Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy

• Published in: Science (American Association for the Advancement of Science) Vol. 319; no. 5864; pp. 810 - 813
• Main Authors: Huang, Bo, Wang, Wenqin, Bates, Mark, Zhuang, Xiaowei
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for the Advancement of Science 08.02.2008; The American Association for the Advancement of Science
• Subjects: Animals; Biological and medical sciences; Carbocyanines; Cell Line; Cercopithecus aethiops; Clathrin; Coated Pits, Cell-Membrane - ultrastructure; Coordinate systems; Cyclic AMP - analogs & derivatives; Exact sciences and technology; Fluorescence; Fluorescent Antibody Technique; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Fundamental areas of phenomenology (including applications); General aspects, investigation technics, apparatus; Geodetic position; Glass; Image Processing, Computer-Assisted; Image reconstruction; tomography; Image resolution; Imaging; Imaging and optical processing; Imaging, Three-Dimensional - instrumentation; Imaging, Three-Dimensional - methods; Innovations; Microscopy; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Microtubules; Microtubules - ultrastructure; Molecules; Nanotechnology; Optics; Physics; Quantum Dots; Scientific apparatus & instruments; Stochastic Processes; Streptavidin; Three dimensional imaging; Tissues, organs and organisms biophysics; Optical microscopy; Fluorophore; Tridimensional image; Nanostructures; Immunofluorescence; Fluorescence microscopy; Image reconstruction; Biomedical imaging
• ISSN: 0036-8075; 1095-9203
• DOI: 10.1126/science.1153529

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.