Super-resolution Capability Research Articles

Fluorescence emission difference with surface plasmon-coupled emission applied in confocal microscopy.

We combined confocal surface plasmon coupled emission microscopy (C-SPCEM) together with fluorescence emission difference (FED) technique to pursuit super-resolution fluorescent image. Solid or hollow point spread function (PSF) for C-SPCEM is achieved with radially-polarized or circularly-polarized illumination. The reason why PSF can be manipulated by the polarization of illumination light is corroborated by the interaction of fluorescent emitter with vector focal field on the plasmonic substrate. After introduction of FED technique, PSF for C-SPECM can shrunk to around λ/4 in full-width half-maximum, which is unambiguously beyond Rayleigh's diffraction limit. The super-resolution capability of C-SPCEM with FED technique is experimentally demonstrated by imaging aggregated fluorescent beads with 150 nm in diameter.

Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.

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.

A simple and cost-effective setup for super-resolution localization microscopy

Single molecule localization microscopy (SMLM) has become a powerful imaging tool for biomedical research, but it is mostly available in imaging facilities and a small number of laboratories due to its high cost. Here, we evaluate the possibility of replacing high-cost components on standard SMLM with appropriate low-cost alternatives and build a simple but high-performance super-resolution SMLM setup. Through numerical simulation and biological experiments, we demonstrate that our low-cost SMLM setup can yield similar localization precision and spatial resolution compared to the standard SMLM equipped with state-of-the-art components, but at a small fraction of their cost. Our low-cost SMLM setup can potentially serve as a routine laboratory microscope with high-performance super-resolution imaging capability.

Superresolved spatially multiplexed interferometric microscopy.

Superresolution capability by angular and time multiplexing is implemented onto a regular microscope. The technique, named superresolved spatially multiplexed interferometric microscopy (S2MIM), follows our previously reported SMIM technique [Opt. Express22, 14929 (2014)OPEXFF1094-408710.1364/OE.22.014929, J. Biomed. Opt.21, 106007 (2016)JBOPFO1083-366810.1117/1.JBO.21.10.106007] improved with superresolved imaging. All together, S2MIM updates a commercially available non-holographic microscope into a superresolved holographic one. Validation is presented for an Olympus BX-60 upright microscope with resolution test targets.

Further resolution enhancement of high-sensitivity laser scanning photothermal microscopy applied to mouse endogenous

Photothermal microscopy has intrinsically super resolution capability due to the bilinear dependence of signal intensity of pump and probe. In the present paper, we have made further resolution improvement of high-sensitivity laser scanning photothermal microscopy by applying non-linear detection. By this, the new method has the following advantages: (1) super resolution with 61% and 42% enhancement from the diffraction limit values of the probe and pump wavelengths, respectively, by a second-order non-linear scheme, (2) compact light source using inexpensive conventional diode lasers, (3) wide applicability to nonfluorescent materials such as gold nanoparticles (GNPs) and hematoxylin-eosin stained biological samples, (4) relative robustness to optical damage, and (5) a high-frame rate using a Galvano mirror. The maximum resolution is determined to be 160 nm in the second-order non-linear detection mode and 270 nm in the linear detection mode by the PT signal of GNPs. The pixel rate and frame rate for 300 × 300 pixel image are 50 μs and 4.5 s, respectively. The pixel and frame rate are shorter than the rates, which are 1 ms and 100 s, respectively, using the piezo-driven stage system.

Super-resolution imaging by microsphere-assisted optical microscopy

Microsphere-assisted optical microscopy achieved the nano-scale resolution for the imaging. Some parts of evanescent waves produced by nano-structures can be transformed into propagating waves with the help of microsphere. In this paper, we develop the equations of the resolution and field of view in microsphere imaging system related to the radius and refractive index of microsphere. Through analysis the highest spatial frequency, the resolution reduces quickly with the increase of the radius when the radius is less than 10 μm. The resolution reduces slowly with the increase of the radius when the radius is more than 10 μm. The resolution improves with the increase of refractive index. The size of the focus spot of microsphere is simulated numerically. The simulations are in good agreement with the theoretical prediction about the microsphere-assisted microscopy. Finally, the super-resolution capability of the microsphere has been verified by the experimental results. The results are useful for the practical design of the microsphere-assisted optical microscopy.

Sparse continuous-field and super-resolution method for direction-of-arrival estimation

In the application of direction-of-arrival (DOA) estimation based on sensor array, bearing-discretization has been a major technique for the sparsity-based methods in the past decade. However, discretization leads to the modeling error of sparse representation, which usually deteriorates the accuracy of DOA estimation. In this paper, a sparse continuous-field and super-resolution method (SCSM) is proposed to overcome the discretization issue. SCSM estimates DOA in the continuous bearing-space by solving a minimization problem, which combines the measurement covariance fitting criterion with the source-sparsity constraint characterized by minimizing an atomic norm. This minimization problem has a unique solution. After obtained the solution uniquely, Prony's method is utilized to estimate the DOA of sources. Therefore, SCSM has the capability to acquire a sparse and accurate estimation of DOA. In addition, it has some other advantages, including excellent super-resolution capability, no requirement of source number, applicability to arbitrary number of snapshots. Numerical results demonstrate the superior performance of the presented method.

SmiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells.

DOA Estimation Using Second-Order Differential of Invariant Noise MUSIC (SODIN-MUSIC)

Sensor arrays are widely used to locate the radiating sources by estimating the direction of arrival (DOA) of their incident signals. Among the most well-known methods employed for DOA estimation is the multiple signal classification (MUSIC). MUSIC provides outstanding performance in estimating DOAs in good propagation environments; however, it fails to estimate the DOAs in severe environments like low signal-to-noise ratio, small number of snapshots, when the incident waves are coming from close angles or when the sources are highly correlated. In this paper, we propose a new approach for DOA estimation with super-resolution capabilities and high accuracy even in the severe environments. The proposed approach is based on computing the second-order differential of invariant noise MUSIC (SODIN-MUSIC) spectrum. SODIN-MUSIC benefits from extracting useful information provided by the second-order differential of MUSIC spectrum and also utilizing the invariance property of noise subspace with respect to changing the power of the emitted signals. In this paper, the proposed SODIN-MUSIC method is developed to estimate both one-dimensional and two-dimensional DOA with any arbitrary array configurations. The simulation results show that SODIN-MUSIC yields superior performance in all distinct environments if compared to both conventional MUSIC and other newly developed MUSIC-based methods.

Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells.

Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson’s disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments.

Single‐look light‐burden superresolution differential SAR tomography

Research and application is spreading of techniques of coherent combination of complex-valued synthetic aperture radar (SAR) data to extract rich information even on complex observed scenes, fully exploiting existing SAR data archives, and new satellites. Among such techniques, SAR tomography stems from multibaseline interferometry to achieve full-3D imaging through elevation beamforming (spatial spectral estimation). The Tomo concept has been integrated with the mature differential interferometry, producing the new differential tomography (Diff-Tomo) processing mode, that allows ‘opening’ the SAR cells in complex non-stationary scenes, resolving multiple heights and slow deformation velocities of layover scatterers. Consequently, the operational capability limit of differential interferometry to the single scatterer case is overcome. Diff-Tomo processing is cast in a 2D baseline-time spectral analysis framework, with sparse sampling. The use of adaptive 2D spectral estimation has demonstrated to allow joint baseline-time processing with reduced sidelobes and enhanced height–velocity resolution at low computational burden. However, this method requires coherent multilooking processing, thus does not produce full range-azimuth resolution products, as it would be desirable for urban applications. A new single-look adaptive Diff-Tomo processor is presented and tested with satellite data, allowing full range-azimuth resolution together with height–velocity sidelobe reduction and superresolution capabilities and the low computational burden.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

Super Resolution Microscopy with Induced Optical Fluctuation

In conventinal microscopy, resolution is fundamentally limited by diffraction barrier. However, as phenomena within size of diffraction limit become important in many field, diffraction barrier becomes a big issue in conventional microscopy. To overcome this, many researchers have developed super-resolution methods using physical principles. Among them, lately, Super Resolution Optical fluctuation Imaging (SOFI) method developed by T. Dertinger et al, provides super-resolution image using statistical analysis of temporal optical fluctuation that comes from the blinking property of fluorophores. Because SOFI only requires opical fluctuation of fluorophores without any special equipment, it can be directly applied to conventional microscopy for super-resolution imaging. However, because intrinsic blinking property of fluorophores is uncontrollable, SOFI has practical challenges and limitations in some applications, including the limitation of available fluorophores and camera speed. To overcome these limitations, we proposed a new concept of SOFI using optical fluctuation induced by the illumination light with random patterns. Because the optical flcutuation does not come from intrinsic blinking property of fluorophore, but comes from the illumination light in this method, the blinking becomes a controllable property using external method. We employ speckle patterns as random patterns because speckle pattern has interesting statistical properties such as correlation. To verify the super-resolution capability of our proposed method, simple model simulation with only two flurophores was analytically solved using equations of SOFI and speckle pattern statistics. Also, to experimentally demonstrate it, diffuser and motorized stage were added in conventional microscopy to generate speckle patterns and statistical properties of speckle patterns. Using simulation and experiment, we theoretically and experimentally demonstrate the super-resolution imaging capability of our proposed method. Furthermore, proposed method gives super-resolution image of samples labelled with non-blinking fluorophores.

Nanoscale shaping and focusing of visible light in planar metal–oxide–silicon waveguides

Focusing light at the nanoscale has become a key factor in super-resolution applications. Dynamic control of this focusing can open new avenues in nanoelectronics and bioimaging, but it requires a platform that merges electronics with super-resolution capabilities. We present a planar metal–oxide–silicon (MOS) platform that allows us to shape, tune, and focus visible light at the nanoscale by compressing the wavelength of light fourfold, resulting in a scaled diffraction limit of 65 nm. We exemplify the control and flexibility by demonstrating nanovortex beams and short-wavelength super-oscillations of light that further enhance resolution toward 35 nm. Our platform achieves focusing strength similar to nanoantennas but without structural hotspots; hence it is possible to scan the focus via optical wavefront-shaping techniques. Super-resolution scanning without mechanical translations in a MOS platform can provide a building block for bioimaging, nanolithography, and lab-on-a-chip applications.

Remote focusing of a light beam

The remote focusing of light in a graded-index medium via mode interference is demonstrated using exact analytical solutions of the wave equation. Strong focusing of a light beam occurs at extremely long distances and this repeats periodically with distance. The high efficiency transfer of a strongly focused subwavelength spot through an optical waveguide over large distances takes place with a period of revival. The simulated intensity distributions of a focused light beam and focusing efficiency (power in a central spot/total power) are presented. The far-field super-resolution imaging capabilities of a graded-index waveguide are also analyzed.

Bayesian segmentation of human facial tissue using 3D MR-CT information fusion, resolution enhancement and partial volume modelling

BackgroundAccurate segmentation of human head on medical images is an important process in a wide array of applications such as diagnosis, facial surgery planning, prosthesis design, and forensic identification. ObjectivesIn this study, a Bayesian method for segmentation of facial tissues is presented. Segmentation classes include muscle, bone, fat, air and skin. MethodsThe method presented incorporates information fusion from multiple modalities, modelling of image resolution (measurement blurring), image noise, two priors helping to reduce noise and partial volume. Image resolution modelling employed facilitates resolution enhancement and superresolution capabilities during image segmentation. Regularization based on isotropic and directional Markov Random Field priors is integrated. The Bayesian model is solved iteratively yielding tissue class labels at every voxel of the image. Sub-methods as variations of the main method are generated by using a combination of the models. ResultsTesting of the sub-methods is performed on two patients using single modality three-dimensional (3D) image (magnetic resonance, MR or computerized tomography, CT) as well as registered MR-CT images with information fusion. Numerical, visual and statistical analyses of the methods are conducted. High segmentation accuracy values are obtained by the use of image resolution and partial volume models as well as information fusion from MR and CT images. The methods are also compared with our Bayesian segmentation method proposed in a previous study. The performance is found to be similar to our previous Bayesian approach, but the presented methods here eliminates ad hoc parameter tuning needed by the previous approach which is system and data acquisition setting dependent. ConclusionsThe Bayesian approach presented provides resolution enhanced segmentation of very thin structures of the human head. Meanwhile, free parameters of the algorithm can be adjusted for different imaging systems and data acquisition settings in a more systematic way as compared with our previous study.

Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods.

Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy are two super-resolution optical microscopy approaches that have rapidly gained popularity in recent years. Both modalities offer super-resolution imaging capabilities with the potential for imaging in multiple colors, three-dimensions, and the possibility to image in live cells. In this review, we focus on the specific advantages and disadvantages of each technique in the context of each other. STORM has been reported to achieve higher spatial resolution when compared to STED, but a lengthy acquisition may be required. STED utilizes relatively higher laser intensities, but is able to generate a super-resolution image immediately after acquisition without the need for any additional data processing. Ultimately, the choice between STORM and STED will depend not only on the specific application, but also on the users' ability to understand and optimize the various parameters ranging from sample preparation to image acquisition, which determine the quality of the final image. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) are two super-resolution microscopy approaches that have rapidly gained popularity in recent years. STORM is based on the precise localization of a large number of individual molecules that together form a super-resolved image (bottom), whereas STED is based on the scanning of two super-imposed light sources which together allow for a super-resolved spot on the sample to be imaged (top). We discuss the specific advantages and disadvantages of each technique and explain the various parameters that affect image quality, which should be taken into consideration when planning experiments.

Sparse synthetic aperture with Fresnel elements (S-SAFE) using digital incoherent holograms.

Creating a large-scale synthetic aperture makes it possible to break the resolution boundaries dictated by the wave nature of light of common optical systems. However, their implementation is challenging, since the generation of a large size continuous mosaic synthetic aperture composed of many patterns is complicated in terms of both phase matching and time-multiplexing duration. In this study we present an advanced configuration for an incoherent holographic imaging system with super resolution qualities that creates a partial synthetic aperture. The new system, termed sparse synthetic aperture with Fresnel elements (S-SAFE), enables significantly decreasing the number of the recorded elements, and it is free from positional constrains on their location. Additionally, in order to obtain the best image quality we propose an optimal mosaicking structure derived on the basis of physical and numerical considerations, and introduce three reconstruction approaches which are compared and discussed. The super-resolution capabilities of the proposed scheme and its limitations are analyzed, numerically simulated and experimentally demonstrated.

A Novel Fast Approach for SAR Tomography: Two-Step Iterative Shrinkage/Thresholding

As an advanced technique, synthetic aperture radar (SAR) tomography makes it possible to overcome the layover problem induced by the intrinsic side-looking geometry of SAR sensors. However, traditional nonparametric spectral estimators, e.g., truncated singular value decomposition, are limited by their poor elevation resolution. On the other hand, the compressive-sensing-based approaches using the basis-pursuit strategy to find an L1-norm minimization solution for SAR tomography (TomoSAR) are extremely time consuming. Therefore, a fast and robust tomographic algorithm with super-resolution capability is needed. In this letter, we propose a new approach for TomoSAR based on two-step iterative shrinkage/thresholding (TWIST). TWIST uses a two-step strategy to speed up the L1-norm minimization procedure and can achieve an exceptionally fast convergence speed for TomoSAR. Experimental studies with simulated signals and a spotlight-mode TerraSAR-X data set were carried out to demonstrate the merits of the proposed TWIST approach in terms of robustness, fast convergence speed, and super-resolution capability.