Super-resolution Imaging Techniques Research Articles

The Architecture of the Presynaptic Release Site.

The architecture of the presynaptic release site is exquisitely designed to facilitate and regulate synaptic vesicle exocytosis. With the identification of some of the building blocks of the active zone and the advent of super resolution imaging techniques, we are beginning to understand the morphological and functional properties of synapses in great detail. Presynaptic release sites consist of the plasma membrane, the cytomatrix, and dense projections. These three components are morphologically distinct but intimately connected with each other and with postsynaptic specializations, ensuring the fidelity of synaptic vesicle tethering, docking, and fusion, as well as signal detection. Although the morphology and molecular compositions of active zones may vary among species, tissues, and cells, global architectural design of the release sites is highly conserved.

Speckle structured illumination endoscopy with enhanced resolution at wide field of view and depth of field

Structured illumination microscopy (SIM) is one of the most widely applied wide field super resolution imaging techniques with high temporal resolution and low phototoxicity. The spatial resolution of SIM is typically limited to two times of the diffraction limit and the depth of field is small. In this work, we propose and experimentally demonstrate a low cost, easy to implement, novel technique called speckle structured illumination endoscopy (SSIE) to enhance the resolution of a wide field endoscope with large depth of field. Here, speckle patterns are used to excite objects on the sample which is then followed by a blind-SIM algorithm for super resolution image reconstruction. Our approach is insensitive to the 3D morphology of the specimen, or the deformation of illuminations used. It greatly simplifies the experimental setup as there are no calibration protocols and no stringent control of illumination patterns nor focusing optics. We demonstrate that the SSIE can enhance the resolution 2–4.5 times that of a standard white light endoscopic (WLE) system. The SSIE presents a unique route to super resolution in endoscopic imaging at wide field of view and depth of field, which might be beneficial to the practice of clinical endoscopy.

Recent development of computational cluster analysis methods for single-molecule localization microscopy images

With the development of super-resolution imaging techniques, it is crucial to understand protein structure at the nanoscale in terms of clustering and organization in a cell. However, cluster analysis from single-molecule localization microscopy (SMLM) images remains challenging because the classical computational cluster analysis methods developed for conventional microscopy images do not apply to pointillism SMLM data, necessitating the development of distinct methods for cluster analysis from SMLM images. In this review, we discuss the development of computational cluster analysis methods for SMLM images by categorizing them into classical and machine-learning-based methods. Finally, we address possible future directions for machine learning-based cluster analysis methods for SMLM data.

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy.

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human HeLa cell line. When combined with the super-resolution technique expansion microscopy (ExM) involving swellable acrylamide hydrogels, fluorescent esters reveal nanoscale features including cytoplasmic membrane-bound compartments and nucleolar densities. We observe differential labelling patterns linked to the biochemical properties of the conjugated dye. Alterations in staining density and compartment specificity were seen depending on the timepoint of application in the ExM protocol. Additional complexity in labelling patterns was detected arising from inter-ester interactions. Our findings raise a number of considerations for the use of fluorescent esters. We demonstrate esters as a useful addition to the repertoire of stains of the cellular proteome, whether applied either on their own to visualise overall cellular morphology, or as counterstains providing ultrastructural context alongside specific target markers like antibodies.

Stimulated Raman versus Inverse Raman: Investigating Depletion Mechanisms for Super-Resolution Raman Microscopy.

Super-resolution fluorescence microscopy has been critical in elucidating the nanoscale structure of biological systems. However, fluorescent labels bring difficulties such as perturbative labeling steps and photobleaching. Thus, label-free super-resolution techniques are of great interest, like our group's 2016 stimulated Raman scattering (SRS) technique, stimulated Raman depletion microscopy (SRDM). Inspired by stimulated emission depletion microscopy, SRDM uses a toroidally shaped beam to deplete the signal formed on the edges of the focal spot, resulting in SRS signal being detected from only a subdiffraction limited region. In initial works, the cause of the depletion was not thoroughly characterized. Here, we conclusively demonstrate suppression mechanisms in SRDM, while also contrasting approaches to super-resolution Raman microscopy on the Stokes and anti-Stokes sides of the spectrum. By monitoring the depletion of both the SRS and inverse Raman scattering (IRS) signal at a range of depletion powers, we observed other four-wave coherent Raman pathways that correspond to the introduction of the femtosecond depletion beam. In addition, we showed the depletion of the IRS signal, paving the way for a super-resolution imaging technique based on IRS, inverse raman depletion microscopy (IRDM). Combined, SRDM and IRDM offer label-free super-resolution imaging over a large spectral range to accommodate a variety of different sample constraints.

Fast structured illumination microscopy via transfer learning with correcting

In microscopic imaging, structured light illumination technology has emerged as a significant super-resolution imaging technique due to its inherent advantages, including rapid imaging speed and low phototoxicity. However, the conventional super-resolution image reconstruction algorithm is still widely used today, despite its slow calculation speed, difficulty in setting parameters, and unpredictable artifact generation. With the advancement of computer technology, image reconstruction and artifact removal can be accomplished rapidly using deep learning. Nowadays, deep learning training requires a large number of data samples, but obtaining a large number of biological samples under structured light illumination is difficult. This paper proposes a new method based on transfer learning that generates a pre-training network using its own simulation data and trains a correction using a small amount of biological sample data to achieve super-resolution imaging of biological samples. Additionally, it can reconstruct sampled wide-field images from 9 to 3 frames, as well as the trained network, which can quickly reconstruct structure illumination microscopy(SIM).

Effects of optical aberrations on localization of MINFLUX super-resolution microscopy.

A novel super-resolution imaging technique based on the minimum photon flux (MINFLUX), can achieve nanometer-scale localization precision and sub-5-nm imaging. However, aberrations can affect the localization performance and degrade the quality of reconstructed images. In this study, we analyze the effects of different low-order aberrations on the MINFLUX system through both theoretical limits and Monte Carlo methods. We report that 1) defocus and spherical aberration have little effect on 2D localization performance, whereas astigmatism and coma have significant negative effects; 2) system aberrations that can be measured in advance cause changes primarily in the magnitude and angular uniformity of localization precision, whereas sample-induced aberrations that cannot be a priori introduce large biases and reduce localization accuracy.

Calcite-Assisted Localization and Kinetics (CLocK) Microscopy.

Localization-based super-resolution imaging techniques have improved the spatial resolution of optical microscopy well below the diffraction limit, yet encoding additional information into super-resolved images, such as anisotropy and orientation, remains a challenge. Here we introduce calcite-assisted localization and kinetics (CLocK) microscopy, a multiparameter super-resolution imaging technique easily integrated into any existing optical microscope setup at low cost and with straightforward analysis. By placing a rotating calcite crystal in the infinity space of an optical microscope, CLocK microscopy provides immediate polarization and orientation information while maintaining the ability to localize an emitter/scatterer with <10 nm resolution. Further, kinetic information an order of magnitude shorter than the integration time of the camera is encoded in the unique point spread function of a CLocK image, allowing for new mechanistic insight into dynamic processes such as single-nanoparticle dissolution and single-molecule surface-enhanced Raman scattering.

Ultrasound super-resolution imaging for the differential diagnosis of thyroid nodules: A pilot study.

Ultrasound imaging provides a fast and safe examination of thyroid nodules. Recently, the introduction of super-resolution imaging technique shows the capability of breaking the Ultrasound diffraction limit in imaging the micro-vessels. The aim of this study was to evaluate its feasibility and value for the differentiation of thyroid nodules. In this study, B-mode, contrast-enhanced ultrasound, and color Doppler flow imaging examinations were performed on thyroid nodules in 24 patients. Super-resolution imaging was performed to visualize the microvasculature with finer details. Microvascular flow rate (MFR) and micro-vessel density (MVD) within thyroid nodules were computed. The MFR and MVD were used to differentiate the benign and malignant thyroid nodules with pathological results as a gold standard. Super-resolution imaging (SRI) technique can be successfully applied on human thyroid nodules to visualize the microvasculature with finer details and obtain the useful clinical information MVD and MFR to help differential diagnosis. The results suggested that the mean value of the MFR within benign thyroid nodule was 16.76 ± 6.82 mm/s whereas that within malignant thyroid was 9.86 ± 4.54 mm/s. The mean value of the MVD within benign thyroid was 0.78 while the value for malignant thyroid region was 0.59. MFR and MVD within the benign thyroid nodules were significantly higher than those within the malignant thyroid nodules respectively (p < 0.01). This study demonstrates the feasibility of ultrasound super-resolution imaging to show micro-vessels of human thyroid nodules via a clinical ultrasound platform. The important imaging markers, such as MVD and MFR, can be derived from SRI to provide more useful clinical information. It has the potential to be a new tool for aiding differential diagnosis of thyroid nodules.

Technologies Enabling Single-Molecule Super-Resolution Imaging of mRNA.

The transient nature of RNA has rendered it one of the more difficult biological targets for imaging. This difficulty stems both from the physical properties of RNA as well as the temporal constraints associated therewith. These concerns are further complicated by the difficulty in imaging endogenous RNA within a cell that has been transfected with a target sequence. These concerns, combined with traditional concerns associated with super-resolution light microscopy has made the imaging of this critical target difficult. Recent advances have provided researchers the tools to image endogenous RNA in live cells at both the cellular and single-molecule level. Here, we review techniques used for labeling and imaging RNA with special emphases on various labeling methods and a virtual 3D super-resolution imaging technique.

Detection and Separation of Close Flaws in Coarse-Grained Materials Using Ultrasonic Image Deconvolution

Ultrasonic inspection of coarse-grained steels is a common challenge in various industrial fields. This task is often difficult because of acoustic scattering that creates structural noise in the ultrasonic signals and images. Therefore, inspections usually use low-frequency probes, which achieve poor resolution with standard delay-and-sum (DAS) imaging techniques, such as the well-known total focusing method (TFM). The purpose of this paper is to evaluate the performance of a super-resolution ultrasonic imaging technique presented by Laroche et al. (IEEE Trans Comput Imaging 7:935–947, 2021) for the inspection of industrial coarse-grained materials. An image deconvolution problem (with spatially varying blur) is formulated, relying on a forward model that links the TFM image to the acoustic reflectivity map. The experiments consider an austenitic-ferritic stainless steel sample insonified using array probes at 3 MHz and 5 MHz placed in contact. The goal is to resolve two close reflectors corresponding to side-drilled holes (SDH) with diameter 0.4 mm spaced by 0.4 mm edge-to-edge and positioned at different depths (10, 20, 30, 40 mm). This configuration corresponds to a critical case where the distance between the two reflectors is much lower than the Rayleigh distance, that is the resolution limit of a DAS imaging system. These are typical cases where DAS images obtained from low-frequency inspections critically lack resolution, but where higher frequency probes cannot be used in practice, because a too week signal-to-noise ratio would affect the detection capability. As predicted by the Rayleigh criterion, TFM is not able to separate the reflectors. The proposed image reconstruction method successfully resolves the majority of the reflectors with a rather accurate distance estimation. In the context of coarse-grained structure inspection, subwavelength reflectors distant from each other by two times less than the resolution limit given by the Rayleigh criterion have been successfully detected and separated. This approach hence enables the use of low-frequency probes, in order to improve the signal-to-noise ratio, while keeping high resolution capability which can be particularly interesting for industrial applications. In particular, the proposed approach shows promising results for the sizing of a real crack in an industrial sample.

Super-resolution stimulated Raman scattering microscopy with the phase-shifted spatial frequency modulation.

We present a unique super-resolution stimulated Raman scattering (SRS) microscopy technique based on phase-shifted spatial frequency modulation (PSFM) under wide-field illumination, permitting super-resolution chemical imaging with single-pixel detection. Through projecting a series of the pump and Stokes laser patterns with varying spatial frequencies onto the sample and combining with the proposed π-phase shift, the higher spatial information can be rapidly retrieved by implementing the fast inverse Fourier-transform on the spatial frequency-encoded SRS data. We have derived the theory of the PSFM-SRS technique for super-resolution imaging. Our further modeling results confirm that PSFM-SRS microscopy provides a ∼2.2-fold improvement in spatial resolution but with a much-reduced laser excitation power density required as compared with conventional point-scan SRS microscopy, suggesting its potential for label-free super-resolution chemical imaging in cells and tissue.

Super-Resolution Airy Disk Microscopy of Individual Color Centers in Diamond

Super-resolution imaging techniques enable nanoscale microscopy in fields such as physics, biology, and chemistry. However, many super-resolution techniques require specialized optical components, such as a helical-phase mask. We present a novel technique, super-resolution Airy disk microscopy, that can be used in a standard confocal microscope without any specialized optics. We demonstrate this technique, in combination with ground state depletion, to image and control nitrogen-vacancy (NV) centers in bulk diamond below the diffraction limit. A greater than 14-fold improvement in resolution compared to the diffraction limit is achieved, corresponding to a spatial resolution of 16.9(8) nm for a 1.3 NA microscope with 589 nm light. We make use of our enhanced spatial resolution to control the spins states of individual NV centers separated from each other by less than the diffraction limit, including pairs sharing the same orientation that are indistinguishable with a conventional electron spin resonance measurement.

Super-resolution fluorescence blinking imaging using compressive sensing

Super-resolution optical fluctuation imaging (SOFI) is an extensively used super-resolution (SR) imaging technique. The sample condition and imaging system are simpler than most SR systems, and the cusp-artifacts limit the resolution of high-order SOFI. To improve the resolution of SOFI, an alternative method is to improve the reconstruction algorithm. Here, compressive sensing (CS) is used in SOFI to improve its resolution. The detailed CS reconstruction algorithm is chosen as multiple measurement vector model sparse Bayesian learning (MSBL). We demonstrate that MSBL can achieve higher than threefold resolution improvement in simulation under certain conditions. The SOFI experiment analysis demonstrates that MSBL can improve the resolution about 2.5-fold compared with the diffraction limit. All results prove that MSBL has a higher resolving ability compared with other algorithms. Our results prove that CS is a broad applicability tool for most of the existing SR microscopy techniques and can be applied in 3D and live-cell SR fluorescence microscopy imaging in the future.

Super-resolved fluorescence imaging of peripheral nerve

Traditional histopathologic evaluation of peripheral nerve employs brightfield microscopy with diffraction limited resolution of ~ 250 nm. Though electron microscopy yields nanoscale resolution of the nervous system, sample preparation is costly and the technique is incompatible with living samples. Super-resolution microscopy (SRM) comprises a set of imaging techniques that permit nanoscale resolution of fluorescent objects using visible light. The advent of SRM has transformed biomedical science in establishing non-toxic means for investigation of nanoscale cellular structures. Herein, sciatic nerve sections from GFP-variant expressing mice, and regenerating human nerve from cross-facial autografts labelled with a myelin-specific fluorescent dye were imaged by super-resolution radial fluctuation microscopy, stimulated emission depletion microscopy, and structured illumination microscopy. Super-resolution imaging of axial cryosections of murine sciatic nerves yielded robust visualization myelinated and unmyelinated axons. Super-resolution imaging of axial cryosections of human cross-facial nerve grafts demonstrated enhanced resolution of small-caliber thinly-myelinated regenerating motor axons. Resolution and contrast enhancement afforded by super-resolution imaging techniques enables visualization of unmyelinated axons, regenerating axons, cytoskeleton ultrastructure, and neuronal appendages of mammalian peripheral nerves using light microscopes.

Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy

Soft X-ray microscopy is a powerful technique for imaging cells with nanometer resolution in their native state without chemical fixation, staining, or sectioning. The studies performed in several laboratories have demonstrated the potential of applying this technique for imaging the internal structures of intact cells. However, it is currently used mainly on synchrotrons with restricted access. Moreover, the operation of these instruments and the associated sample-preparation protocols require interdisciplinary and highly specialized personnel, limiting their wide application in practice. This is why soft X-ray microscopy is not commonly used in biological laboratories as an imaging tool. Thus, a laboratory-based and user-friendly soft X-ray contact microscope would facilitate the work of biologists. A compact, desk-top laboratory setup for soft X-ray contact microscopy (SXCM) based on a laser-plasma soft X-ray source, which can be used in any biological laboratory, together with several applications for biological imaging, are described. Moreover, the perspectives of the correlation of SXCM with other super-resolution imaging techniques based on the current literature are discussed.

Ultrasonic time-reversal-based super resolution imaging for defect localization and characterization

In this paper, time reversal with multiple signal classification (TR-MUSIC) and its phase-coherent form (PC-MUSIC), the two typical ultrasonic time-reversal-based super resolution imaging techniques, have been introduced and explored to image defects in metallic samples. The principles for super resolution imaging technique and defect characterization based on ultrasonic image have been presented firstly. And then both TR-MUSIC and PC-MUSIC are tested with experimental ultrasonic array data acquired from the metallic samples including defects using the full matrix capture (FMC) process. Here, if the size of defect is smaller than the central ultrasonic wavelength, the defect can be regarded as point-like target. On the contrary, if the size of defect is larger than the central ultrasonic wavelength, it can be regarded as extended target. The comparison on super resolution imaging for point-like and extended targets has been investigated. For point-like targets, TR-MUSIC as well as PC-MUSIC can distinguish and locate the position, and the overall performance of TR-MUSIC is better than that of PC-MUSIC. For extended target, TR-MUSIC can only determine the area where the defect occurs. PC-MUSIC can distinguish and assess the length of extended target parallel to the array, the lowest error is 9.62% when the dimension of signal subspace is set to be 10 for the case considered. In addition, the defect characterization based on ultrasonic super-resolved image has been studied, and a sizing strategy is introduced. The length and angle of extended targets oblique to the array can be obtained from the PC-MUSIC images. The worst errors for assessing the length and angel are 9.14% and 13.3% respectively. And for the cases considered in the experiment, the longer the length of extended target, the smaller the error.

Fluorescent Nanoparticles for Super-Resolution Imaging.

Super-resolution imaging techniques that overcome the diffraction limit of light have gained wide popularity for visualizing cellular structures with nanometric resolution. Following the pace of hardware developments, the availability of new fluorescent probes with superior properties is becoming ever more important. In this context, fluorescent nanoparticles (NPs) have attracted increasing attention as bright and photostable probes that address many shortcomings of traditional fluorescent probes. The use of NPs for super-resolution imaging is a recent development and this provides the focus for the current review. We give an overview of different super-resolution methods and discuss their demands on the properties of fluorescent NPs. We then review in detail the features, strengths, and weaknesses of each NP class to support these applications and provide examples from their utilization in various biological systems. Moreover, we provide an outlook on the future of the field and opportunities in material science for the development of probes for multiplexed subcellular imaging with nanometric resolution.

Super-resolution optical microscopy using cylindrical vector beams

AbstractSuper-resolution optical microscopy, which gives access to finer details of objects, is highly desired for fields of nanomaterial, nanobiology, nanophotonics, etc. Many efforts, including tip optimization and illumination optimization etc., have been made in both near-field and far-field super-resolution microscopy to achieve a spatial resolution beyond the diffraction limit. The development of vector light fields opens up a new avenue for super-resolution optical microscopy via special illumination modes. Cylindrical vector beam (CVB) has been verified to enable resolution improvement in tip-scanning imaging, nonlinear imaging, stimulated emission depletion (STED) microscopy, subtraction imaging, superoscillation imaging, etc. This paper reviews recent advances in CVB-based super-resolution imaging. We start with an introduction of the fundamentals and properties of CVB. Next, strategies for CVB based super-resolution imaging are discussed, which are mainly implemented by tight focusing, depletion effect, plasmonic nanofocusing, and polarization matching. Then, the roadmap of super-resolution imaging with CVB illumination in the past two decades is summarized. The typical CVB-based imaging techniques in fields of both near-field and far-field microscopy are introduced, including tip-scanning imaging, nonlinear imaging, STED, subtraction imaging, and superoscillation imaging. Finally, challenges and future directions of CVB-illuminated super-resolution imaging techniques are discussed.

Holographic reconstruction of magnetic field distribution in a Josephson junction from diffraction-like Ic(H) patterns

A general problem of magnetic sensors is a trade-off between spatial resolution and magnetic field sensitivity. With decreasing sensor size its resolution is improved but the sensitivity is deteriorated. Obviation of such the trade-off requires development of super-resolution imaging technique, not limited by the sensor size. Here we present a proof of concept for a super-resolution method of magnetic imaging by a Josephson junction. It is based on a solution of an inverse problem - reconstruction of a local magnetic field distribution within a junction from the dependence of the critical current on an external magnetic field, Ic(H). The method resembles the Fourier-transform holography, with the diffraction-like Ic(H) pattern serving as a hologram. A simple inverse problem solution, valid for an arbitrary symmetric case, is derived. We verify the method numerically and show that the accuracy of reconstruction does not depend on the junction size and is only limited by the field range of the Ic(H) pattern. Finally, the method is tested experimentally using planar Nb Josephson junctions. Super-resolution reconstruction of stray magnetic fields from an Abrikosov vortex, trapped in the junction electrodes, is demonstrated. Thus, our method facilitate both the high field sensitivity and high spatial resolution, obviating the trade-off problem of magnetic sensors. We conclude that the holographic magnetic imaging by a planar Josephson junction can be used in scanning probe microscopy.