Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology Dynamics.

Super-resolution fluorescence imaging has emerged as a potent tool for investigating the nanoscale structure and function of the plasma membrane (PM). Nevertheless, the challenge persists in achieving super-resolution imaging of PM dynamics due to limitations in probe photostability and issues with cell internalization staining. Herein, we report assembly-mediated buffering fluorogenic probes BMP-14 and BMP-16 exhibiting fast PM labeling and extended retention time (over 2 h) on PM. The incorporation of alkyl chains proves effective in promoting the aggregation of BMP-14 and BMP-16 into nonfluorescent nanoparticles to realize fluorogenicity and regulate the buffering capacity to rapidly replace photobleached probes ensuring stable long-term super-resolution imaging of PM. Utilizing these PM-buffering probes, we observed dynamic movements of PM filopodia and continuous shrinkage, leading to the formation of extracellular vesicles (EVs) using structured illumination microscopy (SIM). Furthermore, we discovered two distinct modes of EV fusion: one involving fusion through adjacent lipids and the other through filamentous lipid traction. The entire process of EV fusion outside the PM was dynamically tracked. Additionally, BMP-16 exhibited a unique capability of inducing single-molecule fluorescence blinking when used for cell membrane staining. This property makes BMP-16 suitable for the PAINT imaging of cell membranes.

Solvatochromic Buffering Fluorescent Probe Resolves the Lipid Transport and Morphological Changes during Lipid Droplet Fusion by Super-Resolution Imaging.

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.

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

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 super-resolution 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-lysosome-resolved 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.