An Acid‐Regulated Self‐Blinking Fluorescent Probe for Resolving Whole‐Cell Lysosomes with Long‐Term Nanoscopy

Abstract

Long-term super-resolution imaging appears to be increasingly important for unraveling organelle dynamics at the nanoscale, but is challenging due to the need for highly photostable and environment-sensitive fluorescent probes. Here, we report a self-blinking fluorophore that achieved 12 nm spatial resolution and 20 ms time resolution under acidic lysosomal conditions. This fluorophore was successfully applied in superresolution imaging of lysosomal dynamics over 40 min. The pH dependence of the dye during blinking made the fluorophore sensitive to lysosomal pH. This probe enables simultaneous dynamic and pH recognition of all lysosomes in the entire cell at the single-lysosomeresolved level, which allowed us to resolve whole-cell lysosome subpopulations based on lysosomal distribution, size, and luminal pH. We also observed a variety of lysosome movement trajectories and different types of interactions modes between lysosomes.

Photo-Uncaging Triggers on Self-Blinking to Control Single-Molecule Fluorescence Kinetics for Super-Resolution Imaging

“…This strategy was first elucidated by Urano group by a landmark Sirhodamine derivative (HMSiR), 28,29 and later explored by different groups. [30][31][32][33][34][35] For the stabilizing strategy, hydrogen-bonding functional groups were directly introduced to the fluorophore scaffold, improving the stability of zwitterionic open-ring or leuco close-ring structure. [20][21][22] However, these strategies fail to tune the entire spectrum of the single-molecule blinking kinetics.…”

Photoactivable AIEgen-based Lipid-Droplet-Specific Drug Delivery Model for Live Cell Imaging and Two-Photon Light-Triggered Anticancer Drug Delivery

“…Lipid droplets (LDs) are unique organelles composed of core lipid elements surrounded by an amphipathic lipid layer containing several proteins. − Importantly, the behavior and localization of LDs in cells are closely related to metabolic diseases like obesity, cancer, fatty liver, liver cirrhosis, hyperlipidemia, atherosclerosis, inflammation, and Alzheimer’s. − Recent findings indicate that tumor progression is inextricably linked to LDs, with a higher content of LDs or LD-related proteins observed in cancer cells than in normal cells. , For example, a large number of LDs are found in renal cancers, specifically clear cell renal cell carcinoma, prostate cancer, pancreatic cancer, and colon cancer. − Hence, the tracking and imaging of intracellular LDs are of significant importance for early cancer diagnosis. Several LD probes based on fluorophores, including Nile Red, BODIPY, AIEgens (Aggregation-Induced Emission luminogens), azafluorenone, benzothiadiazole, triphenylamine conjugate with bromobenzylidene (TPA-BI), and Stato-merocyanine, were recently developed. − However, only monitoring the LDs in the live cancer cells is not a solution for cancer ablation . It requires an on-site spatiotemporally controlled delivery of anticancer drugs.…”

A Bright, Photostable, and Far-Red Dye That Enables Multicolor, Time-Lapse, and Super-Resolution Imaging of Acidic Organelles

“…Protein tags that react covalently with a small-molecule fluorophore provide more spectral options and better photostability but still require transfection of the cells under study and thus cannot be used in many nonmodel cell lines. − Small-molecule fluorophores are well suited to address these problems, as they are compatible with virtually any cell type, and the labeling protocol requires only a simple same-day incubation. Indeed, commercially available LysoTracker dyes and recently reported LysoPB Yellow, among others, share these benefits. − However, only a handful of lysosome probes, including LysoTracker Deep Red (LTDR), have been reported that absorb above 600 nm, which is required for long-term live-cell imaging, as higher-energy excitation is cytotoxic. Unfortunately, these dyes lack the photostability required for long-time-lapse SRM. − …”

“…Indeed, commercially available LysoTracker dyes and recently reported LysoPB Yellow, among others, share these benefits. 18 − 26 However, only a handful of lysosome probes, including LysoTracker Deep Red (LTDR), have been reported that absorb above 600 nm, which is required for long-term live-cell imaging, as higher-energy excitation is cytotoxic. Unfortunately, these dyes lack the photostability required for long-time-lapse SRM.…”