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.
Superresolution by localization of quantum dots using blinking statistics
Optics Express, 2005
In microscopy, single fluorescence point sources can be localized with a precision several times greater than the resolution limit of the microscope. We show that the intermittent fluorescence or 'blinking' of quantum dots can analyzed by an Independent Component Analysis so as to identify the light emitted by each individual nanoparticle, localize it precisely, and thereby resolve groups of closely spaced (< λ /30) quantum dots. Both simulated and experimental data demonstrate that this technique is superior to localization based on Maximum Likelihood Estimation of the sum image under the assumption of point emitters. This technique has general application to any emitter with non-Gaussian temporal intensity distribution, including triplet state blinking. When applied to the labeling of structures, a high resolution "image" consisting of individually localized points may be reconstructed leading to the term "Pointillism".
Superlocalization Spectral Imaging Microscopy of a Multicolor Quantum Dot Complex
Analytical Chemistry, 2012
The key factor of realizing super-resolution optical microscopy at the single-molecule level is to separately position two adjacent molecules. An opportunity to independently localize target molecules is provided by the intermittency (blinking) in fluorescence of a quantum dot (QD) under the condition that the blinking of each emitter can be recorded and identified. Herein we develop a spectral imaging based color nanoscopy which is capable of determining which QD is blinking in the multicolor QD complex through tracking the first-order spectrum, and thus, the distance at tens of nanometers between two QDs is measured. Three complementary oligonucleotides with lengths of 15, 30, and 45 bp are constructed as calibration rulers. QD585 and QD655 are each linked at one end. The measured average distances are in good agreement with the calculated lengths with a precision of 6 nm, and the intracellular dual-color QDs within a diffractionlimited spot are distinguished.
Two-Photon 3D FIONA of Individual Quantum Dots in an Aqueous Environment
Nano Letters, 2011
O ne-photon (1P) microscopy of individual quantum dots (QDs) has become routine. 1À3 In contrast, two-photon (2P) microscopy of individual QDs has not had the same success despite the many advantages that 2P microscopy offers: reduced scattering, deep sample penetration, and intrinsic confocality when excited with point excitation. 4 2P microscopy of ensembles of QDs in aqueous samples has been achieved by Larson et al. in 2003. 5 They showed QDs had large absorption cross sections under 2P scan excitation. Nevertheless, individual 2P QD-microscopy has been possible only in artificial environments, such as air-dried samples of QDs, 6 or at cryogenic temperatures. 7 2P microscopy of individual organic-based fluorophores has also been problematic since most fluorophores have very small 2P absorption cross sections, as well as poor photostability. Here we report the application of 2P microscopy to individual QDs in a biological setting with nanometer spatial accuracy in three dimensions, both in solution and in live and fixed cells. We call our technique two-photon fluorescence imaging with one nanometer accuracy (2P FIONA), in analogy with the onephoton technique. 10 With 2P excitation, we are able to achieve three-dimensional (3D) nanometer spatial accuracy, as opposed to the two-dimension FIONA previously achieved with one photon microscopy. We also introduce a fast imaging method using a holographic matrix in excitation and EMCCD in detection that achieves an 80-fold improvement in speed and reduces spherical aberrations.
Spectroscopic super-resolution fluorescence cell imaging using ultra-small Ge quantum dots
Optics express, 2017
We demonstrate a spectroscopic imaging based super-resolution approach by separating the overlapping diffraction spots into several detectors during a single scanning period and taking advantage of the size-dependent emission wavelength in nanoparticles. This approach has been tested using off-the-shelf quantum dots (Invitrogen Qdot) and in-house novel ultra-small (~3 nm) Ge QDs. Furthermore, we developed a method-specific Gaussian fitting and maximum likelihood estimation based on a Matlab algorithm for fast QD localisation. This methodology results in a three-fold improvement in the number of localised QDs compared to non-spectroscopic images. With the addition of advanced ultra-small Ge probes, the number can be improved even further, giving at least 1.5 times improvement when compared to Qdots. Using a standard scanning confocal microscope we achieved a data acquisition rate of 200 ms per image frame. This is an improvement on single molecule localisation super-resolution micros...
Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles
Frontiers in Physiology, 2014
Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies. Keywords: single particle tracking, super-resolution, fluorescent microscopy, quantum dot, nanoparticle www.frontiersin.org July 2014 | Volume 5 | Article 273 | 1 FIGURE 1 | Quantum dot. (A) Schematic drawing of the surface modification of a Qdot. (B) Fluorescence photograph (upper) and spectra (lower) of Qdots of various diameters. The Qdots were excited by a UV light of 365 nm wavelength.
Journal of biophotonics, 2012
Obtaining sub-resolution particle positions in fluorescence microscopy images is essential for single particle tracking and high-resolution localization microscopy. While the localization precision of stationary single molecules or particles is well understood, the influence of particle motion during image acquisition has been largely neglected. Here, we address this issue and provide a theoretical description on how particle motion influences the centroid localization precision, both in case of 2-D and 3-D diffusion. In addition, a novel method is proposed, based on dual-channel imaging, for the experimental determination of the localization precision of moving particles. For typical single particle tracking experiments, we show that the localization precision is approximately two-fold worse than expected from the stationary theory. Strikingly, we find that the most popular localization method, based on the fitting of a Gaussian distribution, breaks down for lateral diffusion. Inst...
Measuring true localization accuracy in super resolution microscopy with DNA-origami nanostructures
New Journal of Physics, 2017
A common method to assess the performance of (super resolution) microscopes is to use the localization precision of emitters as an estimate for the achieved resolution. Naturally, this is widely used in super resolution methods based on single molecule stochastic switching. This concept suffers from the fact that it is hard to calibrate measures against a real sample (a phantom), because true absolute positions of emitters are almost always unknown. For this reason, resolution estimates are potentially biased in an image since one is blind to true position accuracy, i.e. deviation in position measurement from true positions. We have solved this issue by imaging nanorods fabricated with DNA-origami. The nanorods used are designed to have emitters attached at each end in a well-defined and highly conserved distance. These structures are widely used to gauge localization precision. Here, we additionally determined the true achievable localization accuracy and compared this figure of merit to localization precision values for two common super resolution microscope methods STED and STORM.
Shifting molecular localization by plasmonic coupling in a single-molecule mirage
Nature Communications, 2017
Over the last decade, two fields have dominated the attention of sub-diffraction photonics research: plasmonics and fluorescence nanoscopy. Nanoscopy based on single-molecule localization offers a practical way to explore plasmonic interactions with nanometre resolution. However, this seemingly straightforward technique may retrieve false positional information. Here, we make use of the DNA origami technique to both control a nanometric separation between emitters and a gold nanoparticle, and as a platform for super-resolution imaging based on single-molecule localization. This enables a quantitative comparison between the position retrieved from single-molecule localization, the true position of the emitter and full-field simulations. We demonstrate that plasmonic coupling leads to shifted molecular localizations of up to 30 nm: a single-molecule mirage.
Optics Express, 2009
Three-dimensional (3D) particle localization at the nanometer scale plays a central role in 3D particle tracking and 3D localization-based super-resolution microscopy. Here we introduce a localization algorithm that is independent of theoretical models and therefore generally applicable to a large number of experimental realizations. Applying this algorithm and a convertible experimental setup we compare the performance of the two major 3D techniques based on astigmatic distortions and on multiplane detection. In both methods we obtain experimental 3D localization accuracies in agreement with theoretical predictions and characterize the depth dependence of the localization accuracy in detail.