Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain.

Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

General Strategy to Prepare Single-Layered Ag-Au-Pt Nanocrystal Ternary-Coated Biomass Textiles through Polymer-Driven Self-Assembly.

Current metal nanomaterials for developing nanofunctional textiles are mostly based on metal nanoparticles (NPs) that show aqueous instability, a tendency to aggregate, and low chemical affinity to biomass textiles, leading to low nano-metal uptake during finishing, significant declines in function, and nano-pollution. Herein, we demonstrate a strategy to transform metal (Ag, Au, and Pt) NPs into homogenous hyperbranched poly(amide-amine) (HBPAA)-encapsulated NPs showing high water solubility, oxidative resistance, and affinity to biomass materials upon surface capping with HBPAA. The proposed method represents a universal, simple, clean, and efficient self-assembly technology to produce monolayered Ag-Au-Pt ternary-coated biomass textiles. The combination of Ag, Au, and Pt NPs yields a positive potential of approximately +37.12 mV depending on the metal concentration and could simultaneously self-assemble onto natural fibers, including cotton, silk, and wool, through the one-step impregnation of textiles. Increasing the temperature and concentration of the mixture favors the self-assembly process. A mixture of 30-110 mg/L Ag, Au, and Pt NPs could nearly completely anchor onto cotton, silk, and wool textiles after impregnation at 100 °C for 1 h without chemical assistance, thereby indicating the possibility of clean production. As-prepared functional cotton, silk, and wool possessed similarly high antibacterial activities, and a mixture containing over 1500 mg/g NPs inhibited 99% of the Escherichia coli and Staphylococcus aureus in the sample textiles. The developed coating technology is simple, clean, controllable, and broadly applicable; thus, it could be potentially applied in functional textiles.

Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging.

Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies on optical imaging, yet these membranous hubs of bioenergy are exceptionally vulnerable to photodamage. We report that cyclooctatetraene-conjugated cyanine dyes (PK Mito dyes), are ideal mitochondrial probes with remarkably low photodynamic damage for general use in fluorescence cytometry. In contrast, the nitrobenzene conjugate of Cy3 exhibits enhanced photostability but unaffected phototoxicity compared to parental Cy3. PK Mito Red, in conjunction with Hessian-structural illumination microscopy, enables 2000-frame time-lapse imaging with clearly resolvable crista structures, revealing rich mitochondrial dynamics. In a rigorous stem cell sorting and transplantation assay, PK Mito Red maximally retains the stemness of planarian neoblasts, exhibiting excellent multifaceted biocompatibility. Resonating with the ongoing theme of reducing photodamage using optical approaches, this work advocates the evaluation and minimization of phototoxicity when developing imaging probes.