Super-resolution Localization Microscopy Research Articles

Context-aware deep learning enables high-efficacy localization of high concentration microbubbles for super-resolution ultrasound localization microscopy

Ultrasound localization microscopy (ULM) enables deep tissue microvascular imaging by localizing and tracking intravenously injected microbubbles circulating in the bloodstream. However, conventional localization techniques require spatially isolated microbubbles, resulting in prolonged imaging time to obtain detailed microvascular maps. Here, we introduce LOcalization with Context Awareness (LOCA)-ULM, a deep learning-based microbubble simulation and localization pipeline designed to enhance localization performance in high microbubble concentrations. In silico, LOCA-ULM enhanced microbubble detection accuracy to 97.8% and reduced the missing rate to 23.8%, outperforming conventional and deep learning-based localization methods up to 17.4% in accuracy and 37.6% in missing rate reduction. In in vivo rat brain imaging, LOCA-ULM revealed dense cerebrovascular networks and spatially adjacent microvessels undetected by conventional ULM. We further demonstrate the superior localization performance of LOCA-ULM in functional ULM (fULM) where LOCA-ULM significantly increased the functional imaging sensitivity of fULM to hemodynamic responses invoked by whisker stimulations in the rat brain.

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.

Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM

The β-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by “slow” and “fast” diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.

A Brief Introduction to Single-Molecule Fluorescence Methods.

One of the most popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster resonance energy transfer and orientation measurements with fluorescence polarization.

Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca2+-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIMHA-Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Nanodroplet-Based Super-Resolution Ultrasound Localization Microscopy.

Over the past decade, super-resolution ultrasound localization microscopy (SR-ULM) has revolutionized ultrasound imaging with its capability to resolve the microvascular structures below the ultrasound diffraction limit. The introduction of this imaging technique enables the visualization, quantification, and characterization of tissue microvasculature. The early implementations of SR-ULM utilize microbubbles (MBs) that require a long image acquisition time due to the requirement of capturing sparsely isolated microbubble signals. The next-generation SR-ULM employs nanodroplets that have the potential to significantly reduce the image acquisition time without sacrificing the resolution. This review discusses various nanodroplet-based ultrasound localization microscopy techniques and their corresponding imaging mechanisms. A summary is given on the preclinical applications of SR-ULM with nanodroplets, and the challenges in the clinical translation of nanodroplet-based SR-ULM are presented while discussing the future perspectives. In conclusion, ultrasound localization microscopy is a promising microvasculature imaging technology that can provide new diagnostic and prognostic information for a wide range of pathologies, such as cancer, heart conditions, and autoimmune diseases, and enable personalized treatment monitoring at a microlevel.

High-Volume-Rate 3-D Ultrasound Imaging Using Fast-Tilting and Redirecting Reflectors.

Three-dimensional ultrasound imaging has many advantages over 2-D imaging such as more comprehensive tissue evaluation and less operator dependence. However, developing a low-cost and accessible 3-D ultrasound solution with high volume rate and imaging quality remains a challenging task. Recently, we proposed a 3-D ultrasound imaging technique: fast acoustic steering via tilting electromechanical reflectors (FASTER), which uses a fast-tilting acoustic reflector to steer ultrafast plane waves elevationally to achieve high-volume-rate 3-D imaging with conventional 1-D transducers. However, the initial FASTER implementation requires a water tank for acoustic wave conduction and cannot be conveniently used for regular handheld scanning. To address these limitations, here, we developed a novel ultrasound probe clip-on device that encloses a fast-tilting reflector, a redirecting reflector, and an acoustic wave conduction medium. The new FASTER 3-D imaging device can be easily attached to or removed from clinical ultrasound transducers, allowing rapid transformation from 2-D to 3-D imaging. In vitro B-mode studies demonstrated that the proposed method provided comparable imaging quality to conventional, mechanical-translation-based 3-D imaging while offering a much faster volume rate (e.g., 300 versus ∼ 10 Hz). We also demonstrated 3-D power Doppler (PD) and 3-D super-resolution ultrasound localization microscopy (ULM) with the FASTER device. An in vivo imaging study showed that the FASTER device could clearly visualize the 3-D anatomy of the basilic vein. These results suggest that the newly developed redirecting reflector and the clip-on device could overcome key hurdles for future clinical translation of the FASTER 3-D imaging technology.

Advanced Microscopy Techniques for Molecular Biophysics.

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).

Super-Resolution Ultrasound Localization Microscopy Using High-Frequency Ultrasound to Measure Ocular Perfusion Velocity in the Rat Eye.

Imaging of the ocular vasculature can provide new insights into the pathophysiology of ocular diseases. This study proposes a novel high-frequency super-resolution ultrasound localization microscopy (SRULM) technique and evaluates its ability to measure in vivo perfusion changes in the rat eye at elevated intraocular pressure (IOP). A 38.4 MHz center frequency linear array transducer on a VisualSonics Vevo F2 imaging platform was used to collect high frame rate (1 kHz) radiofrequency data of the posterior rat eye following systemic microbubble contrast injection. Following clutter and spatiotemporal non-local means filtering, individual microbubbles were localized and tracked. The microbubble tracks were accumulated over 10,000 frames to generate vascular images quantifying perfusion velocity and direction. Experiments were performed using physiologic relevant controlled flow states for algorithm validation and subsequently performed in vivo on the rat eye at 10 mm Hg IOP increments from 10 to 60 mm Hg. The posterior vasculature of the rat eye, including the ophthalmic artery, long posterior ciliary arteries and their branches, central retinal artery and retinal arterioles and venules were successfully visualized, and velocities quantified at each IOP level. Significant reductions in arterial flow were measured as IOP was elevated. High-frequency SRULM can be used to visualize and quantify the perfusion velocity of the rat eye in both the retrobulbar and intraocular vasculature simultaneously. The ability to detect ocular perfusion changes throughout the depth of the eye may help elucidate the role ischemia has in the pathophysiology of ocular diseases such as glaucoma.

Simple, Economic, and Robust Rail-Based Setup for Super-Resolution Localization Microscopy.

Research during the past 2 decades has showcased the power of single-molecule localization microscopy (SMLM) as a tool for exploring the nanoworld. However, SMLM systems are typically available in specialized laboratories and imaging facilities, owing to their expensiveness as well as complex assembly and alignment procedure. Here, we lay out the blueprint of a sturdy, rail-based, cost-efficient, multicolor SMLM setup that is easy to construct and align in service of simplifying the accessibility of SMLM. We characterize the optical properties of the design and assess its capabilities, robustness, and stability. The performance of the system is assayed using super-resolution imaging of biological samples. We believe that this design will make SMLM more affordable and broaden its availability.

Randomized channel subsampling method for efficient ultrafast ultrasound imaging

Emerging as a novel modality with high spatiotemporal sensitivity, ultrafast ultrasound imaging has been well established and incorporated into clinical apparatus. However, such a mode typically requires large data acquisitions, with more than thousands of frames per second within a relatively long period, which poses high demands on real-time storage and processing hardware. Herein, an image reconstruction approach based on randomized channel subsampling is presented, which significantly reduces the amount of data acquisition while maintaining imaging performance. In vivo datasets from rats were used to evaluate the performance of the proposed method in B-mode imaging, ultrafast Doppler imaging, and super-resolution ultrasound localization microscopy (ULM) under different subsampling conditions. Following a thorough comparison of the contrast-to-noise ratio, signal-to-noise ratio, and visibility of the ultrafast Doppler based small-vessel imaging, the resolution and saturation of ULM imaging were also investigated under different subsampling conditions. The feasibility of the proposed method was demonstrated in task-based functional ultrasound (fUS) imaging with whisker stimulation. A comparison between the results of fUS based on introduced randomized channel subsampling and conventional fUS was also conducted to evaluate the subsampling effects on reconstruction accuracy and cerebral change detection sensitivity.

The MksG nuclease is the executing part of the bacterial plasmid defense system MksBEFG.

Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial 'innate immune system'. Here, we investigated the molecular arrangement of the Corynebacterium glutamicum MksBEFG complex, which is homologous to the MukBEF condensin system. We show here that MksG is a nuclease that degrades plasmid DNA. The crystal structure of MksG revealed a dimeric assembly through its C-terminal domain that is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding ion binding site essential for DNA cleavage in topoisomerases. The MksBEF subunits exhibit an ATPase cycle in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via the polar scaffold protein DivIVA. Introduction of plasmids results in an increase in DNA bound MksG, indicating an activation of the system in vivo.

Live-cell photoactivated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photoactivated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution photoactivated localization microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and ‘traveling’ events that spread between neighboring RyR clusters. Traveling sparks exhibited up to eight distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intracluster RyR recruitment to generate larger events. In contrast, RyR ‘dispersion’ during heart failure facilitates the generation of traveling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

A fundamental understanding of gene regulation requires a quantitative characterization of the spatial organization and dynamics of chromatin. The advent of fluorescence super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) presented a breakthrough to visualize structural features with a resolution of ~20 nm in fixed cells. However, until recently the long acquisition time of super-resolution images prevented high-resolution measurements in living cells due to spreading of localizations caused by chromatin motion. Here, we present a step-by step protocol for our recently developed approach for correlatively imaging telomeres with conventional fluorescence and PALM, in order to obtain time-averaged super-resolution images and dynamic parameters in living cells. First, individual single molecule localizations are assigned to a locus as it moves, allowing to discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. By subtracting the telomere trajectory from the localization of bound molecules, the motion blurring is then corrected, and high-resolution structural characterizations can be made. These structural parameters can also be related to local chromatin motion or larger scale domain movement. This protocol therefore improves the ability to analyze the mobility and time-averaged nanoscopic structure of locus-specific chromatin with single-molecule sensitivity.

Correction Notice: Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

Correction Notice: Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Co-Distribution of Influenza A Hemagglutinin and Matrix Protein M1 at the Plasma Membrane.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

Super-Resolution Ultrasound Localization Microscopy of Microvascular Structure and Flow for Distinguishing Metastatic Lymph Nodes - An Initial Human Study.

Detecting and distinguishing metastatic lymph nodes (LNs) from those with benign lymphadenopathy are crucial for cancer diagnosis and prognosis but remain a clinical challenge. A recent advance in super-resolution ultrasound (SRUS) through localizing individual microbubbles has broken the diffraction limit and tracking enabled in vivo noninvasive imaging of vascular morphology and flow dynamics at a microscopic level. In this study we hypothesize that SRUS enables quantitative markers to distinguish metastatic LNs from benign ones in patients with lymphadenopathy. Clinical contrast-enhanced ultrasound image sequences of LNs from 6 patients with lymph node metastasis and 4 with benign lymphadenopathy were acquired and motion-corrected. These were then used to generate super-resolution microvascular images and super-resolved velocity maps. From these SRUS images, morphological and functional measures were obtained including micro-vessel density, fractal dimension, mean flow speed, and Local Flow Direction Irregularity (LFDI) measuring the variance in local flow direction. These measures were compared between pathologically proven reactive and metastasis LNs. Our initial results indicate that the difference in the indicator of flow irregularity (LFDI) derived from the SRUS images is statistically significant between the two groups. The LFDI is 60% higher in metastatic LNs compared with reactive nodes. This pilot study demonstrates the feasibility of super-resolution ultrasound for clinical imaging of lymph nodes and the potential of using the irregularity of local blood flow directions afforded by SRUS for the characterization of LNs.

ResPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

ResPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

Spatial Organization of Dectin-1 and TLR2 during Synergistic Crosstalk Revealed by Super-resolution Imaging.

Innate immune cells recognize and elicit responses against pathogens by integrating signals from different types of cell-surface receptors. How the receptors interact in the membrane to enable their signaling crosstalk is poorly understood. Here, we reveal the nanoscale organization of TLR2 and Dectin-1, a receptor pair known to cooperate in regulating antifungal immunity, through their synergistic signaling crosstalk at macrophage cell membranes. Using super-resolution single-molecule localization microscopy, we show that discrete noncolocalized nanoclusters of Dectin-1 and TLR2 are partially overlapped during their synergistic crosstalk. Compared to when one type of receptor is activated alone, the simultaneous activation of Dectin-1 and TLR2 leads to a higher percentage of both receptors being activated by their specific ligands and consequently an increased level of tyrosine phosphorylation. Our results depict, in nanoscale detail, how Dectin-1 and TLR2 achieve synergistic signaling through the spatial organization of their receptor nanoclusters.