Engineering Near-Infrared Fluorescent Probes Based on Modulation of Molecular Excited States

Fluorescent dyes have revolutionized the way we study life science and conduct medical diagnostics. Compared with visible wavelengths, near-infrared (NIR) fluorescence has gained significant attention due to its unique properties, such as deep tissue penetration, reduced autofluorescence, and improved signal-to-noise ratios, making it highly desirable for a wide range of in vivo applications including noninvasive sensing, cancer research, and drug delivery. In fluorescence sensing, the absorption of light by a fluorophore and its subsequent transition to an excited state are critical steps. Once in the excited state, the molecule may undergo various relaxation processes including internal conversion, vibrational relaxation, and radiative/nonradiative decay. These processes directly impact the fluorophore's emission wavelength, brightness, photostability, and other properties. Therefore, rational modulation of molecular excited states is vital for achieving effective fluorescence sensing. However, NIR fluorophores with a small S-0-S-1 energy gap, as governed by the energy gap law, exhibit much faster nonradiative deactivation pathway compared to dyes in the visible region. This fast relaxation process makes them more susceptible to interference from molecular aggregation behavior, environmental factors, and so on. Thus, there is often a trade-off effect between achieving a tunable red-shift wavelength and a desirable performance in light-up imaging and quantitative sensing. Overcoming these challenges requires careful engineering of molecular structures and modulation of excited states to achieve the desired balance between extending emission wavelength and performance in NIR bioimaging. In this Account, we present our recent progress in manipulating molecular excited states for the rational design of NIR fluorescent probes. Specifically, we focus on engineering novel molecular building blocks, exploring photophysical mechanisms, and regulating assembly behavior (to inhibit or amplify excited state intramolecular motion in aggregates), aiming to resolve long-standing issues in lighting-up mapping, quantitative sensing, and so on. First, we introduce the monochromophore-based "reliable ratiometric" strategy with additional emission, enabling NIR fluorescence quantification of hypoxia and biomolecules. Second, we demonstrate how to reverse the excited state rotation driving energy, achieving completely overturning the intramolecular charge transfer (ICT) fluorophores' quenching mode into the light-up mode. Third, we discuss the relationship between the NIR chromophore aggregation behavior and excited state relaxation. Through inhibiting or amplifying excited state intramolecular motion, it could well improve imaging fidelity and theranostic outcomes. Finally, we explore future perspectives and challenges of modulation of molecular excited states in dynamic NIR fluorescent bioimaging. It is hoped that this Account provides a deepening of research on molecular excited states and guidance for the development of novel high-performance NIR probes for physiological/pharmacological studies and clinical applications.