Genetik Olarak Kodlanmış Flor Depozisyonu Renklendirilmiş Elektron Mikroskobunu Mümkün Kılıyor

Özet (abstract)

Abstract Nanoscale ultrastructure and molecular distribution determine cell functioning, especially the delicate information processing of neurons. While electron microscopy visualizes subcellular ultrastructures peerlessly, localizing biological molecules at the nanoscale requires compatible molecular markers. Popular fluorescent protein markers suffer from the harsh fixatives necessary for ultrastructure preservation, while most electron-diffracting markers require membrane penetration for successful delivery. Here, we introduce genetically encoded fluorine deposition as an electron microscopy-compatible marker to overcome the limitations. By utilizing a peroxidase APEX2 and a stable, commercially available fluorine-containing substrate, bisphenol AF (BPAF), we succeeded in the targeted deposition of fluorine. Fluorine deposition provided a clear contrast based on its rarity in living systems, survived heavy metal ion staining and Epon embedding by its chemical stability, and avoided obscuring the endogenous structural contrast. The distribution of fluorine and thus the target was mapped with nanoscale secondary ion mass spectrometry (nanoSIMS). NanoSIMS maps molecules carrying a unique element or isotope at suboptical resolution on the same ultrathin section as scanning electron microscopy. As a demonstration, organelles including mitochondria and the α-tubulin molecule, were highlighted in human embryonic kidney cells (HEK 293T) by combined mapping with nanoSIMS and scanning electron microscopy. This specific and easy-to-use fluorine marker opens the possibility of multicolored nanoscale imaging with multiple elements and isotopes.

Yazarların özeti; kaynağından alınmıştır. Chemical & Biomedical Imaging, 2026 · DOI ↗