Fluorescence lifetime imaging (FLIM) technology utilizes photoluminescence lifetime instead of intensity as a detection signal to effectively avoid autofluorescence interference from tissues, thereby providing enhanced imaging accuracy and comprehensive information regarding biochemical and cellular environments. The development of high-performance contrast agents is crucial for advancing FLIM technology. Currently, organic dye molecules are highly favored in FLIM owing to their tunable optical properties, good biocompatibility, and low synthesis costs. However, organic molecules tend to aggregate in biological environments, which renders it difficult to maintain a high fluorescence quantum yield and a long fluorescence lifetime, thus limiting their practical application. Therefore, high-performance dye molecules with satisfactory anti-quenching properties are urgently required to advance FLIM imaging in biomedical research. However, the rational molecular design of optimized fluorescence performance remains challenging owing to insufficient understanding regarding the excited-state dynamics within organic dye molecules. Excited-state dynamics is a critical aspect that correlates the dye molecule structure and macroscopic performance; thus, it determines the photophysical properties of the dye molecules. A comprehensive understanding and elucidation of the excited-state dynamics of dye molecules is crucial for guiding the design of high-performance fluorescent agents for FLIM applications.

Molecular steric hindrance was increased to optimize fluorescence performance. Nonetheless, comprehensive investigations into conformation-related excited-state dynamics allow one to elucidate the fundamental factors affecting fluorescence performance. Two boron-dipyrromethene (BODIPY) dyes with different steric hindrances were synthesized in this study. The core of classical BODIPY was decorated at the meso position with p-methylbenzene and trimethylbenzene to prepare P-BDP and MP-BDP, respectively. The optical properties of P-BDP and MP-BDP, including their spectra, quantum yield, and fluorescence lifetime, were analyzed using steady-state and time-resolved fluorescence spectroscopy. Quantum chemical calculations were performed to reveal the effect of molecular conformation on their optical properties. Furthermore, femtosecond transient absorption spectroscopy was performed to elucidate the effect of molecular conformation on the excited-state dynamics of the dyes, which fundamentally determines the optical properties of the molecules. Selected organic molecules with superior fluorescence performance were encapsulated in an amphiphilic copolymer (Pluronic F-127) to construct nanoparticles (NPs). Additionally, their potential for two-photon fluorescence imaging at the biological level was investigated.

The classic BODIPY features a boron-nitrogen heterocyclic core with a pyrrole ring attached to each side. This coplanar configuration of its tricyclic structure offers a significant π-conjugation effect, which imparts a high molar extinction coefficient. The introduction of the boron bridge not only restricts the free rotation and isomerization of the groups but also enhances the rigidity of the molecular structure, thus endowing BODIPY dyes with excellent fluorescence quantum yield and photostability. BODIPY derivatives, i.e., P-BDP and MP-BDP, with different steric hindrances were synthesized. The molecular structures were confirmed using ¹H NMR and ¹³C NMR spectroscopies. Spectroscopic experiments and quantum chemical calculations indicate that steric hindrance minimally affects the spectral profiles and peaks. However, the sizeable steric hindrance affords an ultrahigh photoluminescence quantum yield (PLQY) of 76.7% for MP-BDP, which significantly surpasses that (41.1%) of P-BDP. Accordingly, MP-BDP exhibits a longer fluorescence lifetime (4.3 ns) than P-BDP (2.8 ns). Quantum chemical calculations indicate that the root-mean-square deviation (RMSQ) value of MP-BDP is 0.05 Å, which is significantly lower than that of P-BDP (RMSD=0.22 Å), thereby indicating that P-BDP undergoes greater conformational changes during the transition from the S₀ to S₁ states. Additionally, the reorganization energy of P-BDP is 824.2 cm^-1, which is much higher than that (659.7 cm^-1) of MP-BDP. This suggests that P-BDP with a lower steric hindrance loses more energy through nonradiative transition channels, thus resulting in a lower fluorescence quantum yield. Furthermore, the results of femtosecond transient absorption spectroscopy confirm that the greater steric hindrance in MP-BDP significantly reduces the vibrational relaxation rate, decreases nonradiative energy loss, and enhances fluorescence performance. More importantly, the larger steric groups contribute significantly in inhibiting aggregation-induced quenching, thus allowing MP-BDP NPs to maintain a long fluorescence lifetime (4.8 ns) and high fluorescence efficiency (38.1%) in practical applications. This characteristic enables MP-BDP NPs to realize excellent two-photon fluorescence lifetime imaging of zebrafish.

This study systematically investigated the effect of steric hindrance on the excited-state dynamics of BODIPY. The result shows that steric hindrance improves the fluorescence efficiency and prolongs the fluorescence lifetime of BODIPY by inhibiting nonradiative energy loss through vibrational relaxation. Steady-state spectroscopy and theoretical calculations show that different meso-substituents exhibit similar configurations in the ground state and do not significantly affect the absorption/emission spectra. However, in the excited state, the substituents in MP-BDP exhibit greater steric hindrance. The results of femtosecond transient absorption spectroscopy show that the greater steric effects in MP-BDP effectively suppress the nonradiative energy loss from the S₁ state via vibrational relaxation, thereby extending the fluorescence lifetime and enhancing the fluorescence efficiency. Additionally, the larger steric groups effectively inhibit aggregation-induced quenching, thus affording successful two-photon fluorescence lifetime imaging at the in-vivo level. This study provides deeper understanding regarding the effect of molecular conformation on fluorescence properties from the perspective of excited-state dynamics, thus providing important theoretical insights for designing efficient BODIPY fluorescent dyes and offering new approaches for developing new two-photon FLIM agents.