We demonstrate the nanometer accuracy of far-field fluorescence localization microscopy at a temperature of 1.8 K using near-infrared and red fluorophores bonded to double-stranded DNA molecules (10.2 nm length). Although each fluorophore was localized with a 1 nm lateral precision by acquiring an image at one axial position within the focal depth of ±0.7 μm, the distance between the two fluorophores on the lateral plane (D xy ) was distributed from 0 to 50 nm. This systematic error was mainly due to detecting with the large focal depth the dipole emission from orientationally fixed fluorophores. Each fluorophore was localized with precisions of ±1 nm (lateral) and simultaneously ±11 nm (axial) by acquiring images every 100 nm in the axial direction from −900 to 900 nm. By correcting the dipole orientation effects, the distribution of D xy was centered around the DNA length. The average and standard deviation of D xy were 10 and 5 nm.

Among imaging techniques, fluorescence microscopy is a unique method to noninvasively image individual molecules in whole cells. If the three-dimensional spatial precision is improved to the angstrom level, various molecular arrangements in the cell can be visualized on an individual basis. We have developed a cryogenic reflecting microscope with a numerical aperture of 0.99 and an imaging stability of 0.05 nm in standard deviation at a temperature of 1.8 K. The key optics to realize the cryogenic performances is the reflecting objective developed by our laboratory. With this cryogenic microscope, an individual fluorescent molecule (ATTO647N) at 1.8 K was localized with standard errors of 0.53 nm (x), 0.31 nm (y), and 0.90 nm (z) when 10 fluorescence photons from the molecule were accumulated in 5 min.

We developed a cryogenic objective mirror [Toratani-Fujiwara (TORA-FUJI) mirror] with a 36-μm field of view (FOV) radius and a 0.93 numerical aperture. The latest reported cryogenic objective mirror (INAGAWA mirror) under a superfluid-helium immersion condition had a nearly maximum numerical aperture (0.99) and was perfectly achromatic. However, its FOV radius was restricted to 1.5 μm, mainly due to coma aberration. In the TORA-FUJI mirror, correcting coma aberration realized the 36-μm FOV radius. In addition, the remaining four Seidel aberrations and the chromatic aberrations were sufficiently corrected. To evaluate the optical performance, the cryogenic fluorescence imaging of individual dyes was performed with the TORA-FUJI mirror at a 685-nm excitation wavelength. This result shows that the TORA-FUJI mirror in superfluid helium at 1.8 K exhibits nearly diffraction-limited performance in the FOV region.

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.