Single-Molecule 3D Nanoscopy for Application to Cryofixed Samples

Under cryogenic conditions below approximately 100 K, the three-dimensional (3D) structures and spatial arrangements of biomolecules are preserved at the angstrom scale in vitrified ice. In addition, the photobleaching rate of fluorophores is reduced by several orders of magnitude, enabling nanometer-scale localization accuracy in the lateral dimension for individual fluorophores conjugated to target biomolecules. However, the axial localization accuracy has remained limited to several tens of nanometers, approximately 25-fold worse than predictions by optical simulations. Here, we demonstrate a cryogenic 3D nanoscopy capable of localizing individual fluorophores with nanometer-scale accuracy in 3D. This system employs multifocal plane detection to achieve near shot-noise-limited localization by minimizing three major sources of error: blinking noise, dipole orientation effects, and background emission. To evaluate the 3D localization accuracy, we conjugated two fluorophores to the end of a 17 nm double-stranded DNA (dsDNA) molecule. To reduce the localization errors due to the dipole orientation of the fluorophores, individual dsDNA molecules were positioned within ± 50 nm of the focal plane of the microscope objective. Under these conditions, the measured 3D distance between fluorophores at 1.9 K was 20 ± 8 nm, consistent with the designed length of DNA. The localization error was comparable to the shot-noise limit.