Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami

Abstract

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.

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

“…In one application, the separation of two dyes with a distance resolution of ~0.04 nm was achieved using a hinged construct [ 30 ]. Although dyes can be precisely positioned on the DNA origami template under cryogenic temperature conditions [ 31 ], in the last few years the impact of the dye’s surrounding environment [ 32 ]—i.e., DNA breathing [ 33 , 34 ], DNA molecular structure [ 35 ], dye-DNA interactions [ 36 , 37 , 38 , 39 ], and linker type [ 40 ]—has been recognized as a crucial factor governing dye orientation relative to the DNA molecule and, consequently, affecting the performance of the particular applications [ 32 , 35 , 36 , 37 , 38 , 39 , 40 ]. All these efforts indicate that much remains to be learned about the DNA molecule [ 41 ], particularly when using it as a functional material for molecular organization, including orientational control.…”

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

“…Another limiting factor on the road to improving the resolution of superresolution microscopies is the size of the fluorophore proper. Last-generation nanoscopies like 2-D MINFLUX ( Balzarotti et al, 2017 ; Eilers et al, 2018 ; Masullo et al, 2021 ), cryogenic nanoscopy ( Furubayashi et al, 2019 ; Furubayashi et al, 2020 ), iterative modulation-enhanced SMLM ( Kalisvaart et al, 2022 ), improved structured-illumination microscopies ( Chen et al, 2018 ; Markwirth et al, 2019 ; Zhanghao et al, 2019 ; Mangeat et al, 2021 ; Qiao et al, 2021 ; Smith et al, 2021 ; Chen et al, 2022 ; Hunter et al, 2022 ; Zhan et al, 2022 ), new high-resolution DNA-PAINT modalities ( Schnitzbauer et al, 2017 ), 3-D MINFLUX ( Gwosch et al, 2020 ; Grabner et al, 2022 ; Gwosch et al, 2022 ) or MINSTED ( Weber et al, 2021 ) are now facing the need to introduce smaller probes to resolve structures at the molecular scale. Successful examples are provided by the recent work in which pyrrolysyl-tRNA synthetase and orthogonal tRNA were matched to introduce clickable amino acids into bacterial and mammalian cell proteins, accomplishing 3-D imaging of β-actin in filopodia with a precision of ∼2 nm ( Mihaila et al, 2022 ).…”