Localization microscopy at doubled precision with patterned illumination

Abstract

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in fieldof-view. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures and tubulin in cells, using DNA-PAINT and STORM imaging. Single-molecule localization microscopy 1,2,3 circumvents the diffraction limit using centroid estimation of sparsely activated, stochastically switching, single-molecule fluorescence images. Improvement over state-of-the-art image resolutions of around 20 nm towards values below 5 nm is desired for truly imaging at the molecular scale. This needs improvements in labelling strategy to reduce linker sizes 4,5,6,7 and methods to overcome low labelling density such as data fusion 8 , but also a step in localization precision. Efforts so far have targeted an increase in the number of detected photons N by chemical engineering of brighter fluorophores 9 , or by avoiding photo-bleaching via e.g. cryogenic techniques 10,11,12. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Deep learning enables structured illumination microscopy with low light levels and enhanced speed

“…3). This could be useful for other advanced imaging techniques that utilize structured illumination, such as repetitive optical selective exposure (ROSE) 22 and SIM-FLUX 23 . These techniques use structured illumination to boost precision of single molecule imaging, requiring the precise estimation of the illumination phase.…”

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

“…Super-resolution microscopy offers up to ~10x higher resolution than conventional diffraction-limited microscopy 11,12 . Improved super-resolution microscopy methods can now localize single emitters with a precision of a few nanometers [31][32][33] , but limitations in labeling efficiency and linkage error have thus far prevented the translation of high localization precision into sub-10-nm spatial resolution. Therefore, the spatial resolution provided by all these inventive methods is currently still too low to unravel the composition and molecular architecture of protein complexes or dense protein networks.…”

Multicolor structured illumination microscopy and quantitative control of polychromatic coherent light with a digital micromirror device

“…This opens the possibility for quantitative, multi-wavelength pattern formation at rates up to 20 kHz for a factor of ~5 lower cost than SLM based units. While SIM imaging rates are ultimately limited by signal-to-noise ratio and phototoxicity, fast control of multi-wavelength pattern formation also provides new avenues in the design of multi-wavelength tomography (41, 42), multi-wavelength optical trapping (43–45), high-speed tracking of photostable fluorescent labels below the diffraction limit (46), and high-speed modulation enhanced localization microscopy in mutiple colors (5, 29, 47). Our work significantly differs from previous SIM approaches using multi-wavelength incoherent LED light sources or multi-wavelength incoherent image projection using a DMD (27, 28).…”