suggests that the design of an ideal NIR-II molecular probe should be considered across four key dimensions: optical performance (O), multiplexed capability (M), in vivo delivery (D), and biological specificity (S). In this Account, we summarize our efforts to address these challenges through molecular engineering within this OMDS framework. First, we discuss strategies for optimizing optical performance by balancing red shift and brightness through control of electronic structure and aggregation behavior. Second, we describe how spectral engineering of lanthanide complexes expands multiplexed capability, enabling excitation-encoded imaging and ratiometric sensing beyond conventional emission-resolved approaches. Third, we highlight molecular design principles for improving in vivo delivery, including tuning charge distribution, hydrophilicity, and scaffold size to achieve programmable pharmacokinetics and favorable biological transport. Finally, we discuss chemigenetic strategies that integrate the tunable photophysics of small-molecule fluorophores with the precision of genetic targeting to achieve selective labeling and dynamic sensing in living systems. Together, these studies establish a molecular design framework that links fluorophore structure to NIR-II optical behavior and biological performance. We anticipate that this OMDS-guided perspective will support the development of the next generation of reliable, application-ready NIR-II molecular probes for biomedical imaging and chemical biology.