Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells.

Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms.

Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit.

Total internal reflection STED microscopy.

Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution.