Super-resolution Imaging Techniques Research Articles

Addressable scanning multifocal structured illumination microscopy using acousto-optic deflectors

Multifocal structured illumination microscopy (MSIM) is a popular super-resolution imaging technique known for its good probe compatibility, low laser power requirements, and improved imaging depth, making it widely applicable in biomedical research. However, the speed of MSIM imaging is typically constrained by the approaches employed to generate and scan the laser foci across the sample. In this study, we propose a flexible two-photon excitation MSIM method using a pair of acousto-optic deflectors. By adopting addressable scanning (AS) and synchronized capturing, MSIM super-resolution imaging can be performed in multiple discrete regions of interest (ROIs) within the field of view. Notably, this AS-MSIM scheme not only enhances the speed of MSIM imaging but also alleviates photobleaching and phototoxicity to biological samples. We demonstrate its potential by achieving super-resolution imaging of selected mitochondria within cells at a frame rate of 4 Hz. Furthermore, we deliberate the possibility of even faster imaging.

Discovery of Limb Brightening in the Parsec-scale Jet of NGC 315 through Global Very Long Baseline Interferometry Observations and Its Implications for Jet Models

We report the first observation of the nearby giant radio galaxy NGC 315 using a global very long baseline interferometry (VLBI) array consisting of 22 radio antennas located across five continents, including high-sensitivity stations, at 22 GHz. Utilizing the extensive u v-coverage provided by the array, coupled with the application of a recently developed superresolution imaging technique based on the regularized maximum-likelihood method, we were able to transversely resolve the NGC 315 jet at parsec scales for the first time. Previously known for its central ridge-brightened morphology at similar scales in former VLBI studies, the jet now clearly exhibits a limb-brightened structure. This finding suggests an inherent limb brightening that was not observable before due to limited angular resolution. Considering that the jet is viewed at an angle of ∼50°, the observed limb brightening is challenging to reconcile with the magnetohydrodynamic models and simulations, which predict that the Doppler-boosted jet edges should dominate over the nonboosted central layer. The conventional jet model that proposes a fast spine and a slow sheath with uniform transverse emissivity may pertain to our observations. However, in this model, the relativistic spine would need to travel at speeds of Γ ≳ 6.0–12.9 along the deprojected jet distance of (2.3–10.8) × 103 gravitational radii from the black hole. We propose an alternative scenario that suggests higher emissivity at the jet boundary layer, resulting from more efficient particle acceleration or mass loading onto the jet edges, and consider prospects for future observations with even higher angular resolution.

Capillary-scale Microvessel Imaging with High-frequency Ultrasound Localization Microscopy in Mouse Brain.

Ultrasound localization microscopy is a super-resolution vascular imaging technique which has garnered substantial interest as a tool for small animal neuroimaging, neuroscience research, and the characterization of vascular pathologies. In the pursuit of increasingly high-fidelity reconstructions of microvasculature, there remains several outstanding questions concerning this sub-diffraction imaging technology, including the accurate reconstruction of microvessels approaching the capillary scale and the pragmatic challenges associated with long data acquisition times. In the context of small animal neurovascular imaging, we posit that increasing the ultrasound imaging frequency is a straightforward approach to enable higher concentrations of microbubble contrast agents, thus increasing the likelihood of microvascular/capillary mapping and decreasing the imaging duration. We demonstrate that higher frequency imaging results in improved ULM fidelity and more efficient microbubble localization due to a smaller microbubble point-spread function that is easier to localize, and which can achieve a higher localizable concentration within the same unit volume of tissue. A select example of in vivo capillary-level vascular reconstruction is demonstrated for the highest frequency imaging probe, which has substantial implications for neuroscientists investigating microvascular function in disease states, regulation, and brain development. High frequency ULM yielding a spatial resolution of 7.1μm, as measured by Fourier ring correlation, throughout the entire depth of the brain, highlighting this technology as a highly relevant tool for neuroimaging research.

STORM Image Denoising and Information Extraction.

Stochastic optical reconstruction microscopy (STORM) is extensively utilized in the fields of cell and molecular biology as a super-resolution imaging technique for visualizing cells and molecules. Nonetheless, the imaging process of STORM is frequently susceptible to noise, which can significantly impact the subsequent image analysis. Moreover, there is currently a lack of a comprehensive automated processing approach for analyzing protein aggregation states from a large number of STORM images. This paper initially applies our previously proposed denoising algorithm, UNet-Att, in STORM image denoising. This algorithm was constructed based on attention mechanism and multi-scale features, showcasing a remarkably efficient performance in denoising. Subsequently, we propose a collection of automated image processing algorithms for the ultimate feature extractions and data analyses of the STORM images. The information extraction workflow effectively integrates automated methods of image denoising, objective image segmentation and binarization, and object information extraction, and a novel image information clustering algorithm specifically developed for the morphological analysis of the objects in the STORM images. This automated workflow significantly improves the efficiency of the effective data analysis for large-scale original STORM images.

Surpassing the Diffraction Limit in Label-Free Optical Microscopy.

Super-resolution optical microscopy has enhanced our ability to visualize biological structures on the nanoscale. Fluorescence-based techniques are today irreplaceable in exploring the structure and dynamics of biological matter with high specificity and resolution. However, the fluorescence labeling concept narrows the range of observed interactions and fundamentally limits the spatiotemporal resolution. In contrast, emerging label-free imaging methods are not inherently limited by speed and have the potential to capture the entirety of complex biological processes and dynamics. While pushing a complex unlabeled microscopy image beyond the diffraction limit to single-molecule resolution and capturing dynamic processes at biomolecular time scales is widely regarded as unachievable, recent experimental strides suggest that elements of this vision might be already in place. These techniques derive signals directly from the sample using inherent optical phenomena, such as elastic and inelastic scattering, thereby enabling the measurement of additional properties, such as molecular mass, orientation, or chemical composition. This perspective aims to identify the cornerstones of future label-free super-resolution imaging techniques, discuss their practical applications and theoretical challenges, and explore directions that promise to enhance our understanding of complex biological systems through innovative optical advancements. Drawing on both traditional and emerging techniques, label-free super-resolution microscopy is evolving to offer detailed and dynamic imaging of living cells, surpassing the capabilities of conventional methods for visualizing biological complexities without the use of labels.

(Invited) Near-Field Optics and Its Applications in Nanoscale Materials: A Review

In this presentation, we first offer an overview of aperture-type scanning near field optical microscopy –a family of super-resolution imaging techniques based on evanescent waves, which can be combined with atomic force microscopy and are capable of subwavelength resolution nano-optical imaging. In the second part of this review, we will discuss a few applications in which our group capitalized on the super-resolution resolving power of SNOM to design specific nano-optical and nano-photonic systems for light harvesting, resistive memory device applications and nanoscale thermo-optical management, as well as to characterize nanoscale luminescent materials. Specific case studies that will be presented include the characterization of weakly photoluminescent and curved carbon dots for memory device applications, the three-dimensional characterization of plasmon-enhanced nanophotonic devices, as well as the development of near-field thermoreflectance imaging for nanophotonic-based thermal management applications. Collectively, our study well represents the versability of SNOM as a unique super-resolution nanophotonic tool for the investigation of light-matter interaction at the nanoscale.

Tuning the excitation laser power in a stochastic optical reconstruction microscope for Alexa Fluor 647 dye in Vectashield mounting media.

Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638nm. An excitation power density of 0.60 kW/cm2 at 638nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.

Optical super-resolution nanothermometry via stimulated emission depletion imaging of upconverting nanoparticles.

From engineering improved device performance to unraveling the breakdown of classical heat transfer laws, far-field optical temperature mapping with nanoscale spatial resolution would benefit diverse areas. However, these attributes are traditionally in opposition because conventional far-field optical temperature mapping techniques are inherently diffraction limited. Optical super-resolution imaging techniques revolutionized biological imaging, but such approaches have yet to be applied to thermometry. Here, we demonstrate a super-resolution nanothermometry technique based on highly doped upconverting nanoparticles (UCNPs) that enable stimulated emission depletion (STED) super-resolution imaging. We identify a ratiometric thermometry signal and maintain imaging resolution better than ~120 nm for the relevant spectral bands. We also form self-assembled UCNP monolayers and multilayers and implement a detection scheme with scan times >0.25 μm2/min. We further show that STED nanothermometry reveals a temperature gradient across a joule-heated microstructure that is undetectable with diffraction limited thermometry, indicating the potential of this technique to uncover local temperature variation in wide-ranging practical applications.

Co-Transplantation-Based Human-Mouse Chimeric Brain Models to Study Human Glial-Glial and Glial-Neuronal Interactions.

Human-mouse chimeric brain models, generated by transplanting human induced pluripotent stem cell (hiPSC)-derived neural cells, are valuable for studying the development and function of human neural cells in vivo. Understanding glial-glial and glial-neuronal interactions is essential for unraveling the complexities of brain function and developing treatments for neurological disorders. To explore these interactions between human neural cells within an intact brain environment, we employe a co-transplantation strategy involving the engraftment of hiPSC-derived neural progenitor cells along with primitive macrophage progenitors into the neonatal mouse brain. This approach creates human-mouse chimeric brains containing human microglia, macroglia (astroglia and oligodendroglia), and neurons. Using super-resolution imaging and 3D reconstruction techniques, we examine the dynamics between human neurons and glia, unveiling human microglia engulfing immature human neurons, microglia pruning synapses of human neurons, and significant interactions between human oligodendrocytes and neurons. Single-cell RNA sequencing analysis of the chimeric brain uncovers a close recapitulation of the human glial progenitor cell population, along with a dynamic stage in astroglial development that mirrors the processes found in the human brain. Furthermore, cell-cell communication analysis highlights significant neuronal-glial and glial-glial interactions, especially the interaction between adhesion molecules neurexins and neuroligins. This innovative co-transplantation model opens up new avenues for exploring the complex pathophysiological mechanisms underlying human neurological diseases. It holds particular promise for studying disorders where glial-neuronal interactions and non-cell-autonomous effects play crucial roles.

Recent Advances in Fluorescent Nanoparticles for Stimulated Emission Depletion Imaging.

Stimulated emission depletion (STED) microscopy, as a popular super-resolution imaging technique, has been widely used in bio-structure analysis and resolving the dynamics of biological processes beyond the diffraction limit. The performance of STED critically depends on the optical properties of the fluorescent probes. Ideally, the probe should process high brightness and good photostability, and exhibit a sensitive response to the depletion beam. Organic dyes and fluorescent proteins, as the most widely used STED probes, suffer from low brightness and exhibit rapid photobleaching under a high excitation power. Recently, luminescent nanoparticles (NPs) have emerged as promising fluorescent probes in biological imaging due to their high brightness and good photostability. STED imaging using various kinds of NPs, including quantum dots, polymer dots, carbon dots, aggregation-induced emission dots, etc., has been demonstrated. This review will comprehensively review recent advances in fluorescent NP-based STED probes, discuss their advantages and pitfalls, and outline the directions for future development.

Celebrating the Birthday of AMPA Receptor Nanodomains: Illuminating the Nanoscale Organization of Excitatory Synapses with 10 Nanocandles.

A decade ago, in 2013, and over the course of 4 summer months, three separate observations were reported that each shed light independently on a new molecular organization that fundamentally reshaped our perception of excitatory synaptic transmission (Fukata et al., 2013; MacGillavry et al., 2013; Nair et al., 2013). This discovery unveiled an intricate arrangement of AMPA-type glutamate receptors and their principal scaffolding protein PSD-95, at synapses. This breakthrough was made possible, thanks to advanced super-resolution imaging techniques. It fundamentally changed our understanding of excitatory synaptic architecture and paved the way for a brand-new area of research. In this Progressions article, the primary investigators of the nanoscale organization of synapses have come together to chronicle the tale of their discovery. We recount the initial inquiry that prompted our research, the preceding studies that inspired our work, the technical obstacles that were encountered, and the breakthroughs that were made in the subsequent decade in the realm of nanoscale synaptic transmission. We review the new discoveries made possible by the democratization of super-resolution imaging techniques in the field of excitatory synaptic physiology and architecture, first by the extension to other glutamate receptors and to presynaptic proteins and then by the notion of trans-synaptic organization. After describing the organizational modifications occurring in various pathologies, we discuss briefly the latest technical developments made possible by super-resolution imaging and emerging concepts in synaptic physiology.

Super-Resolution Imaging of Sub-diffraction-Limited Pattern with Superlens Based on Deep Learning

The development of super-resolution imaging techniques has revolutionized our ability to study the nano-scale world, where objects are often smaller than the diffraction limit of traditional optical microscopes. Super-resolution superlenses have been proposed to solve this problem by manipulating the light wave in the near field. A superlens is a kind of metamaterial-based lens that can enhance the evanescent waves generated by nano-scale objects, utilizing the surface plasmon phenomenon. The superlens allows for the imaging of nano-scale objects that would otherwise be impossible to resolve using traditional lenses. Previous research has shown that nanostructures can be imaged using superlenses, but the exact shape of the superlens must be known in advance, and an analytical calculation is needed to reconstruct the image. Localized plasmon structured illumination microscopy is an approach to achieve super-resolution by imaging the superlens-enhanced evanescent wave with illumination shifts. This study proposes a new approach utilizing a conditional generative adversarial network to obtain super-resolution images of arbitrary nano-scale patterns. To test the efficacy of this approach, finite-difference time-domain simulation was utilized to obtain superlens imaging results. The data from the simulation were then used for deep learning to develop the model. With the help of deep learning, the inverse calculation of complex sub-diffraction-limited patterns can be achieved. The super-resolution feature of the superlens based on deep learning is investigated. The findings of this study have significant implications for the field of nano-scale imaging, where the ability to resolve arbitrary nano-scale patterns will be crucial for advances in nanotechnology and materials science.

Ultra-high spatio-temporal resolution imaging with parallel acquisition-readout structured illumination microscopy (PAR-SIM)

Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational strategies to mitigate phototoxicity during real-time imaging of biological specimens. Traditional efforts to enhance system frame rates have concentrated on processing algorithms, like rolling reconstruction or reduced frame reconstruction, or on investments in costly sCMOS cameras with accelerated row readout rates. In this article, we introduce an approach to elevate SIM frame rates and region of interest (ROI) coverage at the hardware level, without necessitating an upsurge in camera expenses or intricate algorithms. Here, parallel acquisition-readout SIM (PAR-SIM) achieves the highest imaging speed for fluorescence imaging at currently available detector sensitivity. By using the full frame-width of the detector through synchronizing the pattern generation and image exposure-readout process, we have achieved a fundamentally stupendous information spatial-temporal flux of 132.9 MPixels · s−1, 9.6-fold that of the latest techniques, with the lowest SNR of −2.11 dB and 100 nm resolution. PAR-SIM demonstrates its proficiency in successfully reconstructing diverse cellular organelles in dual excitations, even under conditions of low signal due to ultra-short exposure times. Notably, mitochondrial dynamic tubulation and ongoing membrane fusion processes have been captured in live COS-7 cell, recorded with PAR-SIM at an impressive 408 Hz. We posit that this novel parallel exposure-readout mode not only augments SIM pattern modulation for superior frame rates but also holds the potential to benefit other complex imaging systems with a strategic controlling approach.

(Invited) Near-Field Optics and Its Applications in Nanoscale Materials: A Review

In this paper, we first offer an overview of aperture-type scanning near field optical microscopy –a family of super-resolution imaging techniques based on evanescent waves, which can be combined with atomic force microscopy and are capable of subwavelength resolution nano-optical imaging. In the second part of this review, we will discuss a few applications in which our group capitalized on the super-resolution resolving power of SNOM to design specific nano-optical and nano-photonic systems for light harvesting, resistive memory device applications and nanoscale thermo-optical management. Specific case studies that will be presented include the characterization of weakly photoluminescent and curved carbon dots for memory device applications, the three-dimensional characterization of plasmon-enhanced nanophotonic devices, as well as the development of near-field thermoreflectance imaging for nanophotonic-based thermal management applications. Collectively, our study well represents the versability of SNOM as a unique super-resolution nanophotonic tool for the investigation of light-matter interaction at the nanoscale.

ALMA 2D super-resolution imaging of Taurus–Auriga protoplanetary disks: Probing statistical properties of disk substructures

Abstract In the past decade, ALMA observations of protoplanetary disks revealed various substructures including gaps and rings. Their origin of substructures may be probed through statistical studies of their physical properties. We present the analyses of archival ALMA Band 6 continuum data of 43 disks (39 Class II and four Herbig Ae) in the Taurus–Auriga region. We employ a novel 2D super-resolution imaging technique based on sparse modeling to obtain images with high fidelity and spatial resolution. As a result, we have obtained images with spatial resolutions comparable to a few au (${0_{.}^{\prime\prime}02}$–${0_{.}^{\prime\prime}1}$), which is two to three times better than conventional CLEAN methods. All dust disks are spatially resolved, with the radii ranging from 8 to 238 au with a median radius of 45 au. Half of the disks harbor clear gap structures, the radial locations of which show a bimodal distribution with peaks at ≲20 au and ≳30 au. We also see structures indicating weak gaps at all the radii in the disk. We find that the widths of these gaps increase with their depths, which is consistent with the model of planet–disk interactions. The inferred planet mass–orbital radius distribution indicates that the planet distribution is analogous to our solar system. However, planets with Neptune mass or lower may exist in all the radii.

Measuring Presynaptic Calcium Influx at the Drosophila Larval Neuromuscular Junction.

Synaptic transmission plays a critical role in information processing and storage within the nervous system. The triggering of action potentials activates voltage-gated calcium channels at presynaptic active zones, facilitating the calcium-dependent release of synaptic vesicles. Homeostatic mechanisms are crucial in stabilizing synaptic function. At the Drosophila neuromuscular junction, a compensatory increase in presynaptic neurotransmitter release occurs when postsynaptic glutamate receptor function is pharmacologically or genetically impaired, thereby stabilizing synaptic output. This adaptation is known as presynaptic homeostatic potentiation (PHP). Recent advancements, including confocal and super-resolution imaging techniques, have demonstrated an increase in presynaptic calcium influx during both the rapid induction and long-term maintenance of PHP. These observations indicate that the abundance and structural organization of presynaptic calcium channels, along with various active zone components, undergo modifications following the suppression of postsynaptic glutamate receptors. Such findings underscore the critical roles of trafficking and stabilization of presynaptic calcium channels and active zone proteins in homeostatic plasticity. This protocol describes using calcium indicators and confocal imaging methods to measure single-action potential-evoked presynaptic calcium influx during PHP.

A Review of Microsphere Super-Resolution Imaging Techniques.

Conventional optical microscopes are only able to resolve objects down to a size of approximately 200 nm due to optical diffraction limits. The rapid development of nanotechnology has increased the demand for greater imaging resolution, with a need to break through those diffraction limits. Among super-resolution techniques, microsphere imaging has emerged as a strong contender, offering low cost, simple operation, and high resolution, especially in the fields of nanodevices, biomedicine, and semiconductors. However, this technology is still in its infancy, with an inadequate understanding of the underlying principles and the technology's limited field of view. This paper comprehensively summarizes the status of current research, the advantages and disadvantages of the basic principles and methods of microsphere imaging, the materials and preparation processes, microsphere manipulation methods, and applications. The paper also summarizes future development trends.

Instability of excitatory synapses in experimental autoimmune encephalomyelitis and the outcome for excitatory circuit inputs to individual cortical neurons

Synapses are lost on a massive scale in the brain and spinal cord of people living with multiple sclerosis (PwMS), and this synaptic loss extends far beyond demyelinating lesions. Post-mortem studies show the long-term consequences of multiple sclerosis (MS) on synapses but do not inform on the early impacts of neuroinflammation on synapses that subsequently lead to synapse loss. How excitatory circuit inputs are altered across the dendritic tree of individual neurons under neuroinflammatory stress is not well understood. Here, we directly assessed the structural dynamics of labeled excitatory synapses in experimental autoimmune encephalomyelitis (EAE) as a model of immune-mediated cortical neuronal damage. We used in vivo two-photon imaging and a synthetic tissue-hydrogel super-resolution imaging technique to reveal the dynamics of excitatory synapses, map their location across the dendritic tree of individual neurons, and examine neurons at super-resolution for synaptic loss. We found that excitatory synapses are destabilized but not lost from dendritic spines in EAE, starting with the earliest imaging session before symptom onset. This led to changes in excitatory circuit inputs to individual cells. In EAE, stable synapses are replaced by synapses that appear or disappear across the imaging sessions or repeatedly change at the same location. These unstable excitatory inputs occur closer to one another in EAE than in healthy controls and are distributed across the dendritic tree. When imaged at super-resolution, we found that a small proportion of dendritic protrusions lost their presynapse and/or postsynapse. Our finding of diffuse destabilizing effects of neuroinflammation on excitatory synapses across cortical neurons may have significant functional consequences since normal dendritic spine dynamics and clustering are essential for learning and memory.

Facile construction of dual-response super-resolution probes for tracking organelles dynamics.

Super-resolution imaging techniques, such as structured illumination microscopy (SIM), have enabled researchers to obtain nanoscale organelle-level outputs in living systems, but they impose additional stringent requirements on fluorescence probes. However, high-performance, custom-designed SIM probes that can explain underlying biological processes remain unavailable. Herein, a customizable engineering toolkit is developed for the facile assembly of SIM probes suitable for subcellular component detection. This toolkit is used to customize a fluorescent molecule, CPC (coumarin-phenylhydrazine-carboxyl), capable of simultaneously monitoring peroxynitrite (ONOO-) and polarity distribution in mitochondria and lipid droplets (LDs), respectively, through functional ON-OFF mechanisms. The customized CPC molecule demonstrated excellent imaging capabilities under SIM, enabled the successful localization of multiple organelles, and reliably tracked the distribution of different components, thus facilitating the study of the interplay between organelles. Using CPC, the physical transition of intracellular LDs is demonstrated from heterogeneity to homogeneity. This was specifically observed during ferroptosis where the polarity of the LDs increased and their morphology became more contracted. Furthermore, the loss of LDs functionality could not counteract the accumulation of ONOO- within the mitochondria, leading to the decoupling of mitochondrial LDs during ferroptosis. These results confirmed the potential mechanism of LDs dysfunction and decoupling triggered via cumulative mitochondrial oxidative stress during ferroptosis. To summarize, this toolkit will be a powerful tool for examining subtle variations among components during the interplay between different organelles, thus offering novel avenues for understanding and treating related diseases.

Solvatochromic Buffering Fluorescent Probe Resolves the Lipid Transport and Morphological Changes during Lipid Droplet Fusion by Super-Resolution Imaging.

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.