Three-dimensional nano-localization of single fluorescent emitters (original) (raw)

Nanometric axial localization of single fluorescent molecules with modulated excitation

2019

Strategies have been developed in LIDAR to perform distance measurements for non-coherent emission in sparse samples based on excitation modulation. Super-resolution fluorescence microscopy is also striving to perform axial localization but through entirely different approaches. Here we revisit the amplitude modulated LIDAR approach to reach nanometric localization precision and we successfully adapt it to bring distinct advantages to super-resolution microscopy. The excitation pattern is performed by interference enabling the decoupling between spatial and time modulation. The localization of a single emitter is performed by measuring the relative phase of its linear fluorescent response to the known shifting excitation field. Taking advantage of a tilted interfering configuration, we obtain a typical axial localization precision of 7.5 nm over the entire field of view and the axial capture range, without compromising on the acquisition time, the emitter density or the lateral localization precision. The interfering pattern being robust to optical aberrations, this modulated localization (ModLoc) strategy is particularly well suited for observations deep in the samples. Images performed on various biological samples show that the localization precision remains nearly constant up to several micrometers. In the presence of coherent signals, interferometry offers unmatched sensitivity for distance measurements 1. Measuring the relative phase between the excitation in the elastically scattered or reflected signal reaches record precisions. Interferometry has thus naturally been used in some coherent microscopy configurations to obtain nanometric axial localization 2-5. However, in the case of fluorescence microscopy which is today the most widespread technique for cell imaging such an approach remains impossible because of the non- .

Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes

A new form of super-resolution fluorescence microscopy has emerged in recent years, based on the high accuracy localization of individual photo-switchable fluorescent labels. Image resolution as high as 20 nm in the lateral dimensions and 50 nm in the axial direction has been attained with this concept, representing an order of magnitude improvement over the diffraction limit. The demonstration of multicolor imaging with molecular specificity, three-dimensional (3D) imaging of cellular structures, and time-resolved imaging of living cells further illustrates the exciting potential of this method for biological imaging at the nanoscopic scale.

Toward super-resolution fluorescent microscopy of arbitrarily oriented single molecules

Physical review, 2020

We have theoretically developed a microscopy technique to visualize arbitrarily oriented single quantum fluorescent emitters with resolutions beyond the diffraction limit. The lateral resolution of 72 nm, the axial resolution of 103 nm, and the orientational independence degree of 0.9 have been demonstrated. The latter quantifies the dependence of the image intensity maximum of a single quantum emitter on the orientation of its transition dipole moment and can take on possible values from 0 to 1, where 1 corresponds to a completely excluded dependence and 0 indicates that the emitter is absolutely invisible at some orientations. The suggested method is based on orientational stimulated emission depletion microscopy. An elliptically polarized cylindrical vector Bessel beam is suggested as an excitation beam and an elliptically polarized vortex cylindrical vector Bessel beam with the topological charge m = ±1 as the depletion beam to deplete focal-plane fluorescence. A pair of linearly polarized cylindrical vector beams focused in a 4π microscope scheme is assumed to deplete out-of-focal-plane fluorescence. An image of a single quantum emitter can be recorded at four conjugated sets of polarizations of the beams, giving images with different intensity distributions but the same intensity maximum values, and the maximums depicted in the same plot form a rectangle from which one can derive the position and orientation of the emitter. The present study paves the way for super-resolution microscopy of arbitrarily oriented single molecules, which can enhance single molecule counting techniques, studies of polymer materials by guest-fluorescent-molecule probing, and similar applications.

Improved resolution in single-molecule localization microscopy using QD-PAINT

Experimental & Molecular Medicine, 2021

Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric e...

Advances in 3D single particle localization microscopy

APL Photonics

The spatial resolution of conventional optical microscopy is limited by diffraction to transverse and axial resolutions of about 250 nm, but localization of point sources, such as single molecules or fluorescent beads, can be achieved with a precision of 10 nm or better in each direction. Traditional approaches to localization microscopy in two dimensions enable high precision only for a thin in-focus layer that is typically much less than the depth of a cell. This precludes, for example, super-resolution microscopy of extended three-dimensional biological structures or mapping of blood velocity throughout a useful depth of vasculature. Several techniques have been reported recently for localization microscopy in three dimensions over an extended depth range. We describe the principles of operation and typical applications of the most promising 3D localization microscopy techniques and provide a comparison of the attainable precision for each technique in terms of the Cramér-Rao lower bound for high-resolution imaging.

High-Precision Tracking with Non-blinking Quantum Dots Resolves Nanoscale Vertical Displacement

Journal of the American Chemical Society, 2012

Novel non-blinking quantum dots (NBQDs) were utilized in three-dimensional super-localization, highprecision tracking applications under an automated scanning-angle total internal reflection fluorescence microscope (SA-TIRFM). NBQDs were randomly attached to stationary microtubules along the radial axis under gliding assay conditions. By automatically scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves were obtained. Fit with theoretical decay functions, the absolute vertical positions were determined with sub-10-nm localization precision. The emission intensity profile of the NBQDs attached to kinesin-propelled microtubules was used to resolve the self-rotation of gliding microtubules within a small vertical distance of ~50 nm. We demonstrate the applicability of NBQDs in high-precision fluorescence imaging experiments.

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy

Nature Communications, 2015

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-offields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes

Science (New York, N.Y.), 2017

We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolution microscopy, MINFLUX attained ~1-nm precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.