Two highly selective two-photon fluorescent probes for cysteine over homocysteine, N-acetyl-L-cysteine, dithiothreitol, glutathione and other amino acids, and their fluorescent imaging in living cells have been shown.

Lipid droplets are highly associated with obesity, diabetes, inflammatory disorders and cancer. A reliable two-photon dye for specific lipid droplets imaging in live cells and live tissues at ultra-low concentration has rarely been reported. In this work, four new aggregation-induced emission luminogens (AIEgens) based on the naphthalene core were designed and synthesized for specific two-photon lipid droplets staining. The new molecules, namely NAP AIEgens, exhibit large Stokes shift (>110 nm), high solid-state fluorescence quantum yield (up to 30%), good two-photon absorption cross section (45–100 GM at 860 nm), high biocompatibility and good photostability. They could specifically stain lipid droplets at ultra-low concentration (50 nM) in a short time of 15 min. Such ultra-low concentration is the lowest value for lipid droplets staining in live cells reported so far. In vitro and ex vivo two-photon imaging of lipid droplets in live cells and live mice liver tissues were successfully demonstrated. In addition, selective visualization of lipid droplets in live mice liver tissues could be achieved at a depth of about 70 μm. These excellent properties render them as promising candidates for investigating lipid droplets-associated physiological and pathological processes in live biological samples.

Visualizing and tracking mitochondrial dynamic changes is crucially important in the fields of physiology, pathology and pharmacology. Traditional electrostatic-attraction based mitochondrial probes fail to visualize and track the changes due to their leakage from mitochondria when mitochondrial membrane potential (MMP) decreases. Reaction-based MitoTracker probes can realize visualization and tracking of mitochondria changes independent of MMP changes. However, such probes impair mitochondrial proteins and exhibit high cytotoxicity. Therefore, it still remains challenging to explore reaction-free and highly biocompatible probes for visualizing and tracking mitochondrial dynamics independent of MMP fluctuations. Herein we synthesized two reaction-free fluorescent mitochondrial probes ECPI-12 and IVPI-12 bearing a long C₁₂-alkyl chain. These cationic probes can firmly immobilize in the mitochondrial inner membrane by strong hydrophobic interaction between the C₁₂-alkyl chain and lipid bilayer, resulting in high specificity and long-term mitochondrial staining regardless of MMP changes. They also exhibit large two-photon absorption cross-sections and show deep penetration in live tissues in two-photon microscopy. Furthermore, they display excellent biocompatibility and realize in situ and real-time mitophagy tracking in live cells. These excellent properties could make ECPI-12 and IVPI-12 the first selective tools for long-term visualization and tracking of mitochondrial dynamics.