Super-resolution Imaging Techniques Research Articles

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.

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.

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.

Super-Resolution Ultrasound Reveals Cerebrovascular Impairment in a Mouse Model of Alzheimer's Disease.

Increasing evidence has suggested a link between cerebrovascular disease and the cognitive impairment associated with Alzheimer's disease. However, detailed descriptions of microvascular changes across brain regions and how they relate to other more traditional pathology have been lacking. Additionally, the efforts to elucidate the interplay between cerebral microvascular function and Alzheimer's disease progression are complicated by the necessity of probing deep-brain structures since early-stage Alzheimer's disease typically involves hippocampal pathology. The purpose of this study was to examine changes in microvascular dynamics in a mouse model of Alzheimer's disease using cohorts that were age-matched to wild-type controls. Data from both sexes were included in this study. Super-resolution ultrasound localization microscopy revealed microvascular functional and structural features throughout the whole brain depth to visualize and quantify. We found that functional decreases in hippocampal and entorhinal flow velocity preceded structural derangements in regional vascular density. Co-registered histological sectioning confirmed the regionalized perfusion deficits seen on ultrasound imaging, which were co-localized with amyloid beta plaque deposition. In addition to providing global vascular quantifications of deep brain structures with a high local resolution, this technology also permitted velocity-profile analysis of individual vessels and, in some cases, allowed for decoupling of arterial and venous flow contributions. These data suggest that microvascular pathology is an early and pervasive feature of Alzheimer's disease and may represent a novel therapeutic target for this disease.

Label-Retention Expansion Microscopy (LR-ExM) for Enhanced Fluorescent Signals using Trifunctional Probes.

Expansion microscopy (ExM) is a super-resolution imaging technique that bypasses the diffraction limit of conventional optical microscopy (∼250 nm) by enlarging samples with a swellable hydrogel. Combined with various light microscopes, ExM enables an effective resolution ranging from 5 to 70 nm. ExM has now been successfully applied to cell, tissue, and whole-organism samples, providing biologists with a low-cost strategy to visualize samples at the molecular level. However, fluorescence signal loss easily happens for beginners and with early versions of ExM protocols. Here, we describe a protocol called label-retention expansion microscopy (LR-ExM), which can preserve and enhance the signal of ExM imaging via a series of trifunctional probes. These trifunctional probes are antibody-based and easy to prepare, and thus suit the needs of most laboratories. © 2024 Wiley Periodicals LLC. Basic Protocol: LR-ExM using trifunctional probes for enhanced fluorescent signals.

Correlative super-resolution bright-field and fluorescence imaging by microsphere assisted microscopy.

The resolution of fluorescence imaging has been significantly enhanced with the development of super-resolution imaging techniques, surpassing the diffraction limit and reaching sub-diffraction scales of tens of nanometers. However, the resolution of the bright-field images of cells is restricted by the diffraction limit, leading to a significant gap between the resolutions of fluorescence and bright-field imaging, which hinders the research of the precise distribution of intracellular nanostructures. A microsphere superlens offers a promising solution by providing label-free super-resolution imaging capabilities compatible with fluorescence super-resolution imaging. In this study, we used microsphere superlenses to simultaneously enhance the resolution of bright-field and fluorescence imaging, achieving correlated super-resolution bright-field and fluorescence imaging. Compared to conventional bright-field images, we improved the imaging resolution from λ/1.3 to λ/4.2. A correlative super-resolution of mouse skeletal muscle cells was achieved, enabling the clear observation of the precise distribution of nanoparticles in mouse skeletal muscle cells. Furthermore, microsphere superlenses inherit the advantages of optical imaging, which is expected to enable the capturing of ultrafast biological activity within living cells with extremely high temporal resolutions.

PCNA as Protein-Based Nanoruler for Sub-10nm Fluorescence Imaging.

Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.

Real Aperture Radar Super-Resolution Imaging for Sea Surface Monitoring Based on a Hybrid Model

In recent years, super-resolution imaging techniques have been intensely introduced to enhance the azimuth resolution of real aperture scanning radar (RASR). However, there is a paucity of research on the subject of sea surface imaging with small incident angles for complex scenarios. This research endeavors to explore super-resolution imaging for sea surface monitoring, with a specific emphasis on grounded or shipborne platforms. To tackle the inescapable interference of sea clutter, it was segregated from the imaging objects and was modeled alongside I/Q channel noise within the maximum likelihood framework, thus mitigating clutter’s impact. Simultaneously, for characterizing the non-stationary regions of the monitoring scene, we harnessed the Markov random field (MRF) model for its two-dimensional (2D) spatial representational capacity, augmented by a quadratic term to bolster outlier resilience. Subsequently, the maximum a posteriori (MAP) criterion was employed to unite the ML function with the statistical model regarding imaging scene. This hybrid model forms the core of our super-resolution methodology. Finally, a fast iterative threshold shrinkage method was applied to solve this objective function, yielding stable estimates of the monitored scene. Through the validation of simulation and real data experiments, the superiority of the proposed approach in recovering the monitoring scenes and clutter suppression has been verified.

Tandem aberration correction optics (TACO) in wide-field structured illumination microscopy.

Structured illumination microscopy (SIM) is a powerful super-resolution imaging technique that uses patterned illumination to down-modulate high spatial-frequency information of samples. However, the presence of spatially-dependent aberrations can severely disrupt the illumination pattern, limiting the quality of SIM imaging. Conventional adaptive optics (AO) techniques that employ wavefront correctors at the pupil plane are not capable of effectively correcting these spatially-dependent aberrations. We introduce the Tandem Aberration Correction Optics (TACO) approach that combines both pupil AO and conjugate AO for aberration correction in SIM. TACO incorporates a deformable mirror (DM) for pupil AO in the detection path to correct for global aberrations, while a spatial light modulator (SLM) is placed at the plane conjugate to the aberration source near the sample plane, termed conjugate AO, to compensate spatially-varying aberrations in the illumination path. Our numerical simulations and experimental results show that the TACO approach can recover the illumination pattern close to an ideal condition, even when severely misshaped by aberrations, resulting in high-quality super-resolution SIM reconstruction. The TACO approach resolves a critical traditional shortcoming of aberration correction for structured illumination. This advance significantly expands the application of SIM imaging in the study of complex, particularly biological, samples and should be effective in other wide-field microscopies.

Structured illumination contrast transfer function for high resolution quantitative phase imaging.

We report a sub-diffraction resolution imaging of non-fluorescent samples through quantitative phase imaging. This is achieved through a novel application of structured illumination microscopy (SIM), a super-resolution imaging technique established primarily for fluorescence microscopy. Utilizing our contrast transfer function formalism with SIM, we extract the high spatial frequency components of the phase profile from the defocused intensity images, enabling the reconstruction of a quantitative phase image with a frequency spectrum that surpasses the diffraction limit imposed by the imaging system. Our approach offers several advantages including a deterministic, phase-unwrapping-free algorithm and an easily implementable, non-interferometric setup. We validate the proposed technique for high-resolution phase imaging through both simulation and experimental results, demonstrating a two-fold enhancement in resolution. A lateral resolution of 0.814 µm is achieved for the phase imaging of human cheek cells using a 0.42 NA objective lens and an illumination wavelength of 660 nm, highlighting the efficacy of our approach for high-resolution quantitative phase imaging.

Super-resolution imaging of folate receptor alpha on cell membranes using peptide-based probes

Folate receptor alpha (FRα) is a vital membrane protein which have great association with cancers and involved in various biological processes including folate transport and cell signaling. However, the distribution and organization pattern of FRα on cell membranes remains unclear. Previous studies relied on antibodies to recognize the proteins. However, multivalent crosslinking and large size of antibodies confuse the direct observation to some extent. Fortunately, the emergence of peptide, which are small-sized and monovalent, has supplied us an unprecedented choice. Here, we applied fluorophore-conjugated peptide probe to recognize the FRα and study the distribution pattern of FRα on cell membrane using dSTORM super-resolution imaging technique. FRα were found to organized as clusters on cell surface with different sizes. And they have a higher expression level and formed larger clusters on various cancer cells than normal cells, which hinted that its specific distribution could be utilized for cancer diagnosis. Furthermore, we revealed that the lipid raft and cortical actin as restrictive factors for the FRα clustering, suggesting a potential assembly mechanism insight into FRα clustering on cell membrane. Collectively, our work clarified the morphology distribution and clustered organization of FRα with peptide probes at the nanometer scale, which paves the way for further revealing the relationship between the spatial organization and functions of membranal proteins.

Three-dimensional super resolution ultrasound imaging with a multi-frequency hemispherical phased array.

High resolution imaging of the microvasculature plays an important role in both diagnostic and therapeutic applications in the brain. However, ultrasound pulse-echo sonography imaging the brain vasculatures has been limited to narrow acoustic windows and low frequencies due to the distortion of the skull bone, which sacrifices axial resolution since it is pulse length dependent. To overcome the detect limit, a large aperture 256-module sparse hemispherical transmit/receive array was used to visualize the acoustic emissions of ultrasound-vaporized lipid-coated decafluorobutane nanodroplets flowing through tube phantoms and within rabbit cerebral vasculature in vivo via passive acoustic mapping and super resolution techniques. Nanodroplets were vaporized with 55kHz burst-mode ultrasound (burst length=145μs, burst repetition frequency=9-45Hz, peak negative acoustic pressure=0.10-0.22MPa), which propagates through overlying tissues well without suffering from severe distortions. The resulting emissions were received at a higher frequency (612 or 1224kHz subarray) to improve the resulting spatial resolution during passive beamforming. Normal resolution three-dimensional images were formed using a delay, sum, and integrate beamforming algorithm, and super-resolved images were extracted via Gaussian fitting of the estimated point-spread-function to the normal resolution data. With super resolution techniques, the mean lateral (axial) full-width-at-half-maximum image intensity was 16±3 (32±6)μm, and 7±1 (15±2)μm corresponding to ∼1/67 of the normal resolution at 612 and 1224kHz, respectively. The mean positional uncertainties were ∼1/350 (lateral) and ∼1/180 (axial) of the receive wavelength in water. In addition, a temporal correlation between nanodroplet vaporization and the transmit waveform shape was observed, which may provide the opportunity to enhance the signal-to-noise ratio in future studies. Here, we demonstrate the feasibility of vaporizing nanodroplets via low frequency ultrasound and simultaneously performing spatial mapping via passive beamforming at higher frequencies to improve the resulting spatial resolution of super resolution imaging techniques. This method may enable complete four-dimensional vascular mapping in organs where a hemispherical array could be positioned to surround the target, such as the brain, breast, or testicles.

Passive superresolution imaging of incoherent objects

The need to observe objects that are smaller than the diffraction limit has led to the development of various superresolution techniques. However, most such techniques require active interaction with the sample, which may not be possible in multiple practical scenarios. The recently developed technique of Hermite–Gaussian imaging (HGI) achieves superresolution by passively observing the light coming from an object. This approach involves decomposing the incoming field into the Hermite–Gaussian basis of spatial modes and measuring the amplitude or intensity of each component. From these measurements, the original object can be reconstructed. However, implementing HGI experimentally has proven to be challenging, and previous achievements have focused on coherent imaging or parameter estimation of simple objects. In this paper, we implement interferometric HGI in the incoherent regime and demonstrate a three-fold improvement in the resolution compared to direct imaging. We evaluate the performance of our method under different noise levels. Our results constitute a step towards powerful passive superresolution imaging techniques in fluorescent microscopy and astronomy.

Localization Free Super-Resolution Microbubble Velocimetry Using a Long Short-Term Memory Neural Network.

Ultrasound localization microscopy is a super-resolution imaging technique that exploits the unique characteristics of contrast microbubbles to side-step the fundamental trade-off between imaging resolution and penetration depth. However, the conventional reconstruction technique is confined to low microbubble concentrations to avoid localization and tracking errors. Several research groups have introduced sparsity- and deep learning-based approaches to overcome this constraint to extract useful vascular structural information from overlapping microbubble signals, but these solutions have not been demonstrated to produce blood flow velocity maps of the microcirculation. Here, we introduce Deep-SMV, a localization free super-resolution microbubble velocimetry technique, based on a long short-term memory neural network, that provides high imaging speed and robustness to high microbubble concentrations, and directly outputs blood velocity measurements at a super-resolution. Deep-SMV is trained efficiently using microbubble flow simulation on real in vivo vascular data and demonstrates real-time velocity map reconstruction suitable for functional vascular imaging and pulsatility mapping at super-resolution. The technique is successfully applied to a wide variety of imaging scenarios, include flow channel phantoms, chicken embryo chorioallantoic membranes, and mouse brain imaging. An implementation of Deep-SMV is openly available at https://github.com/chenxiptz/SR_microvessel_velocimetry, with two pre-trained models available at https://doi.org/10.7910/DVN/SECUFD.

Application of Super-resolution SPEED Microscopy in the Study of Cellular Dynamics.

Super-resolution imaging techniques have broken the diffraction-limited resolution of light microscopy. However, acquiring three-dimensional (3D) super-resolution information about structures and dynamic processes in live cells at high speed remains challenging. Recently, the development of high-speed single-point edge-excitation subdiffraction (SPEED) microscopy, along with its 2D-to-3D transformation algorithm, provides a practical and effective approach to achieving 3D subdiffraction-limit information in subcellular structures and organelles with rotational symmetry. One of the major benefits of SPEED microscopy is that it does not rely on complex optical components and can be implemented on a standard, inverted epifluorescence microscope, simplifying the process of sample preparation and the expertise requirement. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside submicrometer biological channels or cavities at high spatiotemporal resolution. The collected data are then subjected to postlocalization 2D-to-3D transformation to obtain 3D super-resolution structural and dynamic information. In recent years, SPEED microscopy has provided significant insights into nucleocytoplasmic transport across the nuclear pore complex (NPC) and cytoplasm-cilium trafficking through the ciliary transition zone. This Review focuses on the applications of SPEED microscopy in studying the structure and function of nuclear pores.

Super-resolution computational saturated absorption microscopy.

Imaging beyond the diffraction limit barrier has attracted wide attention due to the ability to resolve previously hidden image features. Of the various super-resolution microscopy techniques available, a particularly simple method called saturated excitation microscopy (SAX) requires only simple modification of a laser scanning microscope: The illumination beam power is sinusoidally modulated and driven into saturation. SAX images are extracted from the harmonics of the modulation frequency and exhibit improved spatial resolution. Unfortunately, this elegant strategy is hindered by the incursion of shot noise that prevents high-resolution imaging in many realistic scenarios. Here, we demonstrate a technique for super-resolution imaging that we call computational saturated absorption (CSA) in which a joint deconvolution is applied to a set of images with diversity in spatial frequency support among the point spread functions (PSFs) used in the image formation with saturated laser scanning fluorescence microscopy. CSA microscopy allows access to the high spatial frequency diversity in a set of saturated effective PSFs, while avoiding image degradation from shot noise.

Superresolution Fluorescence Microscopy of Platelet Subcellular Structures as a Potential Tumor Liquid Biopsy.

Blood-based tumor liquid biopsies are promising as an alternative or complement to tissue biopsies due to their noninvasiveness, convenience, and safety, and there is still a great demand for the discovery of new biomarkers for these biopsies. Here, nanoscale distribution patterns of subcellular structures in platelets, as imaged by structured illumination superresolution fluorescence microscopy, as a new type of potential biomarker for tumor liquid biopsies are presented. A standardized protocol for platelet sample preparation and developed an automated high-throughput image analysis workflow is established. The diagnostic capability based on the statistical analysis of 280000 superresolution images of individual platelets from a variety of tumor patients, benign mass patients, and healthy volunteers (n = 206) is explored. These results suggest that the nanoscale distribution patterns of α-granules in platelets have the potential to be biomarkers for several cancers, including glioma and cervical, endometrial, and ovarian cancers, facilitating not only diagnosis but also therapeutic monitoring. This study provides a promising novel type of platelet parameter for tumor liquid biopsies at the subcellular level rather than the existing cellular or molecular level and opens up a new avenue for clinical applications of superresolution imaging techniques.

Super-resolution dynamic tracking of cellular lipid droplets employing with a photostable deep red fluorogenic probe.

Lipid droplets (LDs) are critical organelles involved in many physiological processes in eukaryotic cells. To visualize and study LDs, particular the small/nascent LDs, the emerging super-resolution fluorescence imaging techniques with nanoscale resolution would be much more powerful in comparison to the conventional confocal/wide-field imaging techniques. However, directly limited by the availability of advanced LDs probes, super-resolution fluorescence imaging of LDs is a practically challenging task. In this context, a superior LDs fluorescent probe named Lipi-Deep Red is newly developed for structured illumination microscopy (SIM) super-resolution imaging. This fluorescent probe features with the advantages of strong deep red/NIR emission, fluorogenic character, high LDs specificity, and outstanding photostability. These advantages enable the fluorescent probe to be finely applied in SIM super-resolution imaging, e.g. time-lapse imaging (up to 1000 frames) to monitor the LDs dynamics at nanoscale (159nm), two-color time-lapse imaging to discover the nearby contact/interaction between LDs and mitochondria. Consequently, the fusion processes of LDs are impressively visualized at a high spatial and temporal resolution. Two kinds of contact models between LDs and mitochondria (dynamic contact and stable contact) newly proposed in the recent literatures are successfully revealed.

Low-cost acoustic force trap in a microfluidic channel

A low-cost glass-based microfluidic flow cell with a piezo actuator is built using off-the-shelf parts (total cost €9 per device) to apply acoustophoretic force on polystyrene micro-beads. The main challenge in the fabrication of these devices was to ensure their leak tightness, which we solved using double-sided tape and nail polish. Beads with 1.5 μm diameter flowing in a 100 μm deep channel were trapped at 7.5 MHz using a 23.7 peak-to-peak voltage (Vpp) sinusoidal input. The trap located at 50 ± 0.1 μm depth was measured to have a stiffness of approximately 0.6 pN/μm. With this simple device we can trap and control the axial position of micrometer scale objects, which allows for the manipulation of beads and cells. We intend to use the device for force spectroscopy on micro-bead tethered DNA. This can be combined with super-resolution imaging techniques to study mechanics and binding of protein structures along a DNA strand as a function of induced tension.

Vertical Emitting Nanowire Vector Beam Lasers.

Due to the peculiar structured light field with spatially variant polarizations on the same wavefront, vector beams (VBs) have sparked research enthusiasm in developing advanced super-resolution imaging and optical communications techniques. A compact VB nanolaser is intriguing for VB applications in miniaturized photonic integrated circuits. However, determined by the diffraction limit of light, it is a challenge to realize a VB nanolaser in the subwavelength scale because the VB lasing modes should have laterally structured distributions. Here, we demonstrate a VB nanolaser made from a 300 nm thick InGaAs/GaAs nanowire (NW). To select the high-order VB lasing mode, a standing NW as-grown from the selective-area-epitaxial (SAE) growth process is utilized, which has a bottom donut-shaped interface with the silicon oxide growth substrate. With this donut-shaped interface as one of the reflective mirrors of the nanolaser cavity, the VB lasing mode has the lowest threshold. Experimentally, a single-mode VB lasing mode with a donut-shaped amplitude and azimuthally cylindrical polarization distribution is obtained. Together with the high yield and uniformity of the SAE-grown NWs, our work provides a straightforward and scalable path toward cost-effective co-integration of VB nanolasers on potential photonic integrated circuits.