Single-molecule Localization Method Research Articles

Ultrafast Super-Resolution Imaging Exploiting Spontaneous Blinking of Static Excimer Aggregates.

Single-molecule localization methods have been popularly exploited to obtain super-resolved images of biological structures. However, the low blinking frequency of randomly switching emission states of individual fluorophores greatly limits the imaging speed of single-molecule localization microscopy (SMLM). Here we present an ultrafast SMLM technique exploiting spontaneous fluorescence blinking of cyanine dye aggregates confined to DNA framework nanostructures. The DNA template guides the formation of static excimer aggregates as a "light-harvesting nanoantenna", whereas intermolecular excitation energy transfer (EET) between static excimers causes collective ultrafast fluorescence blinking of fluorophore aggregates. This DNA framework-based strategy enables the imaging of DNA nanostructures with 12.5-fold improvement in speed compared to conventional SMLM. Further, we demonstrate the use of this strategy to track the movement of super-resolved DNA nanostructures for over 20 min in a microfluidic system. Thus, this ultrafast SMLM holds great potential for revealing the dynamic processes of biomacromolecules in living cells.

Theoretical study of the properties of multiphoton MINFLUX and RASTMIN super-resolution imaging

Fluorescence microscopy is an important tool for studying cellular physiological functions, and the precise localization of the individual fluorescent molecules is crucial for resolution. In recent years, researchers have proposed various single-molecule localization methods with molecular-level precision. Here, we conducted simulation calculations and feasibility studies on two imaging techniques of minimal emission fluxes (MINFLUX) and RASTer scanning a minimum of light (RASTMIN) for single-photon and multiphoton imaging. Our results indicated that the single-photon excitation wavelength had little influence on the localization precision of MINFLUX and RASTMIN, but multiphoton MINFLUX and RASTMIN significantly improved the center localization precision. However, in multiphoton MINFLUX, the average localization precision was slightly compromised. In contrast, benefiting from the advantages of raster scanning, the average localization precision of multiphoton RASTMIN was superior to that of single-photon RASTMIN. Furthermore, to address the issues of reduced average localization accuracy with multiphoton MINFLUX, we propose an increase in the number of scanning points to enhance the performance of multiphoton MINFLUX imaging.

Single-molecule localization microscopy based on denoising, interpolation and local maxima.

A single fluorescent molecule is highly likely to be located at the center pixel position of a raw image diffused spot in an ideal situation. Even if the molecule and the center pixel position do not completely overlap, they are very close. A single-molecule localization method based on denoising, interpolation and local maxima (DIL) is proposed. The low-resolution raw image is denoised and interpolated, and a new image with a pixel size equal to that of the super-resolution image is attained. The local maxima of the new image are extracted. With this method, it is found that the local maxima positions can be regarded as the fluorescent molecule positions. Simulation results demonstrate that the DIL single-molecule localization accuracy reaches ∼18 nm when the Gaussian noise variance is equal to 0.01. Experimental results demonstrate that the DIL localization methodology is comparable to the Gaussian fitting algorithm and is faster.

An alternative to MINFLUX that enables nanometer resolution in a confocal microscope

Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1–2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures.

Pulsed Interleaved MINFLUX.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.

Molecular resolution imaging by repetitive optical selective exposure.

We introduce an interferometric single-molecule localization method for super-resolution fluorescence microscopy. Fluorescence molecules are located by the intensities of multiple excitation patterns of an interference fringe, providing around a twofold improvement in the localization precision compared with the conventional imaging with the same photon budget. We demonstrate this technique by resolving nanostructures down to 5 nm in size over a large 25 × 25 μm2 field of view.

Fast Frequency-Domain Compressed Sensing Analysis for High-Density Super-Resolution Imaging Using Orthogonal Matching Pursuit

Single-molecule localization methods play a vital role in a localization-based super-resolution fluorescence microscopy. However, it is difficult for conventional localization schemes based on the Gaussian fitting to locate overlapped high-density fluorescent emitters. Currently, in the spatial domain, the compressive-sensing-based algorithm (CSSTORM) can localize high-emitter-density images. However, the computational cost of this approach is extremely high, which limits its practical application. Here, we propose an alternative frequency-domain compressed sensing (FD-CS) technique for fast super-resolution imaging. Unlike the CSSTORM method, which is a measurement matrix based on the point spread function, a Fourier dictionary designed in the frequency domain and orthogonal matching pursuit is used to reliably recover the original signal. The simulation and experimental results prove that the FD-CS is 1000 times faster than CSSTORM with CVX and ten times faster than that with L1-Homotopy with almost the same localization accuracy and recall rate. This drastic reduction in computational time should allow the compressed sensing approach to be routinely applied to a super-resolution image analysis.

Imaging and reconstruction of cell cortex structures near the cell surface

Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

HyVolution—the smart path to confocal super-resolution

Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.

Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations.

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

Structure–function relationships and supramolecular organization of the EGFR (epidermal growth factor receptor) on the cell surface

Dimerization and higher-order oligomerization are believed to play an important role in the activation of the EGFR (epidermal growth factor receptor). Understanding of the process has been limited by the lack of availability of suitable methods for the measurement in cells of distances in the range 10-100nm, too short for imaging methods and too long for spectroscopic methods such as FRET. In the present article, we review the current state of our knowledge of EGFR oligomerization, and describe results from a new single-molecule localization method that has allowed the quantitative characterization of the distribution of EGFR-EGFR distances in cells. Recent data suggest the involvement of cortical actin in regulating the formation of EGFR complexes.

Actin restructuring duringSalmonella typhimuriuminfection investigated by confocal and super-resolution microscopy

We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy

Single-molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single-molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive index mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here we show a method to obtain accurate, depth-dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth-dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 μm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane.

Super-resolution mbPAINT for optical localization of single-stranded DNA.

We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as "motion blur" point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.

Design and experimental demonstration of high-efficiency double-helix point spread function phase plate

Double helix point spread function possesses continuously rotating invariant features with defocus, and it can be used for performing nano-resolution image of thick samples and for studying singleparticle tracking by combining the single-molecule localization method. However, the inadequacy of the original double helix point spread function is that the transfer function efficiency is very low, so it is difficult to achieve photon limited applications for the bio-imaging. The double-helix point spread function is optimized by introducing an iterative algorithm so that the constraints of the point spread function can be optimized best in three different domains: the Laguerre-Gauss modal plane, the spatial domain, and the Fourier domain. The simulation results show that the peak intensity of high efficiency double helix point spread function is 30 times higher than the original one. The design and fabrication of the phase plate are also proposed. The experimental results are in good agreement with the theoretical predictions, thus proving the feasibility of this method.

DNA and chromatin imaging with super‐resolution fluorescence microscopy based on single‐molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.