研究目的
To develop and characterize ultrastructure expansion microscopy (U-ExM) for the visualization of preserved ultrastructures by optical microscopy, enabling near-native expansion of diverse structures in vitro and in cells, and to combine it with super-resolution microscopy to unveil details of ultrastructural organization.
研究成果
U-ExM preserves ultrastructural details and can be used to visualize the molecular architecture of diverse multiprotein complexes. It alleviates antibody competition and prevents fluorophore loss, improving structural integrity. The method, combined with STED imaging, can unveil the chirality of the centriole, a feature previously revealed only by super-resolution microscopy.
研究不足
The study acknowledges that the preservation of molecular architecture of organelles by ExM and MAP protocols remains unclear. The method requires careful optimization to enable isotropic expansion of molecular assemblies.
1：Experimental Design and Method Selection:
The study involved the development of U-ExM, an extension of expansion microscopy, to visualize preserved ultrastructures. The methodology included immunolabeling, physical expansion of samples, and imaging by confocal and super-resolution microscopy.
2：Sample Selection and Data Sources:
Isolated Chlamydomonas centrioles were used as reference structures due to their characteristic ninefold microtubule triplet-based symmetry. Human U2OS cells and Chlamydomonas reinhardtii cells were also used.
3：List of Experimental Equipment and Materials:
The study utilized a Leica TCS SP8 confocal microscope, a commercial STED microscope (Expert Line, Abberior-Instruments, Germany), and an inverted microscope (Zeiss Axio Observer.Z1) for dSTORM imaging. Reagents included FA, SA, AA, BIS, and various antibodies.
4：Experimental Procedures and Operational Workflow:
The workflow involved sample preparation, immunolabeling, gelation, expansion, and imaging. Different protocols (ExM, MAP, U-ExM) were tested and compared.
5：Data Analysis Methods:
Data analysis included measuring centriole diameter, isotropic expansion, and ninefold symmetry using Fiji software. Statistical analyses were performed using one-way ANOVA and unpaired two-tailed t-tests.
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