Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.
- MeSH
- algoritmy MeSH
- antigeny CD4 genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- fluorescenční barviva MeSH
- fluorescenční mikroskopie metody statistika a číselné údaje MeSH
- Jurkat buňky MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- mutantní proteiny genetika metabolismus MeSH
- optické zobrazování metody statistika a číselné údaje MeSH
- shluková analýza MeSH
- T-lymfocyty imunologie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
- Názvy látek
- antigeny CD4 MeSH
- fluorescenční barviva MeSH
- membránové proteiny MeSH
- mutantní proteiny MeSH
