Taihe Zhang,Lu Gao,Hong Du,Xiaoyu Huang,Wei Ge,Haisen Ta,Fu Wang
The cell plasma membrane, serving as the fundamental barrier separating cells from the extracellular environment, is a complex and dynamic structure that is crucial to cell biology. The super-resolution observation of plasma membrane morphology and dynamics in living cells, facilitated by stimulated emission depletion (STED) microscopy, holds significant importance for advancing the understanding of plasma membrane functions. Due to the photobleaching and unclear target, the development of plasma membrane nanoprobes suitable for STED imaging remains challenging. In this study, we present red fluorescent carbon dots (R-CDs) characterized by amphiphilicity and photostability. As carbon-based nanomaterials, R-CDs exhibit the targeting ability of the cell plasma membrane, resulting in a superior imaging signal-to-noise ratio. Furthermore, R-CDs enable STED imaging of plasma membranes in living cells, achieving a resolution enhancement of at least 3-fold compared to confocal imaging. Leveraging the excellent performance of R-CDs, the dynamic behavior of microstructures such as vesicles on the plasma membrane was successfully observed through time-lapse STED imaging under nanoscale. Hence, R-CDs, as fluorescent nanoprobes with good photostability and biocompatibility, demonstrate significant potential for long-term STED imaging of live-cell plasma membranes and the exploration of plasma membrane dynamics.
Stimulated emission depletion (STED) nanoscopy is an emerging super-resolution imaging platform for the study of the cellular structure. Developing suitable fluorescent probes of small size, good photostability, and easy functionalization is still in demand. Herein, we introduce a new type of surface-engineered gold nanoclusters (Au NCs) that are ultrasmall (1.7 nm) and ultrabright (QY = 60%) for STED bioimaging. A rigid shell formed by l-arginine (l-Arg) and 6-aza-2-thiothymine (ATT) on the Au NC surface enables not only its strong fluorescence in aqueous solution but also its easy chemical modification for specific biomolecule labeling. Au NCs show remarkable performance as STED nanoprobes, including high depletion efficiency, good photobleaching resistance, and low saturation intensity. Super-resolution imaging has been achieved with these Au NCs, and targeted nanoscopic imaging of cellular tubulin has been demonstrated. Moreover, the circular structure of lysosomes in live cells has been revealed. As a Au NC is also an ideal probe for electron microscopy, dual imaging of Aβ42 aggregates with the single labeling probe of Au NCs has been realized in correlative light and electron microscopy (CLEM). This work reports, for the first time, the application of Au NCs as a novel probe in STED and CLEM imaging. With their excellent properties, Au NCs show promising potential for nanoscale bioimaging.
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