Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels ("multilevelSTED") optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.
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http://dx.doi.org/10.1038/srep26725 | DOI Listing |
Adv Mater
January 2025
Department of Chemistry, National University of Singapore, Singapore, 117543, Singapore.
Conjugated oligoelectrolytes (COEs) are lipid bilayer spanning optical reporters that hold promise for delineating spatiotemporal changes in subcellular compartments. However, their ability to probe a broader range of biological processes remains restricted due to the lack of environmentally-responsive chemical functionalities. Herein, the study reports a novel COE, namely COE-KP, for monitoring spatiotemporal changes in the endolysosomal vesicles.
View Article and Find Full Text PDFSci Rep
November 2024
Centre for Cell Imaging, Institute of Systems, Molecular and Integrative Biology, Liverpool, L69 7ZB, UK.
Most embedding media for live and fixed samples were not designed for microscopy and have issues including long polymerization times, peak of toxicity toward the sample during the sol-gel transition, and irreversibility of this transition. Gels derived from biological sources are widely used in microscopy, but their precise composition is ill-defined and can vary between batches. Non-physiological temperatures and/or specific enzymatic solutions are often needed to revert the gel back to the sol state to allow sample recovery.
View Article and Find Full Text PDFMicroscopes generally achieve better 2D imaging compared to 3D, and super-resolution microscopes often aggravate such axial-lateral resolution mismatch. A prime example is vortex phase-based stimulated emission depletion (STED) fluorescence microscopy, which only narrows the point-spread function laterally, thereby increasing the point-spread function (PSF) anisotropy. In this study, we developed a semi-analytical theory based on the Nijboer-Zernike expansion, developed a set of metrics and performed experiments to establish the merits of the alternative, bivortex phase-based, coherent-hybrid STED.
View Article and Find Full Text PDFBiomaterials
March 2025
Department of Chemistry, and Department of Chemical and Biological Engineering, The Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, 999077, PR China; School of Science and Engineering, Shenzhen Institute of Aggregate Science and Technology, The Chinese University of Hong Kong, Shenzhen (CUHK-Shenzhen), Guangdong, 518172, PR China. Electronic address:
Organic photosensitizers (PSs) play important roles in phototheranostics, and contribute to the fast development of precision medicine. However, water-soluble and highly emissive organic PSs, especially those emitting in the short-wave infrared region (SWIR), are still challenging. Also, it's difficult to prepare self-reporting PSs for visualizing the treatment via stimulated emission depletion (STED) nanoscopy.
View Article and Find Full Text PDFSmall Methods
December 2024
Department of Materials Science and Engineering, University of Utah, Salt Lake City, UT, 84112, USA.
Mitochondria are essential organelles that not only undergo dynamic morphological changes but also exhibit functional activities such as mitochondrial membrane potential (MMP). While super-resolution techniques such as stimulated emission depletion (STED) nanoscopy can visualize the ultrastructure of mitochondria and the MMP probe can monitor mitochondria function, few dyes meet both demands. Here, a small molecule (MitoPDI-90) based on perylene diimide with cationic groups is reported and used for mitochondrial STED imaging and MMP indication.
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