Super-resolution Microscopy Imaging Research Articles

Contrast enhancement of microsphere-assisted super-resolution imaging in dark-field microscopy

We report a method of boosting the imaging contrast of microsphere-assisted super-resolution visualization by utilizing dark-field illumination (DFI). We conducted experiments on both 10-µm-diameter silica (SiO2) microspheres with refractive index n ∼ 1.46 under no and partial immersion in ethyl alcohol (n ∼ 1.36) and 20-µm-diameter barium titanate glass (BTG, n ∼ 1.9) microspheres with full immersion to show the super-resolution capability. We experimentally demonstrated that the imaging contrast and uniformity were extraordinarily improved in the DFI mode. The intensity profiles in the visualization also numerically confirm the enhanced sharpness for a better imaging quality when applying DFI.

Focus engineering based on analytical formulae for tightly focused polarized beams with arbitrary geometric configurations of linear polarization.

Highly confined electromagnetic fields with controllable intensity profiles and polarization orientations are greatly desired in many fields. In this paper, we report on the generation of highly confined fields through tightly focused locally linearly polarized beams. Using the Richards-Wolf vectorial diffraction method, we derive and build integrated analytical formulae for calculating the tightly focused field of polarized beams with arbitrary geometric configurations of linear polarization. Based on the analytical model, the focusing properties of four types of polarized light beams, i.e., linearly, azimuthally, radially, and spatially variant polarized beams, with locally linear states of polarization are investigated numerically and discussed in detail. By manipulating the radial and azimuthal indices and initial phases, we obtain a tunable three-dimensional optical cage, multifoci, optical needles, and channels in the focal volume of a high-numerical-aperture objective lens. These peculiar properties may find applications in fields such as optical trapping and manipulation of nanoparticles and super-resolution microscopy imaging.

Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms.

Heteromeric interactions between the catalytically impaired human epidermal growth factor receptor (HER3/ERBB3) and its catalytically active homologs EGFR and HER2 are essential for their signaling. Different ligands can activate these receptor pairs but lead to divergent signaling outcomes through mechanisms that remain largely unknown. We used stochastic optical reconstruction microscopy (STORM) with pair-correlation analysis to show that EGF and neuregulin (NRG) can induce different extents of HER3 clustering that are dependent on the nature of the coexpressed HER receptor. We found that the presence of these clusters correlated with distinct patterns and mechanisms of receptor phosphorylation. NRG induction of HER3 phosphorylation depended on the formation of the asymmetric kinase dimer with EGFR in the absence of detectable higher-order oligomers. Upon EGF stimulation, HER3 paralleled previously observed EGFR behavior and formed large clusters within which HER3 was phosphorylated via a noncanonical mechanism. HER3 phosphorylation by HER2 in the presence of NRG proceeded through still another mechanism and involved the formation of clusters within which receptor phosphorylation depended on asymmetric kinase dimerization. Our results demonstrate that the higher-order organization of HER receptors is an essential feature of their ligand-induced behavior and plays an essential role in lateral cross-activation of the receptors. We also show that HER receptor ligands exert unique effects on signaling by modulating this behavior.

Single particle maximum likelihood reconstruction from superresolution microscopy images.

Point localization superresolution microscopy enables fluorescently tagged molecules to be imaged beyond the optical diffraction limit, reaching single molecule localization precisions down to a few nanometers. For small objects whose sizes are few times this precision, localization uncertainty prevents the straightforward extraction of a structural model from the reconstructed images. We demonstrate in the present work that this limitation can be overcome at the single particle level, requiring no particle averaging, by using a maximum likelihood reconstruction (MLR) method perfectly suited to the stochastic nature of such superresolution imaging. We validate this method by extracting structural information from both simulated and experimental PALM data of immature virus-like particles of the Human Immunodeficiency Virus (HIV-1). MLR allows us to measure the radii of individual viruses with precision of a few nanometers and confirms the incomplete closure of the viral protein lattice. The quantitative results of our analysis are consistent with previous cryoelectron microscopy characterizations. Our study establishes the framework for a method that can be broadly applied to PALM data to determine the structural parameters for an existing structural model, and is particularly well suited to heterogeneous features due to its single particle implementation.

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

SummaryThe active zone (AZ) matrix of presynaptic terminals coordinates the recruitment of voltage-gated calcium channels (VGCCs) and synaptic vesicles to orchestrate neurotransmitter release. However, the spatial organization of the AZ and how it controls vesicle fusion remain poorly understood. Here, we employ super-resolution microscopy and ratiometric imaging to visualize the AZ structure on the nanoscale, revealing segregation between the AZ matrix, VGCCs, and putative release sites. Long-term blockade of neuronal activity leads to reversible AZ matrix unclustering and presynaptic actin depolymerization, allowing for enrichment of AZ machinery. Conversely, patterned optogenetic stimulation of postsynaptic neurons retrogradely enhanced AZ clustering. In individual synapses, AZ clustering was inversely correlated with local VGCC recruitment and vesicle cycling. Acute actin depolymerization led to rapid (5 min) nanoscale AZ matrix unclustering. We propose a model whereby neuronal activity modulates presynaptic function in a homeostatic manner by altering the clustering state of the AZ matrix.

Single Objective Light-Sheet Microscopy for High-Speed Whole-Cell 3D Super-Resolution

Single molecule super-resolution (SM-SR) in 3D throughout a whole cell is difficult because wide-field illumination cannot be targeted to the in-focus plane. This results in high background fluorescence that severely degrades detection and localization precision. We have developed a technique, termed Single Objective Light-Sheet Microscopy (SO-LSM) that uses a common, single objective, inverted epi-fluorescence microscope along with a reflective surface to generate illumination only in the image focal plane. We use a reflective surface, placed at an angle of 45° on a cover slip to reflect the beam. This surface forms the side wall of a microfluidic channel incorporated into a microfluidic device. A light-sheet is generated through the objective and reflected such that it illuminates the in-focus plane of the cell. The light-sheet is scanned through the cell for whole-cell imaging and an astigmatic lens in the emission path enables the 3D localization of individual emitters, creating a whole-cell 3D super-resolution image. SO-LSM provides improved localization accuracy due to background reduction. An additional benefit is reduced photobleaching of un-imaged, out-of-focus fluorophores. Moreover, the light-sheet confines the laser light in one dimension, which increases the excitation intensity, which makes the probes photo-switch faster thereby significantly reducing the aquisition time. The microfluidics device provides a closed environment that allows for fast and automated buffer exchange, which is utilized to maintain a reduced oxygen environment needed for many SM-SR probes. SO-LSM is easily implemented on a regular wide-field fluorescence microscope. In conclusion, SO-LSM increases image quality due to better localization of more emitters, acquired in less time then widefield illumination. The development of this microscope and its imaging and analysis routines is therefore of great value to investigators seeking a straightforward, inexpensive method for whole-cell 3D super-resolution microscopy and high-speed live cell imaging.

Intracellular Delivery of Membrane Impermeable Photostable Fluorescent Probes into Living Cells for Super-Resolution Microscopy

Specific labeling of the cellular target with fluorophore is one of the fundamental requirement in all fluorescence imaging techniques including single molecule and super-resolution microscopy techniques. While extracellular targets may be tagged with virtually any kind of probes, intracellular labeling of living cells is limited to the use of fluorescent proteins and limited selection of membrane permeable dyes. Here we show that pore forming proteins can be used to temporarily permeabilize the cells and allow delivery of various fluorescent probes, ranging from organic dyes (<1 kDa) to fluorescent immunoglobulin antibody (∼150 kDa), for specific labeling of intracellular targets for live fluorescence cell imaging. We demonstrate that permeabilized cells can efficiently be recovered to carry on normal cellular processes shown by nuclear translocation of nanobody-labeled p65 proteins in response to chemical stimulation. One of the most photostable but membrane impermeable organic fluorophore, Atto 647N, has been delivered into cells to label actin fibers and kinesin dimers and to perform super-resolution fluorescence microscopy imaging dSTORM and single molecule tracking, respectively. This technique opens up a large number of stable, but otherwise membrane impermeable fluorescence labeling probes that are available for investigating transfected and endogenous intracellular targets.

A Quantitative Platform for Super-Resolution Microscopy Imaging

Pointillistic super-resolution microscopy techniques provide nanometer scale spatial resolution and single molecule sensitivity. Thus, they are excellent tools for probing the lateral organization of molecules, especially in biological environments. Additional information on such organization can be obtained by subjecting super-resolution data to quantitative analyses. These powerful approaches are able to quantify the extent to which molecules localize into clusters and the size/occupancy of clusters. However, quantitative super-resolution approaches typically encounter two major challenges. First, it is difficult to determine optimum imaging parameters and characterize, under these conditions, the fluorescent probe's biophysical properties. Second, it is challenging to identify efficient labeling strategies for detecting endogenous proteins, particularly those that yield stoichiometric and site specific labeling. To efficiently characterize fluorescent probes, we have developed an easily implementable nano-biology assay. Using chemistry compatible with super-resolution imaging (i.e. producing minimal background signal), proteins were covalently immobilized to glass surfaces with specific orientations. Interrogating such surfaces revealed important features of both optical highlighter proteins (paGFP and pamCherry1) and proteins labeled with organic fluorophores. To efficiently label endogenous receptors, we leveraged a unique cyclic peptide called meditope, which binds within the Fab framework of engineered monoclonal antibodies. A fluorescent meditope was complexed with the engineered trastuzumab Fab. This complex was used to image both SK-BR-3 and BT-474 cells, where we delineated the distribution of epidermal growth factor receptor 2 (HER2). According to our results, the majority of HER2 receptors were monomers as expected. However, a fraction of receptors were organized as oligomers, and more significantly in BT-474 cells that are oncogene-addicted. This demonstrates that our method is capable of detecting subtle differences in endogenous receptor organization and thus offers a powerful platform for quantitative super-resolution imaging.

Extracting microtubule networks from superresolution single-molecule localization microscopy data.

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization-based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts.

Abstract 13129: Acute Inhibition of Sodium Channel Beta Subunit (β1) -mediated Adhesion is Highly Proarrhythmic

Introduction: The sodium channel auxiliary subunit and cell adhesion molecule β1 (SCN1b) is enriched at cardiomyocyte intercalated disks (ID) along with the pore-forming subunit Na V 1.5. We recently demonstrated that the perinexus, an Na V 1.5-rich ID nanodomain located near connexin43 (Cx43) gap junctions (GJ), is important for cell-to-cell electrical coupling. Disrupting close membrane apposition within the perinexus was highly proarrhythmic. Hypothesis: β1-mediated adhesion is key to close membrane apposition within the perinexus; therefore, inhibiting β1-mediated adhesion may disrupt perinexal structure, impair conduction and precipitate arrhythmias. Methods and Results: Na V 1.5 and β1 localization within ID nanodomains was quantitatively assessed from super-resolution STochastic Optical Reconstruction Microscopy (STORM) images of en face IDs from adult guinea pig ventricles. Na V 1.5 and β1 were located 63±15 and 75±25 nm from Cx43 GJ; 31% of both Na V 1.5 and β1 localized to GJ perinexi. β1-mediated adhesion was acutely inhibited by βadp1, a novel peptide mimetic of β1’s putative extracellular adhesion domain. βadp1 decreased cellular adhesion as assessed by Electric Cell-Substrate Impedance Spectroscopy (ECIS) in β1-overexpressing 1610 cells in a dose-dependent manner but not in native 1610 cells. Perinexal intermembrane spacing as measured by Transmission Electron Microscopy (TEM) increased 3-fold from 17±1 nm in vehicle controls (0.25% DMSO) to 49±5 nm (p&lt;0.05) in 100 μM βadp1-treated hearts; however, intermembrane spacing at other ID locations was unaltered (22±1 vs. 24±1 nm). Optical mapping revealed preferential slowing of transverse conduction (8.0±0.7 vs. 16.5±1.2 cm/s, p&lt;0.05) and increased anisotropy (3.9±0.2 vs. 2.6±0.2, p&lt;0.05) in βadp1-treated hearts relative to vehicle controls. Further, 3 of 4 βadp1-treated hearts exhibited spontaneous ventricular tachycardias. Conclusions: These data support a role for β1-mediated adhesion in maintaining close apposition between Na V 1.5-rich perinexal membranes. Importantly, acute inhibition of β1-mediated adhesion resulted in disruption of close membrane apposition within the GJ perinexus and precipitated severe conduction slowing and spontaneous arrhythmias.

Superresolution microscopy imaging based on full-wave modeling and image reconstruction

Computational imaging, or post-processing of images, allows the resolution limit of optical microscopy to be exceeded. Here we present an image-inversion approach to improve the resolution of a confocal laser scanning microscope (CLSM). The method combines a full-wave modeling of CLSM and an inverse reconstruction algorithm. The inverse reconstruction is cast into an optimization problem, where the distribution of refractive index of objects that yields the best match between computed images and experimental images is identified. The reconstructed image is a quantitative image based on the distribution of refractive index. Experimental results demonstrate a 35 nm edge-to-edge resolution using a two-disk pattern with 105 nm disk diameter. The proposed computational imaging approach greatly improves the resolution, without the need to change the existing microscopy system, at moderate computational cost.

Super-Resolution Real Imaging in Microsphere-Assisted Microscopy.

Microsphere-assisted microscopy has received a lot of attention recently due to its simplicity and its capability to surpass the diffraction limit. However, to date, sub-diffraction-limit features have only been observed in virtual images formed through the microspheres. We show that it is possible to form real, super-resolution images using high-refractive index microspheres. Also, we report on how changes to a microsphere’s refractive index and size affect image formation and planes. The relationship between the focus position and the additional magnification factor is also investigated using experimental and theoretical methods. We demonstrate that such a real imaging mode, combined with the use of larger microspheres, can enlarge sub-diffraction-limit features up to 10 times that of wide-field microscopy’s magnification with a field-of-view diameter of up to 9 μm.

Improved superresolution microscopy imaging by sparse deconvolution with an interframe penalty

Penalized regression with a combination of sparseness and an interframe penalty is explored for image deconvolution in wide‐field single‐molecule fluorescence microscopy. The aim is to reconstruct superresolution images, which can be achieved by averaging the positions and intensities of individual fluorophores obtained from the analysis of successive frames. Sparsity of the fluorophore distribution in the spatial domain is obtained with an L0‐norm penalty on estimated fluorophore intensities, effectively constraining the number of fluorophores per frame. Simultaneously, continuity of the fluorophore localizations in the time mode is obtained by penalizing the total numbers of pixel status changes between successive frames. We implemented the interframe penalty in a sparse deconvolution algorithm (sparse image deconvolution and reconstruction) for improved imaging of densely labeled biological samples. For simulated and real biological data, we show that more accurate estimates of the final superresolution images of cellular structures can be obtained.

Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm.

Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D.

Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex.

The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout‐controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse.

Nanometer resolved single-molecule colocalization of nuclear factors by two-color super resolution microscopy imaging

In order to study the detailed assembly and regulation mechanisms of complex structures and machineries in the cell, simultaneous in situ observation of all the individual interacting components should be achieved. Multi-color Single-Molecule Localization Microscopy (SMLM) is ideally suited for these quantifications. Here, we build on previous developments and thoroughly discuss a protocol for two-color SMLM combining PALM and STORM, including sample preparation details, image acquisition and data postprocessing analysis. We implement and evaluate a recently proposed colocalization analysis method (aCBC) that allows single-molecule colocalization quantification with the potential of revealing fine, nanometer-scaled, structural details of multicomponent complexes. Finally, using a doubly-labeled nuclear factor (Beaf-32) in Drosophila S2 cells we experimentally validate the colocalization quantification algorithm, highlight its advantages and discuss how using high molecular weight fluorescently labeled tags compromises colocalization precision in two-color SMLM experiments.

High- and Super-Resolution Microscopy Imaging of the NK Cell Immunological Synapse.

Recent advances in imaging technology have enabled significant advances in the study of NK cell cytotoxic effector function through quantitative analysis of the NK cell immunological synapse. This can include the use of high- and super-resolution microscopy to quantify dynamics of cytoskeletal elements and the role they play in the regulation and execution of NK cell directed secretion. Here we describe a protocol for the recapitulation of the NK cell lytic synapse on glass, the acquisition of microscopy images, and suggested approaches for the processing and analysis of microscopy data.

基于随机脉冲编码的超分辨显微成像算法研究

基于随机脉冲编码的超分辨显微成像算法研究

DNA Superresolution Structure of Reed-Sternberg Cells Differs Between Long-Lasting Remission Versus Relapsing Hodgkin's Lymphoma Patients.

Recent developments in microscopy have led to superresolution microscopy images of cells. Structured illumination microscopy was used before to reveal new details in the DNA structure and the structure of the DNA-free space in the DAPI-stained cell nuclei of the Hodgkin's lymphoma HDLM-2 cell line. This study extends this technology to primary pre-treatment classical Hodgkin's lymphoma samples of ten patients. Significant differences in both the DNA structure and the structure of the DNA-free space were detected between lymphocytes and malignant cells. Both types of structures were similar for lymphocytes of different patients. When the patients were un-blinded and grouped based on their clinical outcome, either non-relapsed or relapsed, a significant difference in the DNA structure of their Reed-Sternberg (RS) cells was found. Since, RS cells develop from mono-nucleated Hodgkin (H) cells, these data suggest distinct architectural restructuring of nuclei during RS cell formation in patients going to long-lasting remission versus relapse. J. Cell. Biochem. 117: 1633-1637, 2016. © 2015 Wiley Periodicals, Inc.