Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton

• Published in: Nature methods Vol. 9; no. 2; pp. 185 - 188
• Main Authors: Xu, Ke, Babcock, Hazen P, Zhuang, Xiaowei
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.02.2012; Nature Publishing Group
• Subjects: 631/1647/245/2221; 631/1647/328/2238; 631/535; 631/80/128/1276; Actin; Actins - chemistry; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; brief-communication; Cell Line; Cytoskeleton; Cytoskeleton - chemistry; Fluorescence microscopy; Intermediate filament proteins; Life Sciences; Microscopy; NMR; Nuclear magnetic resonance; Physiological aspects; Proteomics; United States
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/nmeth.1841

The use of dual-objective detection with astigmatism-based three-dimensional stochastic optical reconstruction microscopy (STORM) imaging improves resolution more than twofold and removes noise in resulting super-resolution images. This allowed detailed fluorescence imaging of distinctive features of the three-dimensional actin cytoskeleton ultrastructure with single-filament resolution in cells. By combining astigmatism imaging with a dual-objective scheme, we improved the image resolution of stochastic optical reconstruction microscopy (STORM) and obtained <10-nm lateral resolution and <20-nm axial resolution when imaging biological specimens. Using this approach, we resolved individual actin filaments in cells and revealed three-dimensional ultrastructure of the actin cytoskeleton. We observed two vertically separated layers of actin networks with distinct structural organizations in sheet-like cell protrusions.