Localization microscopy at doubled precision with patterned illumination

Abstract

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny fieldof-view and a lack of practical parallelism. 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. Main textSingle-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 by chemical engineering of brighter fluorophores 9 , or by avoiding photo-bleaching via e.g. cryogenic techniques 10,11,12 . These improvements scale the localization precision with ( % ) ⁄ , with the fluorescence emission wavelength, and the microscope objective numerical aperture 13 .Recently, a new concept called MINFLUX was proposed 14 , in which a doughnut illumination spot is shifted over an area of size ~50 nm, and the position of a single molecule in the scan range is determined by triangulation based on the detected photon count for the different doughnut positions.

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.…”

Nanometric axial localization of single fluorescent molecules with modulated excitation

“…Localization by modulating the excitation have been used in the early days of single emitter tracking along the lateral direction to probe myosin V stepping mechanism 38 with gold nanobeads 39 . Recently, this strategy has been adapted to single molecule transverse localization for SMLM by various groups [40][41][42] . Similarly, MINFLUX 43 technique is based on a structured illumination to localize the molecule but the estimator and the illumination pattern in the shape of a doughnut have been optimized to reach unmatched precision for a specific point (albeit breaking the translational invariance) 44 .…”

Quantum limits for precisely estimating the orientation and wobble of dipole emitters

“…Since the first observation of single molecules [1], scientists and engineers have worked tirelessly to quantify precisely their positions [2][3][4] and orientations [5][6][7][8][9] to probe dynamic processes within soft matter at the nanoscale. Two fundamental challenges confront these experiments: the optical diffraction limit, i.e., the finite numerical aperture of the imaging system, and Poisson shot noise associated with photon counting.…”