Structures and dynamics in live bacteria revealed by super-resolution fluorescence microscopy
Contributors: Zhuang, Xiaowei; Xie, Xiaoliang S.; Samuel, Aravinthan
Published: 2012
Language: eng
Description: Light microscopy, in particular fluorescence microscopy, is a widely used imaging method in biological research due to its noninvasive nature and molecular specificity. The resolution of conventional fluorescence microscopy is limited to a few hundred nanometers by the diffraction of light, leaving many biological structures too small to be optically resolved. Stochastic Optical Reconstruction Microscopy (STORM) technique overcomes this limit by localizing single photoswitchable fluorophores separated in time. We further extended the then two-dimensional capability to three-dimensional (3D) STORM by determining both axial and lateral positions of individual fluorophores with nanometer accuracy using optical astigmatism. Iterative, stochastic activation of photo-switchable probes enables high-precision 3D localization of each probe and thus the construction of a 3D image without scanning the sample. We achieved an image resolution of 20 - 30 nm in the lateral dimensions and 50 - 60 nm in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.
Notes: Thesis Ph.D. Harvard University 2012