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

TLDR

Recent advances in far‑field fluorescence microscopy have achieved near‑molecular lateral resolution of 20–30 nm, yet 3D nanoscale imaging remains a challenge. The authors demonstrate 3D stochastic optical reconstruction microscopy (STORM) that uses optical astigmatism to localize individual fluorophores in three dimensions with nanometer accuracy. By iteratively stochastically activating photoswitchable probes and exploiting optical astigmatism, the method achieves high‑precision 3D localization without scanning the sample. The approach delivers 20–30 nm lateral and 50–60 nm axial resolution, allowing the 3D morphology of nanoscopic cellular structures to be resolved.

Abstract

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.