Super-resolution Imaging Techniques Research Articles

Dual color DMD-SIM by temperature-controlled laser wavelength matching.

Structured illumination microscopy (SIM) is a fast and gentle super-resolution fluorescence imaging technique, featuring live-cell compatible excitation light levels and high imaging speeds. To achieve SIM, spatial modulation of the fluorescence excitation light is employed. This is typically achieved by interfering coherent laser beams in the sample plane, which are often created by spatial light modulators (SLMs). Digital micromirror devices (DMDs) are a form of SLMs with certain advantages, such as high speed, low cost and wide availability, which present certain hurdles in their implementation, mainly the blazed grating effect caused by the jagged surface structure of the tilted mirrors. Recent works have studied this effect through modelling, simulations and experiments, and laid out possible implementations of multi-color SIM imaging based on DMDs. Here, we present an implementation of a dual-color DMD based SIM microscope using temperature-controlled wavelength matching. By carefully controlling the output wavelength of a diode laser by temperature, we can tune two laser wavelengths in such a way that no opto-mechanical realignment of the SIM setup is necessary when switching between both wavelengths. This reduces system complexity and increases imaging speed. With measurements on nano-bead reference samples, as well as the actin skeleton and membrane of fixed U2OS cells, we demonstrate the capabilities of the setup.

Precision Mapping of Amyloid-β Binding Reveals Perisynaptic Localization and Spatially Restricted Plasticity Deficits.

Secreted amyloid-β (Aβ) peptide forms neurotoxic oligomeric assemblies thought to cause synaptic deficits associated with Alzheimer’s disease (AD). Soluble Aβ oligomers (Aβo) directly bind to neurons with high affinity and block plasticity mechanisms related to learning and memory, trigger loss of excitatory synapses and eventually cause cell death. While Aβo toxicity has been intensely investigated, it remains unclear precisely where Aβo initially binds to the surface of neurons and whether sites of binding relate to synaptic deficits. Here, we used a combination of live cell, super-resolution and ultrastructural imaging techniques to investigate the kinetics, reversibility and nanoscale location of Aβo binding. Surprisingly, Aβo does not bind directly at the synaptic cleft as previously thought but, instead, forms distinct nanoscale clusters encircling the postsynaptic membrane with a significant fraction also binding presynaptic axon terminals. Synaptic plasticity deficits were observed at Aβo-bound synapses but not closely neighboring Aβo-free synapses. Thus, perisynaptic Aβo binding triggers spatially restricted signaling mechanisms to disrupt synaptic function. These data provide new insight into the earliest steps of Aβo pathology and lay the groundwork for future studies evaluating potential surface receptor(s) and local signaling mechanisms responsible for Aβo binding and synapse dysfunction.

Optical superoscillation technologies beyond the diffraction limit

Optical superoscillations are rapid, subwavelength spatial variations of the intensity and phase of light, occurring in complex electromagnetic fields formed by the interference of several coherent waves. The discovery of superoscillations stimulated a revision of the limits of classical electromagnetism — in particular, the studies of phenomena such as unlimitedly small energy hotspots, phase singularities, energy backflow, anomalously high wavevectors and their intriguing similarities to the evanescent plasmonic fields on metals. In recent years, the understanding of superoscillatory light has led to the development of superoscillatory lensing, imaging and metrology technologies. Dielectric, metallic and metamaterial nanostructured superoscillatory lenses have been introduced that are able to create hotspots smaller than allowed by conventional lenses. Far-field, label-free, non-intrusive deeply subwavelength super-resolution imaging and metrology techniques that exploit high light localization and rapid variation of phase in superoscillatory fields have also been developed, including new approaches based on artificial intelligence. We review the fundamental properties of superoscillatory optical fields and examine emerging technological applications.

Deciphering the functional nano-anatomy of the tripartite synapse using stimulated emission depletion microscopy.

A major challenge for studying neuron-astrocyte communication lies in visualizing the tripartite synapse, which is the physical site where astrocytic processes contact and interact with neuronal synapses. While conventional light microscopy cannot resolve the anatomical details of the tripartite synapse, electron microscopy only provides ultrastructural snapshots that tell us little about its living state and dynamics. Stimulated emission depletion (STED) microscopy is a super-resolution fluorescence imaging technique that can provide live images of tripartite synapses with nanoscale spatial resolution. It is compatible with physiology experiments and imaging in the intact brain in vivo, opening up new opportunities to link the nanoscale structure of the tripartite system with functional readouts of neurons and astrocytes or even behavior. In this review, we first summarize the findings and insights from previous studies addressing the structure-function relationship of the tripartite synapse using conventional imaging techniques. We then explain the basic principle of STED microscopy and the main challenges facing its application to live-tissue imaging of fine astrocytic processes. We summarize insights from our recent STED studies, which revealed new aspects of the structure and physiology of the tripartite synapse and the surrounding extracellular space. Finally, we discuss how the STED approach and other advanced optical techniques can illuminate the role of astrocytes for brain physiology and animal behavior.

Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization.

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.

[Comparison and progress review of various super-resolution fluorescence imaging techniques].

所见即所得是生命科学研究的中心哲学,贯穿在不断认识单个分子、分子复合体、分子动态行为和整个分子网络的历程中。活的动态的分子才是有功能的,这决定了荧光显微成像在生命科学研究中成为不可替代的工具。但是当荧光成像聚焦到分子水平的时候,所见并不能给出想要得到的。这个障碍是由于受光学衍射极限的限制,荧光显微镜无法在衍射受限的空间内分辨出目标物。超分辨荧光成像技术突破衍射极限的限制,在纳米尺度至单分子水平可视化生物分子,以前所未有的时空分辨率研究活细胞结构和动态过程,已成为生命科学研究的有力工具,并逐渐应用到材料科学、催化反应过程和光刻等领域。超分辨成像技术原理不同,其具有的技术性能各异,限制了各自特定的技术特色和应用范围。目前主流的超分辨成像技术包括3种:结构光照明显微镜技术(structured illumination microscopy, SIM)、受激发射损耗显微技术(stimulated emission depletion, STED)和单分子定位成像技术(single molecule localization microscopy, SMLM)。这些显微镜采用不同的复杂技术,但是策略却是相同和简单的,即通过牺牲时间分辨率来提升衍射受限的空间内相邻两个发光点的空间分辨。该文通过对这3种技术的原理比较和在生物研究中的应用进展介绍,明确了不同超分辨成像技术的技术优势和适用的应用方向,以方便研究者在未来研究中做合理的选择。

Cd-free InP/ZnSeS quantum dots for ultrahigh-resolution imaging of stimulated emission depletion.

Stimulated emission depletion (STED) nanoscopy is a promising super-resolution imaging technique for microstructure imaging; however, the performance of super-resolution techniques critically depends on the properties of the fluorophores (photostable fluorophores) used. In this study, a suitable probe for improving the resolution of STED nanoscopy was investigated. Quantum dots (QDs) typically exhibit good photobleaching resistance characteristics. In comparison with CdSe@ZnS QDs and CsPbBr3 QDs, Cd-free InP/ZnSeS QDs have a smaller size and exhibit an improved photobleaching resistance. Through imaging using InP/ZnSeS QDs, we achieved an ultrahigh resolution of 26.1 nm. Furthermore, we achieved a 31 nm resolution in cell experiments involving InP/ZnSeS QDs. These results indicate that Cd-free InP/ZnSeS QDs have significant potential for application in fluorescent probes for STED nanoscopy.

Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons.

Gonadal steroid 17β-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.

Super-Resolution Imaging of the Actin Cytoskeleton in Living Cells Using TIRF-SIM.

Super-resolution (SR) imaging techniques have advanced rapidly in recent years, but only a subset of these techniques is gentle enough to be used by cell biologists to study living cells with minimal photodamage. Our research is focused on studies of the dynamic remodeling of the actin cytoskeleton in living pancreatic beta cells during insulin secretion. These studies require super-resolution light microscopic techniques that are gentle enough to record rapid changes of the actin cytoskeleton in real time. In this chapter, we describe an SR technique that breaks the diffraction limit of the conventional light microscope called TIRF-SIM. Using this SR techniques, we have been able to show that (1) microvilli on pancreatic beta cells translocate in the plane of the plasma membrane and (2) the cortical actin network reorganizes when cells are stimulated to secrete insulin. We describe the FIJI plugins that were used to process and analyze the TIRF-SIM images to obtain quantitative data.

Visualization of Subatomic Movements in Nanostructures.

Electron microscopy, scanning probe, and optical super-resolution imaging techniques with nanometric resolution are now routinely available but cannot capture the characteristically fast (MHz-GHz frequency) movements of micro-/nano-objects. Meanwhile, optical interferometric techniques can detect high-frequency picometric displacements but only with diffraction-limited lateral resolution. Here, we introduce a motion visualization technique, based on the spectrally resolved detection of secondary electron emission from moving objects, that combines picometric displacement sensitivity with the nanometric spatial (positional/imaging) resolution of electron microscopy. The sensitivity of the technique is quantitatively validated against the thermodynamically defined amplitude of a nanocantilever's Brownian motion. It is further demonstrated in visualizing externally driven modes of cantilever, nanomechanical photonic metamaterial, and MEMS device structures. With a noise floor reaching ∼1 pm/Hz1/2, it can provide for the study of oscillatory movements with subatomic amplitudes, presenting new opportunities for the interrogation of motion in functional structures across the materials, bio- and nanosciences.

Imaging the Heterogeneous Localization of a Single Molecule.

Single-molecule localization allows determining the underlying biological and biochemical processes and promotes the development of super-resolution imaging techniques. Here, we present an optical technique of tracking the motion of a single nanoparticle linked to a substrate via a biomolecule tether to reveal the localization of single biomolecules and the transient states of single nanoparticle switching between specific binding pairs. The affinities, steric hindrance, and conformational variation of a single-molecule binding pair uncover the dynamic details and intrinsic mechanism of binding processes with high specificity and accuracy (a few nanometers). The application of tracking motions of single soft liposomes on different modified surfaces was further demonstrated, which revealed the characteristic behavior related to surface chemistry. Our results show that the trajectory of nanoscale liposomes loaded with small-drug molecules is linked to the compositional inhomogeneity, which provides a route for thorough comprehension of the fundamental biotechnological process.

Multimodal single-molecule microscopy with continuously controlled spectral resolution

Color is a fundamental contrast mechanism in fluorescence microscopy, providing the basis for numerous imaging and spectroscopy techniques. Building on spectral imaging schemes that encode color into a fixed spatial intensity distribution, here, we introduce continuously controlled spectral-resolution (CoCoS) microscopy, which allows the spectral resolution of the system to be adjusted in real-time. By optimizing the spectral resolution for each experiment, we achieve maximal sensitivity and throughput, allowing for single-frame acquisition of multiple color channels with single-molecule sensitivity and 140-fold larger fields of view compared with previous super-resolution spectral imaging techniques. Here, we demonstrate the utility of CoCoS in three experimental formats, single-molecule spectroscopy, single-molecule Förster resonance energy transfer, and multicolor single-particle tracking in live neurons, using a range of samples and 12 distinct fluorescent markers. A simple add-on allows CoCoS to be integrated into existing fluorescence microscopes, rendering spectral imaging accessible to the wider scientific community.

Super-Resolution Image Reconstruction Based on Single-Molecule Localization Algorithm

Fluorescence imaging is an important and efficient tool in cell biology and biomedical research. In order to observe the dynamics of biological macromolecules such as DNA, RNA and proteins in live cells, it is extremely necessary to surpass the Abbe diffraction limit in microscopic imaging. Single-molecule localization microscopy (SMLM) is a sort of super-resolution imaging technique that can obtain a large number of images of sparse fluorescent molecules by the use of photoswitchable fluorescent probes and single-molecule localization technology. The center positions of fluorescent molecules in the images are precisely located, and then the entire sample pattern is reconstructed with super resolution. In this paper, we present a single-molecule localization algorithm (SMLA) that is based on blind deconvolution and centroid localization (BDCL) method. Single-molecule localization and image reconstruction of 15,000/9990 frames of original images of tubulins are accomplished. In addition, this fluorophore localization algorithm is used to localize high particle-density images. The results show that our BDCL-SMLA method is a reasonable attempt and useful method for SMLM imaging when the imaging system is unknown.

Super-resolution optical fluctuation imaging—fundamental estimation theory perspective

We provide a quantitative analysis of super-resolution imaging techniques which exploit temporal fluctuations of luminosity of the sources in order to beat the Rayleigh limit. We define an operationally justified resolution gain figure of merit, that allows us to connect the estimation theory concepts with the ones typically used in the imaging community, and derive fundamental resolution limits that scale at most as the fourth-root of the mean luminosity of the sources. We fine-tune and benchmark the performance of state-of-the-art methods, focusing on the cumulant-based image processing techniques (known under the common acronym stochastic optical fluctuation imaging), taking into account the impact of limited photon number and sampling time.

Nitroso-Caged Rhodamine: A Superior Green Light-Activatable Fluorophore for Single-Molecule Localization Super-Resolution Imaging

The evolution of super-resolution imaging techniques, especially single-molecule localization microscopy, demands the engineering of switchable fluorophores with labeling functionality. Yet, the switching of these fluorophores depends on the exterior conditions of UV light and enhancing buffers, which is bioincompatible for living-cell applications. Herein, to surpass these limitations, a nitroso-caging strategy is employed to cage rhodamines into leuco forms, which for the first time, is discovered to uncage highly bright zwitterions by green light. Further, clickable construction grants the specificity and versatility for labeling various components in living cells. The simultaneous photoactivation and excitation of these novel probes allow for single-laser super-resolution imaging without any harmful additives. Super-resolution imaging of microtubules in fixed cells or mitochondria and the distribution of glycans and H2B proteins in living cells are achieved at a molecular scale with robust integrity. We envision that our nitroso-caging probes would set a platform for the development of future visible-activatable probes.

TAOK2 is an ER kinase that catalyzes the dynamic tethering of ER to microtubules

The endoplasmic reticulum (ER) extensively associates with the microtubule cytoskeleton. Although integral to its many functions, mechanisms through which the ER tethers to microtubules and regulates its coupling dynamics with the cytoskeleton are poorly understood. Herein we report that TAOK2 is an ER-resident multifunctional protein kinase required for tethering of ER membranes to microtubules. Using biochemical and super-resolution imaging techniques, we show that TAOK2 is a multipass membrane-spanning kinase localized in distinct ER domains via four transmembrane and amphipathic helices. Using in vitro and cellular assays, we find that TAOK2 directly binds microtubules with high affinity. We define the minimal TAOK2 determinants that induce ER-microtubule tethering. While TAOK2 knockout cells exhibit decreased ER stability and disrupted movement of ER along growing microtubule plus-ends, motor-mediated ER motility is not affected. ER-microtubule tethering is tightly regulated by the catalytic activity of TAOK2 in interphase and mitotic cells. Perturbation of TAOK2 activity leads to defects in ER morphology and cell division. Our study identifies TAOK2 as an ER-microtubule tether, and reveals a kinase-regulated mechanism for control of ER dynamics critical for cell growth and division.

Super Resolution Digital Image Correlation (SR-DIC): an Alternative to Image Stitching at High Magnifications

BackgroundHigh-resolution Digital Image Correlation (DIC) measurements have previously been produced by stitching of neighboring images, which often requires short working distances. Separately, the image processing community has developed super resolution (SR) imaging techniques, which improve resolution by combining multiple overlapping images.ObjectiveThis work investigates the novel pairing of super resolution with digital image correlation, as an alternative method to produce high-resolution full-field strain measurements.MethodsFirst, an image reconstruction test is performed, comparing the ability of three previously published SR algorithms to replicate a high-resolution image. Second, an applied translation is compared against DIC measurement using both low- and super-resolution images. Third, a ring sample is mechanically deformed and DIC strain measurements from low- and super-resolution images are compared.ResultsSR measurements show improvements compared to low-resolution images, although they do not perfectly replicate the high-resolution image. SR-DIC demonstrates reduced error and improved confidence in measuring rigid body translation when compared to low resolution alternatives, and it also shows improvement in spatial resolution for strain measurements of ring deformation.ConclusionsSuper resolution imaging can be effectively paired with Digital Image Correlation, offering improved spatial resolution, reduced error, and increased measurement confidence.

Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation

Localization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters. These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. This sub-diffraction localization precision is theoretically bounded by the number of photons emitted per molecule and by the sensor noise. These parameters can be estimated from the raw images. Alternatively, the resolution can be estimated from a rendered image of the localizations. Here, we show how the rendering of localization datasets can influence the resolution estimation based on decorrelation analysis. We demonstrate that a modified histogram rendering, termed bilinear histogram, circumvents the biases introduced by Gaussian or standard histogram rendering. We propose a parameter-free processing pipeline and show that the resolution estimation becomes a function of the localization density and the localization precision, on both simulated and state-of-the-art experimental datasets.

Super-resolution structured illumination microscopy: past, present and future.

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

Optical amplification for astronomical imaging at higher resolution

Heisenberg’s uncertainty principle tells us that it is impossible to determine simultaneously the position of a photon crossing a telescope’s aperture and its momentum. Super-resolution imaging techniques rely on modification of the observed sample or on entangling photons. In astronomy, we have no access to the object, but resolution may be improved by optical amplification. Unfortunately, spontaneous emission contributes noise and negates the possible gain from stimulated emissions. We show that it is possible to increase the weight of the stimulated photons by considering photon statistics and observe an improvement in resolution. Most importantly, we demonstrate a method that can apply for all imaging purposes.