Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.
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.
Huang
Bo
B
Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
Wang
Wenqin
W
Bates
Mark
M
Zhuang
Xiaowei
X
D000818:Animals; D002232:Carbocyanines; D002460:Cell Line; D002522:Chlorocebus aethiops; D002966:Clathrin; D003034:Coated Pits, Cell-Membrane / Q000648:ultrastructure*; D000242:Cyclic AMP / Q000031:analogs & derivatives; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008870:Microtubules / Q000648:ultrastructure*; D036103:Nanotechnology; D045663:Quantum Dots; D013269:Stochastic Processes; D019809:Streptavidin
D002232:Carbocyanines; D002966:Clathrin; D005456:Fluorescent Dyes; C089187:cyanine dye 3; C067297:8-aminohexylamino cAMP; D019809:Streptavidin; D000242:Cyclic AMP