Cryogenic Colocalization Microscopy for Nanometer‐Distance Measurements

Abstract

The main limiting factor in spatial resolution of localization microscopy is the number of detected photons. Recently we showed that cryogenic measurements improve the photostability of fluorophores, giving access to Angstrom precision in localization of single molecules. Here, we extend this method to colocalize two fluorophores attached to well-defined positions of a double-stranded DNA. By measuring the separations of the fluorophore pairs prepared at different design positions, we verify the feasibility of cryogenic distance measurement with sub-nanometer accuracy. We discuss the important challenges of our method as well as its potential for further improvement and various applications.

Coherent Interaction of Light and Single Molecules in a Dielectric Nanoguide

“…Figures 4(c) and 4(d) show spatiospectral maps of the resonance and Stokes-shifted fluorescence along more than 15μm of the nanoguide and within a spectral range of about 3 GHz. Considering that one can localize the center of the dipole moment of each molecule with angstrom precision through the analysis of its image point-spread function [41], the data in Fig. 4 illustrate the ability to address each molecule through high-resolution spectral and spatial information.…”

Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy

“…Single-molecule experiments that reach subnanometer precision in two dimensions require careful estimation of detector nonuniformity and dipole mislocalization errors [25,53], and we expect that the extension of these ultraprecise experiments to three dimensions will also require that field-dependent aberrations be carefully corrected. Another relevant trend is the increased use of sCMOS rather than EMCCD detectors for single-molecule localization [54].…”

Optical tracing of multiple charges in single-electron devices

“…Unlike room-temperature localization microscopy, cryogenic spectroscopy measurements can handle molecular concentrations as high as 10 4 per cubic micrometer. Furthermore, given the unlimited photon budget, the position of each molecule can be determined with an accuracy better than one nanometer [29,30].…”

“…2(b). Note that although these localization inaccuracies are much smaller than the optical diffraction limit, they are now routinely accessible in cryogenic conditions using single-molecule localization techniques [30]. The positions of electrons or, strictly-speaking, the metallic grains that are charged by these electrons, are recalculated based on a least-square optimization routine.…”