Super-resolution Optical Microscopy Research Articles

Plasmonic Metasurfaces for Superposition of Profile-Tunable Tightly Focused Vector Beams and Generation of the Structured Light

Vector beams (VBs) and their superposition have found important applications in versatile fields such as optical communications, super-resolution microscopy and quantum information, and metasurfaces have enabled the miniaturization and integration of the optical systems manipulating the vector beams, providing potential applications to subwavelength regimes. In this work, we propose a metasurface to realize the superposition of profile-tunable tightly focused VBs, with the novel structured light fields generated. The metasurface is composed of two sets of orthogonal-nanoslit pairs arranged on the inner and outer rings. By realizing the chiral conversion of circularly polarized light with the slit-pairs which act as half-wave plates, and by creating helical phase profiles of optical vortices with the geometrical phase of rotational nano-slit pairs, two focused Bessel VBs are formed. By finely varying the diameters of two sets of rings, the doughnuts of the two Bessel VBs of different orders are tuned to be of the same size, and the superposition of the two VBs is realized. The theoretical analyses of the superimposed fields were presented, the FDTD simulations were performed to optimize the designed metasurfaces, and the experimental measurements were carried out to validate feasibility of the metasurface. The novel and interesting characteristics of the superposed fields different from those of the conventional VBs were demonstrated. This work will be of significance for classical and quantum applications of VBs in various fields.

SOFFLFM: Super-resolution optical fluctuation Fourier light-field microscopy

Fourier light-field microscopy (FLFM) uses a microlens array (MLA) to segment the Fourier plane of the microscopic objective lens to generate multiple two-dimensional perspective views, thereby reconstructing the three-dimensional (3D) structure of the sample using 3D deconvolution calculation without scanning. However, the resolution of FLFM is still limited by diffraction, and furthermore, it is dependent on the aperture division. In order to improve its resolution, a super-resolution optical fluctuation Fourier light-field microscopy (SOFFLFM) was proposed here, in which the super-resolution optical fluctuation imaging (SOFI) with the ability of super-resolution was introduced into FLFM. SOFFLFM uses higher-order cumulants statistical analysis on an image sequence collected by FLFM, and then carries out 3D deconvolution calculation to reconstruct the 3D structure of the sample. The theoretical basis of SOFFLFM on improving resolution was explained and then verified with the simulations. Simulation results demonstrated that SOFFLFM improved the lateral and axial resolution by more than [Formula: see text] and 2 times in the second- and fourth-order accumulations, compared with that of FLFM.

Antibody Functionalization of Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticle Probes for Advanced Intracellular Labeling and Optical Super Resolution Microscopy

Fluorescent labeling of cellular substructures is commonly performed using antibody–organic dye conjugates. Organic dyes do not exhibit ideal optical properties in terms of brightness and photostability, however, in particular when it comes to advanced optical super-resolution microscopy (SRM) applications. Here, we demonstrate the efficient conjugation of widely available secondary antibodies and cationic species to ultrasmall (sub-10 nm) fluorescent silica core─poly(ethylene glycol) shell (core–shell) aluminosilicate nanoparticles (aC′ dots) encapsulating different color dyes for specific targeting and high-quality fluorescence imaging of structures of the cytoskeleton (tubulin and actin) and nucleus, respectively. We show that the different color aC′ dots provide enhanced brightness and photostability relative to their parent dyes. As recently discovered, we further demonstrate that they exhibit photo-induced blinking with low ON–OFF duty cycles enabling optical SRM, for example, in the form of stochastic optical reconstruction microscopy (STORM), without the need for complex imaging setups or cocktails. After carefully optimizing Ab–aC′ dot conjugation as well as cell structure labeling protocols in fixed and permeabilized HeLa and MDA-MB-231 cells, we demonstrate three-color STORM and exemplify improved resolution compared to standard antibody–dye conjugates. This work paves the way to next-generation multifunctional optical probes based on ultrasmall silica nanoparticle platforms for advanced applications in bioimaging, nanomedicine, and beyond.

Near-field optical microscopy toward its applications for biological studies.

Near-field scanning optical microscopy (NSOM) is a super-resolution optical microscopy based on nanometrically small near-field light at a metallic tip. It can be combined with various types of optical measurement techniques, including Raman spectroscopy, infrared absorption spectroscopy, and photoluminescence measurements, which provides unique analytical capabilities to a variety of scientific fields. In particular, to understand nanoscale details of advance materials and physical phenomena, NSOM has been often adopted in the fields of material science and physical chemistry. However, owing to the recent critical developments showing the great potential for biological studies, NSOM has also recently gained much attention in the biological field. In this article, we introduce recent developments made in NSOM, aiming at biological applications. The drastic improvement in the imaging speed has shown a promising application of NSOM for super-resolution optical observation of biological dynamics. Furthermore, stable imaging and broadband imaging were made possible owing to the advanced technologies, which provide a unique imaging method to the biological field. As NSOM has not been well exploited in biological studies to date, several rooms need to be explored to determine its distinct advantages. We discuss the possibility and perspective of NSOM for biological applications. This review article is an extended version of the Japanese article, Development of Near-field Scanning Optical Microscopy toward Its Application for Biological Studies, published in SEIBUTSU BUTSURI Vol. 62, p. 128-130 (2022).

Laser heating with doughnut-shaped beams

Doughnut-shaped laser beams have applications in laser-based additive manufacturing, laser heating of diamond anvil cells, and optical super-resolution microscopy. In applications like additive manufacturing and heating of diamond anvil cells, a doughnut-shaped beam is frequently used to obtain a more uniform temperature profile relative to that generated by a conventional Gaussian beam. Conversely, in super-resolution microscopy, the doughnut-shaped beam serves to enhance spatial resolution and heating is an undesirable side effect that can cause thermal damage. Here, we develop analytical expressions for the temperature rise induced by a doughnut-shaped laser beam both alone and in combination with a Gaussian beam. For representative, experimentally determined beam radii and a wide range of thermal properties, we find that a doughnut-shaped beam results in a peak temperature rise no more than 90% and often less than 75% of that for a Gaussian beam with the same total power. Meanwhile, the region of the sample surface that reaches 80% of the maximum temperature rise is at least 1.5 times larger for a doughnut-shaped beam than for a Gaussian beam. When doughnut-shaped and Gaussian beams are applied simultaneously, the ratio of the maximum temperature rise for the two beams combined vs a Gaussian beam alone can be up to 2.5 times lower than the ratio of the doughnut-shaped vs the Gaussian beam power. For applications like super-resolution microscopy that require high doughnut-shaped laser beam powers, the doughnut-shaped beam intensity profile is thus advantageous for minimizing the total peak temperature rise when applied together with a Gaussian beam.

Caught in Action: Visualizing Dynamic Nanostructures Within Supramolecular Systems Chemistry.

Supramolecular systems chemistry has been an area of active research to develop nanomaterials with life-like functions. Progress in systems chemistry relies on our ability to probe the nanostructure formation in solution. Often visualizing the dynamics of nanostructures which transform over time is a formidable challenge. This necessitates a paradigm shift from dry sample imaging towards solution-based techniques. We review the application of state-of-the-art techniques for real-time, in situ visualization of dynamic self-assembly processes. We present how solution-based techniques namely optical super-resolution microscopy, solution-state atomic force microscopy, liquid-phase transmission electron microscopy, molecular dynamics simulations and other emerging techniques are revolutionizing our understanding of active and adaptive nanomaterials with life-like functions. This Review provides the visualization toolbox and futuristic vision to tap the potential of dynamic nanomaterials.

Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy

BackgroundRecently, bacterial extracellular vesicles (EVs) have been considered to play crucial roles in various biological processes and have great potential for developing cancer therapeutics and biomedicine. However, studies on bacterial EVs have mainly focused on outer membrane vesicles released from gram-negative bacteria since the outermost peptidoglycan layer in gram-positive bacteria is thought to preclude the release of EVs as a physical barrier.ResultsHere, we examined the ultrastructural organization of the EV produced by gram-positive bacteria using super-resolution stochastic optical reconstruction microscopy (STORM) at the nanoscale, which has not been resolved using conventional microscopy. Based on the super-resolution images of EVs, we propose three major mechanisms of EV biogenesis, i.e., membrane blebbing (mechanisms 1 and 2) or explosive cell lysis (mechanism 3), which are different from the mechanisms in gram-negative bacteria, despite some similarities.ConclusionsThese findings highlight the significant role of cell wall degradation in regulating various mechanisms of EV biogenesis and call for a reassessment of previously unresolved EV biogenesis in gram-positive bacteria.

Optical Fingerprint of Flat Substrate Surface and Marker-Free Lateral Displacement Detection with Angstrom-Level Precision.

We report that flat substrates such as glass coverslips with surface roughness well below 0.5nm feature notable speckle patterns when observed with high-sensitivity interference microscopy. We uncover that these speckle patterns unambiguously originate from the subnanometer surface undulations, and develop an intuitive model to illustrate how subnanometer nonresonant dielectric features could generate pronounced interference contrast in the far field. We introduce the concept of optical fingerprint for the deterministic speckle pattern associated with a particular substrate surface area and intentionally enhance the speckle amplitudes for potential applications. We demonstrate such optical fingerprints can be leveraged for reproducible position identification and marker-free lateral displacement detection with an experimental precision of 0.22nm. The reproducible position identification allows us to detect new nanoscopic features developed during laborious processes performed outside of the microscope. The demonstrated capability for ultrasensitive displacement detection may find applications in the semiconductor industry and superresolution optical microscopy.

Steerable photonic jet for super-resolution microscopy

A promising technique in optical super-resolution microscopy is the illumination of the sample by a highly localized beam, a photonic jet (also called photonic nanojet). We propose a method of computation of incident field amplitude and phase profiles that produce photonic jets at desired locations in the near field after interaction with a fixed micro-scale dielectric lens. We also describe a practical way of obtaining the incident field profiles using spatial light modulators. We expect our photonic jet design method to work for a wide range of lens shapes, and we demonstrate its application numerically using two-dimensional micro-lenses of circular and square cross-sections. We furthermore offer a theoretical analysis of the resolution of photonic jet design, predicting among other that a larger lens can produce a narrower photonic jet. Finally, we give both theoretical and numerical evidence that the waist width of the achieved designed jets is increasing linearly and slowly over a large interval of radial distances. With uniform plane wave illumination, the circular two-dimensional micro-lens produces a similar-sized jet at a fixed radial distance, while the square lens does not form a jet at all. We expect our steerable optical photonic jet probe to enable highly localized adaptive real-time measurements and drive advances in super-resolution optical microscopy and scatterometry, as well as fluorescence and Raman microscopy. Our relatively weak peak jet intensity allows application in biology and health sciences, which require high resolution imaging without damaging the sample bio-molecules.

Three-dimensional visualization of the cardiac ryanodine receptor clusters and the molecular-scale fraying of dyads.

Clusters of ryanodine receptor calcium channels (RyRs) form the primary molecular machinery of intracellular calcium signalling in cardiomyocytes. While a range of optical super-resolution microscopy techniques have revealed the nanoscale structure of these clusters, the three-dimensional (3D) nanoscale topologies of the clusters have remained mostly unresolved. In this paper, we demonstrate the exploitation of molecular-scale resolution in enhanced expansion microscopy (EExM) along with various 2D and 3D visualization strategies to observe the topological complexities, geometries and molecular sub-domains within the RyR clusters. Notably, we observed sub-domains containing RyR-binding protein junctophilin-2 (JPH2) occupying the central regions of RyR clusters in the deeper interior of the myocytes (including dyads), while the poles were typically devoid of JPH2, lending to a looser RyR arrangement. By contrast, peripheral RyR clusters exhibited variable co-clustering patterns and ratios between RyR and JPH2. EExM images of dyadic RyR clusters in right ventricular (RV) myocytes isolated from rats with monocrotaline-induced RV failure revealed hallmarks of RyR cluster fragmentation accompanied by breaches in the JPH2 sub-domains. Frayed RyR patterns observed adjacent to these constitute new evidence that the destabilization of the RyR arrays inside the JPH2 sub-domains may seed the primordial foci of dyad remodelling observed in heart failure.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Correlative nano-imaging of metals and proteins in primary neurons by synchrotron X-ray fluorescence and STED super resolution microscopy: Experimental validation

BackgroundIt is becoming increasingly clear that biological metals such as iron, copper or zinc are involved in synaptic functions, and in particular in the mechanisms of synaptogenesis and subsequent plasticity. Understanding the role of metals on synaptic functions is a difficult challenge due to the very low concentration of these elements in neurons and to the submicrometer size of synaptic compartments. New methodTo address this challenge we have developed a correlative nano-imaging approach combining metal and protein detection. First, stimulated emission depletion (STED) microscopy, a super resolution optical microscopy technique, is applied to locate fluorescently labeled proteins. Then, synchrotron radiation induced X-ray fluorescence (SXRF) is performed on the same regions of interest, e.g. synaptic compartments. ResultsWe present here the principle scheme that allows this correlative nano-imaging and its experimental validation. We applied this correlative nano-imaging to the study of the physiological distribution of metals in synaptic compartments of primary rat hippocampal neurons. We thus compared the nanometric distribution of metals with that of synaptic proteins, such as PSD95 or cytoskeleton proteins. Comparison with existing method(s)Compared to correlative imaging approaches currently used to characterize synaptic structures, such as electron microscopy correlated with optical fluorescence, our approach allows for ultra-sensitive detection of trace metals using highly focused synchrotron radiation beams. ConclusionWe provide proof-of-principle for correlative imaging of metals and proteins at the synaptic scale and discuss the present limitations and future developments in this area.

Generation of axially splitted ultra-long multiple optical needles/optical tubes using generalized cylindrical vector Bessel Gaussian beam phase modulated by annular Walsh function filter

Based on vector diffraction theory and annular Walsh function filter, axially splitted ultra-long multiple optical needles/optical tubes of electric as well as magnetic field distributions are numerically generated by generalized cylindrical vector Bessel Gaussian beam phase modulated by annular Walsh function filter. This multiple optical structures can be conveniently tailored by properly tuning the initial phase of the Cylindrical vector beam (CVB) and Walsh order of the annular Walsh function filter. Furthermore, the effect of energy flux density (Poynting vector) as well as Spin angular momentum(SAM) distribution in the focal region are theoretically analyzed. We expect that such an axially splitted focal systems could be applicable in optical trapping and manipulation of multiple particles, optical super resolution microscopy etc.

Single Image Capture of Bioactive Ion Crosstalk within Inter-Organelle Membrane Contacts at Nanometer Resolution.

Rapid bioactive ion exchange is a form of communication that regulates a wide range of biological processes. Despite advances in super-resolution optical microscopy, visualizing ion exchange remains challenging due to the extremely fast nature of these events. Here, a "converting a dynamic event into a static image construction" (CDtSC) strategy is developed that uses the color transformation of a single dichromatic molecular probe to visualize bioactive ion inter-organelle exchange in live cells. As a proof of concept, a reactive sulfur species (RSS) is analyzed at the mitochondria-lysosome contact sites (MLCs). A non-toxic and sensitive probe based on coumarin-hemicyanine structure is designed that responds to RSS localized in both mitochondria and lysosomes while fluorescing different colors. Using this probe, RSS give-and-take at MLCs is visualized, thus providing the first evidence that RSS is involved in inter-organelle contacts and communication. Taken together, the CDtSC provides a strategy to visualize and analyze rapid inter-organelle ion exchange events in live cells at nanometer resolution.

Super-resolution optical microscopy using cylindrical vector beams

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

Correlative Assembly of Subsynaptic Nanoscale Organizations During Development.

Nanoscale organization of presynaptic proteins determines the sites of transmitter release, and its alignment with assemblies of postsynaptic receptors through nanocolumns is suggested to optimize the efficiency of synaptic transmission. However, it remains unknown how these nano-organizations are formed during development. In this study, we used super-resolution stochastic optical reconstruction microscopy (STORM) imaging technique to systematically analyze the evolvement of subsynaptic organization of three key synaptic proteins, namely, RIM1/2, GluA1, and PSD-95, during synapse maturation in cultured hippocampal neurons. We found that volumes of synaptic clusters and their subsynaptic heterogeneity increase as synapses get matured. Synapse sizes of presynaptic and postsynaptic compartments correlated well at all stages, while only more mature synapses demonstrated a significant correlation between presynaptic and postsynaptic nano-organizations. After a long incubation with an inhibitor of action potentials or AMPA receptors, both presynaptic and postsynaptic compartments showed increased synaptic cluster volume and subsynaptic heterogeneity; however, the trans-synaptic alignment was intact. Together, our results characterize the evolvement of subsynaptic protein architectures during development and demonstrate that the nanocolumn is organized more likely by an intrinsic mechanism and independent of synaptic activities.

Intense vector supercontinuum radiation from femtosecond filamentation.

Intense vector supercontinuum (SC) radiation with spatial polarization is obtained by using 800nm femtosecond vector laser beams in the air. The SC generated by azimuthally, radially, cylindrically polarized beams, and higher-order vector beams are investigated, respectively. The results show that the SC generated by vector beams is greatly enhanced compared to that by a Gaussian beam. The energy density of SC radiation reaches the order of 1µJ/nm in a bandwidth of 258 nm from 559 nm to 817 nm and 0.1 µJ/nm from 500 nm to 559 nm. Furthermore, by checking the polarization distribution of SC in different wavelengths from visible to near-infrared bands, we find that the SC maintains nearly the same polarization distribution as pump pulses. This work provides an effective and convenient way to generate powerful SC vector beams which may facilitate potential applications including optical communication, micro/nano-fabrication, and super-resolution microscopy.

Differential Effects of Vascular Endothelial Growth Factor on Glycocalyx of Endothelial and Tumor Cells Uncovered by Super‐resolution Optical Microscopy and Potential Targets for Tumor Metastasis

On the surface of every mammalian cell, there is a matrix‐like glycocalyx (GCX) consisting of proteoglycans and glycosaminoglycans (GAGs). Disruption of endothelial cell (EC) GCX by vascular endothelial growth factor (VEGF, VEGF‐A165), a tumor secretion, was found to be an early event in tumor cell (TC) metastasis across vascular barriers. But how the TC secretion VEGF affects its own GCX is unknown although bulkier GCX of tumor cells was found to enhance its metastasis. To investigate the VEGF effect on TC GCX and to elucidate the ultrastructural organization of EC and TC GCX and their alteration by VEGF, we employed super‐resolution stochastic optical reconstruction microscopy (STORM) to observe the spatio‐chemical organizations of the heparan sulfate (HS) and hyaluronic acid (HA), two representative GAGs of GCX, on human cerebral microvascular endothelial cells (hCMEC) and malignant breast cancer cells MDA‐MB‐231 (MB231). We found that HS and HA have distinct organizations on hCMEC and MB231. Only HS of hCMEC is perpendicular to the cell surface, while HA of hCMEC, as well as HS and HA of MB231 all lie in the same plane as the cell surface where they appear to weave into a 2D network covering the cell. We also found that VEGF significantly reduces the length and coverage of HS on hCMEC but does not change the thickness and coverage of HA on hCMEC. On the contrary, VEGF significantly enhances the coverage of HS and HA on MB231 although it does not alter the thickness. The differential effects of VEGF on the GCX of TC and that of EC may favor TC adhesion and transmigration across EC barriers for their metastasis.

Engineering Optically Active Defects in Hexagonal Boron Nitride Using Focused Ion Beam and Water

Hexagonal boron nitride (hBN) has emerged as a promising material platform for nanophotonics and quantum sensing, hosting optically active defects with exceptional properties such as high brightness and large spectral tuning. However, precise control over deterministic spatial positioning of emitters in hBN remained elusive for a long time, limiting their proper correlative characterization and applications in hybrid devices. Recently, focused ion beam (FIB) systems proved to be useful to engineer several types of spatially defined emitters with various structural and photophysical properties. Here we systematically explore the physical processes leading to the creation of optically active defects in hBN using FIB and find that beam–substrate interaction plays a key role in the formation of defects. These findings are confirmed using transmission electron microscopy, which reveals local mechanical deterioration of the hBN layers and local amorphization of ion beam irradiated hBN. Additionally, we show that, upon exposure to water, amorphized hBN undergoes a structural and optical transition between two defect types with distinctive emission properties. Moreover, using super-resolution optical microscopy combined with atomic force microscopy, we pinpoint the exact location of emitters within the defect sites, confirming the role of defected edges as primary sources of fluorescent emission. This lays the foundation for FIB-assisted engineering of optically active defects in hBN with high spatial and spectral control for applications ranging from integrated photonics, to nanoscale sensing, and to nanofluidics.

Chip-based multimodal super-resolution microscopy for histological investigations of cryopreserved tissue sections

Histology involves the observation of structural features in tissues using a microscope. While diffraction-limited optical microscopes are commonly used in histological investigations, their resolving capabilities are insufficient to visualize details at subcellular level. Although a novel set of super-resolution optical microscopy techniques can fulfill the resolution demands in such cases, the system complexity, high operating cost, lack of multi-modality, and low-throughput imaging of these methods limit their wide adoption for histological analysis. In this study, we introduce the photonic chip as a feasible high-throughput microscopy platform for super-resolution imaging of histological samples. Using cryopreserved ultrathin tissue sections of human placenta, mouse kidney, pig heart, and zebrafish eye retina prepared by the Tokuyasu method, we demonstrate diverse imaging capabilities of the photonic chip including total internal reflection fluorescence microscopy, intensity fluctuation-based optical nanoscopy, single-molecule localization microscopy, and correlative light-electron microscopy. Our results validate the photonic chip as a feasible imaging platform for tissue sections and pave the way for the adoption of super-resolution high-throughput multimodal analysis of cryopreserved tissue samples both in research and clinical settings.

A Review of Clinical Applications for Super-resolution Ultrasound Localization Microscopy.

Microvascular structure and hemodynamics are important indicators for the diagnosis and assessment of many diseases and pathologies. The structural and functional imaging of tissue microvasculature in vivo is a clinically significant objective for the development of many imaging modalities. Contrast-enhanced ultrasound (CEUS) is a popular clinical tool for characterizing tissue microvasculature, due to the moderate cost, wide accessibility, and absence of ionizing radiation of ultrasound. However, in practice, it remains challenging to demonstrate microvasculature using CEUS, due to the resolution limit of conventional ultrasound imaging. In addition, the quantification of tissue perfusion by CEUS remains hindered by high operator-dependency and poor reproducibility. Inspired by super-resolution optical microscopy, super-resolution ultrasound localization microscopy (ULM) was recently developed. ULM uses the same ultrasound contrast agent (i.e. microbubbles) in CEUS. However, different from CEUS, ULM uses the location of the microbubbles to construct images, instead of using the backscattering intensity of microbubbles. Hence, ULM overcomes the classic compromise between imaging resolution and penetration, allowing for the visualization of capillary-scale microvasculature deep within tissues. To date, many in vivo ULM results have been reported, including both animal (kidney, brain, spinal cord, xenografted tumor, and ear) and human studies (prostate, tibialis anterior muscle, and breast cancer tumors). Furthermore, a variety of useful biomarkers have been derived from using ULM for different preclinical and clinical applications. Due to the high spatial resolution and accurate blood flow speed estimation (approximately 1 mm/s to several cm/s), ULM presents as an enticing alternative to CEUS for characterizing tissue microvasculature in vivo. This review summarizes the principles and present applications of CEUS and ULM, and discusses areas where ULM can potentially provide a better alternative to CEUS in clinical practice and areas where ULM may not be a better alternative. The objective of the study is to provide clinicians with an up-to-date review of ULM technology, and a practical guide for implementing ULM in clinical research and practice.