Variable immersion microscopy with a high numerical aperture.

Three-dimensional (3D) optical microscopy with a high numerical aperture (NA) remains challenging for thick biological specimens owing to aberrations arising from interface refractions. We developed a variable immersion lens (VIL) to passively minimize these aberrations. A VIL is a high-NA concentric meniscus lens and was used in combination with an aberration-corrected high-NA reflecting objective (TORA-FUJI mirror). Wave-optics simulation at a wavelength of 488 nm showed that a VIL microscope enables diffraction-limited 1.2-NA imaging in water (refractive index of 1.34) at a depth of 0.3 mm by minimizing aberrations due to refraction of a sample interface. Another aberration due to the refractive index mismatching between a mounting medium, and an object can also be corrected by the VIL system, because various fluids with different refractive indices can be used as mounting media for the VIL. As a result of correcting the two aberrations at the same time, we experimentally demonstrated that a 6 µm diameter fluorescent bead can be imaged to the true dimensions in 3D.

Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami.

Far-field fluorescence localization nanoscopy of individual fluorophores at a temperature of 1.8 K was demonstrated using DNA origami as a one-nanometer-accurate scaffold. Red and near-infrared fluorophores were modified to the scaffold, and the fluorophores were 11 or 77 nm apart. We performed the localization nanoscopy of these two fluorophores at 1.8 K with a far-field fluorescence microscope. Under the cryogenic conditions, the fluorophores were perfectly immobilized and their photobleaching was drastically suppressed; consequently, the lateral spatial precision (a measure of reproducibility) was increased to 1 nm. However, the lateral spatial accuracy (a measure of trueness) remained tens of nanometers. We observed that the fluorophore centroids were laterally shifted as a function of the axial position. Because the orientation of the transition dipole of the fluorophores was fixed under cryogenic conditions, the anisotropic emission from the single fixed dipole had led to the lateral shift. This systematic error due to the dipole-orientation effect could be corrected by the three-dimensional localization of the individual fluorophores with spatial precisions of (lateral) 1 nm and (axial) 17 nm. In addition, the xy-error arising from the three-dimensional (3D) orientation of the scaffold with the two fluorophores 11 nm apart was estimated to be 0.3 nm. As a result, the individual fluorophores on the DNA origami were localized at the designed position, and the lateral spatial accuracy was quantified to be 4 nm in the standard error.