Super-resolution Measurements Research Articles

Membrane phase separation drives responsive assembly of receptor signaling domains.

Plasma membrane heterogeneity has been tied to a litany of cellular functions and is often explained by analogy to membrane phase separation; however, models based on phase separation alone fall short of describing the rich organization available within cell membranes. Here we present comprehensive experimental evidence motivating an updated model of plasma membrane heterogeneity in which membrane domains assemble in response to protein scaffolds. Quantitative super-resolution nanoscopy measurements in live B lymphocytes detect membrane domains that emerge upon clustering B cell receptors (BCRs). These domains enrich and retain membrane proteins based on their preference for the liquid-ordered phase. Unlike phase-separated membranes that consist of binary phases with defined compositions, membrane composition at BCR clusters is modulated through the protein constituents in clusters and the composition of the membrane overall. This tunable domain structure is detected through the variable sorting of membrane probes and impacts the magnitude of BCR activation.

Super-resolved three-dimensional near-field mapping by defocused imaging and tracking of fluorescent emitters

Abstract Near-field optics is essential in many nanotechnology applications, such as implementing sensitive biosensing and imaging systems with extreme precision. Understanding optical near-fields at the nanoscale has so attracted the considerable research interest, which use a variety of analytical approaches, most notably near-field scanning microscopy. Here, we show defocused point localization mapped accumulation (DePLOMA), which can overcome many weaknesses of conventional analytical methods. DePLOMA is based on imaging fluorescence emitters at an out-of-focal plane. The acquisition, collection, and accumulation of the position and fluorescence intensity of emitters moving above nanostructures can generate three-dimensional near-field maps of light distribution. The idea enables super-resolution liquid-phase measurements, as demonstrated by reconstruction of near-field created by nanoslits with a resolution determined by emitter size. We employed fluorescent emitters with a radius of 50 and 100 nm for confirmation. The axial resolution was found to be enhanced by more than 6 times above that of diffraction-limited confocal laser scanning microscopy when DePLOMA was used.

Localization of Nonblinking Point Sources Using Higher-Order-Mode Detection and Optical Heterodyning: Developing a Strategy for Extending the Scope of Molecular, Super-resolution Imaging.

While the stochastic, "blinking" nature of fluorescent systems has enabled the super-resolution of their localization by the fitting of their point-spread functions (PSFs), this strategy cannot be exploited for similar resolution of "nonblinking" systems, such as those that might be encountered in a coherent Raman experiment. An alternative method for subdiffraction-limited imaging lies in the exploitation of optical heterodyning. For example, if a Gaussian PSF (a TEM00 mode) of a point emitter is displaced with respect to the origin of the optical system, photons in the higher-order TEM modes carry information about that displacement. Information concerning the displacement can be extracted from photons in these higher-order modes. These photons can be collected by optical heterodyning, which exploits the large gain in a detector's response to an optical signal from an emitter coupled to a local oscillator, which is prepared in the TEM of interest, e.g., TEM10. We have generalized and developed the heterodyning technique to localize point emitters via the detection of higher-order spatial modes. We have developed a theoretical approach to find a practical estimation limit of the localization parameters using a realistic model that accounts for shot noise, background noise, and Gaussian noise. To demonstrate the applicability of the method, we designed experiments in which a laser is a surrogate for one and two point emitters. Using the Fisher information and its accompanying Cramér-Rao lower bound, we demonstrate super-resolution localization in these cases: we show that objects can be localized to roughly 2-3 orders of magnitude of their point-spread function's size for a given optical system. Finally and most importantly, it is suggested that the results will ultimately be generalizable to multiple emitters and, most importantly, to "nonblinking" molecular systems, which will be essential for broadening the scope of super-resolution measurements beyond the limits of fluorescence-based techniques.

A Programmable DNA-Silicification-Based Nanocavity for Single-Molecule Plasmonic Sensing.

Plasmonic nanocavities are highly desirable for optical sensing because of their singular ability to confine light into deep subwavelength volumes. Yet, it remains profoundly challenging to fabricate structurally resilient nanocavities with high fidelity, and to obtain direct, noninvasive visualization of the plasmonic hotspots within such constructs. Herein, highly precise and robust nanocavities, entitled DNA-silicified template for Raman optical beacon (DNA-STROBE), are engineered by using silicified DNA scaffolds for spatial organization of discrete plasmonic nanoparticles. In addition to substantially enhancing structural stability and chemical inertness, DNA silicification significantly improves nanogap control, resulting simultaneously in large and controlled local electromagnetic field enhancement. The ultrasmall mode volume of the DNA-STROBE constructs promotes single-molecule occupancy enabling surface-enhanced Raman spectroscopy (SERS) observations of single-molecule activity even at elevated background concentration, significantly relaxing the restrictive pico- to nanomolar molecular concentration condition typically required for such investigations. Additionally, leveraging super-resolution SERS measurements allows noninvasive and diffraction-unlimited spatial profiling of otherwise unresolvable plasmonic hotspots. The highly programmable and reproducible nature of the DNA-STROBE, coupled with its quantitative label-free molecular readouts, provides a versatile platform with applications across the spectrum of nanophotonics and biomedical sciences.

Super-resolution FRET measurements

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems, as well as to investigate nanomaterials. In the last years, the integration of super-resolution methods with FRET measurements has generated advances in two fronts. On the one hand, FRET-based probes have enhanced super-resolution imaging. On the other, the development of super-resolved FRET imaging methods has allowed the visualization of molecular interaction patterns with higher spatial resolution, less averaging and higher dynamic range. Here, we review these advances and discuss future perspectives, including the possible integration of FRET with next generation super-resolution techniques capable of reaching true molecular-scale spatial resolution.

Application of Superresolution Radial Fluctuation (SRRF) Imaging to Measurement of Single-Molecule DNA Hybridization Kinetics

Superresolution radial fluctuations (SRRF) is a novel algorithm based superresolution imaging technique that incorporates radial intensity gradients and temporal intensity fluctuations to calculate and project SRRF images with resolution up to 2x below the diffraction limit in real time. SRRF, has been used for imaging sub-diffraction intracellular structures and for performing time-resolved superresolution measurements of live cell dynamics. However, no application to single-molecule localization and tracking techniques has been shown. Here, we characterize the capabilities of the SRRF algorithm on measuring DNA hybridization kinetics from a DNA capture surface via single-molecule localization and tracking. We find that SRRF data has some obvious advantages such as a substantial increase in the signal-to-background ratio of single-molecule data. This is a side effect of the image radiality transform which yields low radiality values for regions which contain randomized intensity values on the distance scales of the radiality calculation. This is particularly usefully when measuring hybridization kinetics with slow on rates which necessitate higher solution phase concentrations and resulting in higher background levels. We also find that with sparse data such as these, molecule localization can be performed by calculation of the 1st moment centroids of the SRRF point spread functions (PSF) which can deliver spatial resolution similar to non-linear fitting of the optical PSF but with much faster computational times. Finally, we explore the ability for SRRF to resolve individual molecules in high DNA surface density conditions which result in many overlapping optical PSFs. We find that measuring single-molecule data following the SRRF radiality transform allows for resolving much higher densities of molecules than diffraction limited images thus allowing for faster collection of statistically significant population data.

Rapid whole-heart CMR with single volume super-resolution

BackgroundThree-dimensional, whole heart, balanced steady state free precession (WH-bSSFP) sequences provide delineation of intra-cardiac and vascular anatomy. However, they have long acquisition times. Here, we propose significant speed-ups using a deep-learning single volume super-resolution reconstruction, to recover high-resolution features from rapidly acquired low-resolution WH-bSSFP images.MethodsA 3D residual U-Net was trained using synthetic data, created from a library of 500 high-resolution WH-bSSFP images by simulating 50% slice resolution and 50% phase resolution. The trained network was validated with 25 synthetic test data sets. Additionally, prospective low-resolution data and high-resolution data were acquired in 40 patients. In the prospective data, vessel diameters, quantitative and qualitative image quality, and diagnostic scoring was compared between the low-resolution, super-resolution and reference high-resolution WH-bSSFP data.ResultsThe synthetic test data showed a significant increase in image quality of the low-resolution images after super-resolution reconstruction. Prospectively acquired low-resolution data was acquired ~× 3 faster than the prospective high-resolution data (173 s vs 488 s). Super-resolution reconstruction of the low-resolution data took < 1 s per volume. Qualitative image scores showed super-resolved images had better edge sharpness, fewer residual artefacts and less image distortion than low-resolution images, with similar scores to high-resolution data. Quantitative image scores showed super-resolved images had significantly better edge sharpness than low-resolution or high-resolution images, with significantly better signal-to-noise ratio than high-resolution data. Vessel diameters measurements showed over-estimation in the low-resolution measurements, compared to the high-resolution data. No significant differences and no bias was found in the super-resolution measurements in any of the great vessels. However, a small but significant for the underestimation was found in the proximal left coronary artery diameter measurement from super-resolution data. Diagnostic scoring showed that although super-resolution did not improve accuracy of diagnosis, it did improve diagnostic confidence compared to low-resolution imaging.ConclusionThis paper demonstrates the potential of using a residual U-Net for super-resolution reconstruction of rapidly acquired low-resolution whole heart bSSFP data within a clinical setting. We were able to train the network using synthetic training data from retrospective high-resolution whole heart data. The resulting network can be applied very quickly, making these techniques particularly appealing within busy clinical workflow. Thus, we believe that this technique may help speed up whole heart CMR in clinical practice.

Predicting three-dimensional genome organization with chromatin states.

We introduce a computational model to simulate chromatin structure and dynamics. Starting from one-dimensional genomics and epigenomics data that are available for hundreds of cell types, this model enables de novo prediction of chromatin structures at five-kilo-base resolution. Simulated chromatin structures recapitulate known features of genome organization, including the formation of chromatin loops, topologically associating domains (TADs) and compartments, and are in quantitative agreement with chromosome conformation capture experiments and super-resolution microscopy measurements. Detailed characterization of the predicted structural ensemble reveals the dynamical flexibility of chromatin loops and the presence of cross-talk among neighboring TADs. Analysis of the model’s energy function uncovers distinct mechanisms for chromatin folding at various length scales and suggests a need to go beyond simple A/B compartment types to predict specific contacts between regulatory elements using polymer simulations.

Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

Super-resolution imaging within reach

Although several optical techniques have been developed to overcome the resolution limit in microscopy, the optical imaging of subwavelength features is still a real challenge (difficulty to build, or risks of phototoxicity). Microsphere-assisted microscopy has recently made super-resolution imaging accessible to scientists. An easy-to-implement optical setup to perform full-field and contactless super-resolution measurements using transparent microspheres is presented. We show that this rather simple imaging system (in comparison with the usual optical nanoscopes) makes it possible to achieve a lateral resolution of 200 nm in air and, thus, to visualize nanostructured media and biological elements.

How to Calibrate Your CCD in the Dark? a Precise CCD Calibration Method

Single particle tracking and super-resolution microscopy experiments require good knowledge of CCD pixels’ gains in terms of photoelectrons per ADU (Analog to Digital Unit, output of the CCD camera) registered by the read-out electronics of the CCD camera. This is because the observed CCD output in a given time period produced by light with constant intensity follows, ignoring other noise sources, statistics of photoelectrons produced by incident photons. It is common practice to calibrate the gain of a CCD camera using a constant intensity light source to get the gain in photoelectron per ADU. This procedure requires a stable light source and it is not always possible to achieve a truly uniform and stable illumination. We propose an easier calibration method that uses dark noise as the calibration source for a CCD camera; the method requires that the dark noise level is stable during the calibration data taking. The proposed method works as demonstrated under assumptions that statistics of photoelectrons and thermal electrons follow Poisson distribution and CCD pixel gains in electrons per ADU are the same for the both sources of electrons. We will show that these assumptions are valid and will demonstrate that it is possible to calibrate individual gain of each pixel at ∼1% level or better using real and simulated data. As a byproduct, the proposed calibration method provides properties of individual pixel, not only its gain but also other properties such as linearity in low photon intensity and dark noise level. This information gives assessment of quality of the CCD camera. All the necessary software is developed using freely available software package Octave similar to Matlab. This method might improve the localization of fluorescent molecules in super-resolution and particle tracking measurements.

Superresolution measurements in vivo: imaging Drosophila embryo by photoactivated localization microscopy.

Visualization and quantification of supramolecular assemblies in cells are essential to understand the design principles of cells and tissues. The advent of photoactivated localization microscopy (PALM) and related techniques has offered unprecedented information on protein supramolecular assemblies in 3-D with a spatial resolution of a few tens of nanometers. Yet application of PALM microscopy for in vivo studies remains challenging. This chapter describes how to implement PALM microscopy for quantitative analysis of intercellular adhesion in the Drosophila embryo. Our protocol describes the sample preparation, the imaging setup, and the acquisition procedure. We also discuss how to proceed with quantitative analysis of data. Initially designed and implemented for Drosophila embryo imaging of intercellular adhesion, this protocol can be readily adapted to other structures than adhesions and other organisms such as Zebrafish or Caenorhabditis elegans.

Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging.

In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.

Super-resolution compressive imaging with anamorphic optics

A new imaging technique that combines compressive sensing and super-resolution techniques is presented. Compressive sensing is accomplished by capturing optically a set of Radon projections. Super-resolution measurements are simply taken by introducing a slanted two-dimensional array in the optical system. The goal of the technique is to overcome resolution limitation that occurs in imaging scenarios where dense pixels sensors with large number of pixels are not available or cannot be used. With the presented imaging technique, owing to the compressive sensing approach, we were able to reconstruct images with significantly more number of pixels than measured, and owing to the super-resolution design we have been able to achieve resolution significantly beyond that limited by the sensor's pixels size.

Super-resolution measurements related to uncertainty relations in optical and biological fluorescence systems

Super-resolution effects in optical and fluorescence biological systems are analyzed, and their relations with uncertainty relations are discussed. Super-resolutions obtained in the optical systems, including especially NSOM, SIM, hyperlens and Pendry lens, are related to an increase of the spatial frequencies in the object plane, leading to very small effective space uncertainties, and thus the resolution is increased far beyond the Abbe limit. Super-resolution measurements obtained in the fluorescent biological systems STED, FPALM and RESOLFT are related to space-momentum uncertainty relation. An example of a four-level STED system is analyzed, in analogy to a four-level laser system, but the special space dependence of the STED light is taken into account, restricting the fluorescence from extremely small volume, and thus extremely high resolution is obtained. Localization of individual molecules is described by the FPALM method where, by illuminating the fluorescent molecule by a ‘read out’ laser, many fluorescent photons are emitted by the same molecule before it decays or transmitted to non-fluorescent states. The relation between this effect and the space-momentum uncertainty relation is discussed. While in the STED method very high laser intensities are needed, in its variant method known as RESOLFT, the super-resolution measurements can be obtained by much weaker light intensities. This new method is analyzed, and the relation between this method and space-momentum uncertainty relation is explained.

Super-Resolution Raman Spectroscopy by Digital Image Processing

We demonstrate the results of a strain (stress) evaluation obtained from Raman spectroscopy measurements with the super-resolution method (the so-called super-resolution Raman spectroscopy) for a Si substrate with a patterned SiN film (serving as a strained Si sample). To improve the spatial resolution of Raman spectroscopy, we used the super-resolution method and a high-numerical-aperture immersion lens. Additionally, we estimated the spatial resolution by an edge force model (EFM) calculation. One- and two-dimensional stress distributions in the Si substrate with the patterned SiN film were obtained by super-resolution Raman spectroscopy. The results from both super-resolution Raman spectroscopy and the EFM calculation were compared and were found to correlate well. The best spatial resolution, 70 nm, was achieved by super-resolution Raman measurements with an oil immersion lens. We conclude that super-resolution Raman spectroscopy is a useful method for evaluating stress in miniaturized state-of-the-art transistors, and we believe that the super-resolution method will soon be a requisite technique.

Conformation and Energy Transfer in Single Conjugated Polymers

In contrast to the detailed understanding of inorganic materials, researchers lack a comprehensive view of how the properties of bulk organic materials arise from their individual components. For conjugated polymers to eventually serve as low cost semiconductor layers in electronic devices, researchers need to better understand their functionality. For organics, traditional materials science measurements tend to destroy the species of interest, especially at low concentrations. However, fluorescence continues to be a remarkably flexible, relatively noninvasive tool for probing the properties of individual molecules and allows researchers to carry out a broad range of experiments based on a relatively simple concept. In addition, the sensitivity of single-molecule spectroscopy allows researchers to see the properties of an individual component that would be masked in the bulk phase. In this Account, we examine several photophysical properties of different conjugated polymers using single-molecule spectroscopy. In these experiments, we probed the relationship between the conformation of single conjugated polymer chains and the distance scale and efficiency of energy transfer within the polymer. Recent studies used polarization anisotropy measurements on single polymer chains to study chain folding following spin-casting from solution. This Account summarizes the effects of monomer regioregularity and backbone rigidity, by comparing a regiorandom phenylene vinylene (MEH-PPV) with both a regiorandom and regioregular thiophene (P3HT). Synthesis of novel polymers allowed us to explore the role of different conformation-directing inclusions in a PPV backbone. We showed that these inclusions control the conformation of individual chains and that molecular dynamics can predict these structural effects. In situ solvent vapor annealing studies explored the dynamics of polymer chains as well as the effect of solvent evaporation on the structural equilibrium of the polymer. We observed that a slower rate of solvent evaporation results in a narrow population of highly ordered polymer chains. These highly ordered single chains serve as a model system to probe the effect of conformation on energy transfer following excitation in single MEH-PPV polymer chains in two distinct experiments. In the first, we correlated the anisotropy of the fluorescence emission of individual chains with the anisotropy of their fluorescence excitation. Using this data, we derived a model for energy transfer in a conjugated polymer, simulating chromophores along a chain, coupled via Förster energy transfer. In the second experiment, super-resolution measurements demonstrated the ability of single-molecule spectroscopy to directly visualize energy transfer along a polymer chain embedded in a model device environment. A capacitive device allowed for controlled localization of hole polarons onto the polymer chain. These positive charges subsequently quenched local excitations, providing insight into the range of energy transfer in these single polymer molecules. As researchers continue to characterize conjugated polymer films and develop methods for creating multichain systems, single-molecule techniques will provide a greater understanding of how polymer morphology influences interchain interactions and will lead to a richer description of the electronic properties of bulk conjugated polymer films.

Super-Resolution Tracking and Imaging of IgE Receptors during Stimulation by Antigen

In mast cells, cross-linking of IgE bound to FceR1 receptors with multivalent antigen initiates cell activation and consequent inflammatory responses. Clustered IgE-FceRI interacts with early signaling partners such as Lyn, Syk, and LAT, initiating a signaling cascade. Our previous scanning electron microscopy (SEM) studies quantified the nanoscale co-redistribution of FceRI with Lyn, Syk, and LAT within the plasma membrane upon antigen stimulation. Here we use super-resolution localization microscopy to localize proteins on the cell surface with sub-diffraction resolution using a TIR fluorescence microscope. We present, in snapshots, the time-dependence of IgE receptor reorganization after antigen addition using chemically fixed cells and quantify the extent of clustering using pair autocorrelation functions. We have validated our methods for quantifying and correcting for artifactual clustering due to probe over-counting through two-color experiments where individual IgE proteins are labeled with one of two species of distinguishable fluorescent probe and imaged at sub-diffraction resolution in multiple color channels. Here clustering is quantified using pair cross-correlation functions. Results of one- and two-color super-resolution measurements agree well with conclusions from SEM experiments. We have also extended this technique to imaging living cells at room temperature to measure IgE receptor redistribution in real time. We quantify the organization and diffusion behavior of proteins undergoing stimulated responses with the excellent statistics afforded by the technique. Live cell experiments are conducted under a variety of stimulation conditions, such as in the presence of drugs that perturb membrane physical state or affect actin cytoskeleton assembly. Our results confirm that super-resolution imaging is an effective tool for quantitative imaging of cellular components at the nanoscale. Our ongoing studies are providing new information to clarify the physical basis for spatial assembly of specific proteins in the plasma membrane during early signaling events.

THE KEPLER PIXEL RESPONSE FUNCTION

Kepler seeks to detect sequences of transits of Earth-size exoplanets orbiting solar-like stars. Such transit signals are on the order of 100 ppm. The high photometric precision demanded by Kepler requires detailed knowledge of how the Kepler pixels respond to starlight during a nominal observation. This information is provided by the Kepler pixel response function (PRF), defined as the composite of Kepler's optical point-spread function, integrated spacecraft pointing jitter during a nominal cadence and other systematic effects. To provide sub-pixel resolution, the PRF is represented as a piecewise-continuous polynomial on a sub-pixel mesh. This continuous representation allows the prediction of a star's flux value on any pixel given the star's pixel position. The advantages and difficulties of this polynomial representation are discussed, including characterization of spatial variation in the PRF and the smoothing of discontinuities between sub-pixel polynomial patches. On-orbit super-resolution measurements of the PRF across the Kepler field of view are described. Two uses of the PRF are presented: the selection of pixels for each star that maximizes the photometric signal-to-noise ratio for that star, and PRF-fitted centroids which provide robust and accurate stellar positions on the CCD, primarily used for attitude and plate scale tracking. Good knowledge of the PRF has been a critical component for the successful collection of high-precision photometry by Kepler.

Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence.

The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and--close to the coverslip--can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles ('vesicles') of approximately 300 nm diameter in the "footprint" region of living neuroendocrine cells grown on the interface.