Ultrafast 3-D Transcutaneous Super Resolution Ultrasound Using Row-Column Array Specific Coherence-Based Beamforming and Rolling Acoustic Sub-aperture Processing: In Vitro, in Rabbit and in Human Study.

OBJECTIVE: This study aimed to realise 3-D super-resolution ultrasound imaging transcutaneously with a row-column array which has far fewer independent electronic channels and a wider field of view than typical fully addressed 2-D matrix arrays. The in vivo image quality of the row-column array is generally poor, particularly when imaging non-invasively. This study aimed to develop a suite of image formation and post-processing methods to improve image quality and demonstrate the feasibility of ultrasound localisation microscopy using a row-column array, transcutaneously on a rabbit model and in a human. METHODS: To achieve this, a processing pipeline was developed which included a new type of rolling window image reconstruction, which integrated a row-column array specific coherence-based beamforming technique with acoustic sub-aperture processing. This and other processing steps reduced the 'secondary' lobe artefacts, and noise and increased the effective frame rate, thereby enabling ultrasound localisation images to be produced. RESULTS: Using an in vitro cross tube, it was found that the procedure reduced the percentage of 'false' locations from ∼26% to ∼15% compared to orthogonal plane wave compounding. Additionally, it was found that the noise could be reduced by ∼7 dB and the effective frame rate was increased to over 4000 fps. In vivo, ultrasound localisation microscopy was used to produce images non-invasively of a rabbit kidney and a human thyroid. CONCLUSION: It has been demonstrated that the proposed methods using a row-column array can produce large field of view super-resolution microvascular images in vivo and in a human non-invasively.

Environmentally dependent and independent control of 3D cell shape.

How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment. Environmental sensitivity metrics calculated for small molecules and gene knockdowns identify interactions between the environment and cellular factors that are important for morphogenesis. We show that the Rho guanine nucleotide exchange factor (RhoGEF) TIAM2 contributes to shape determination in environmentally independent ways but that non-muscle myosin II, microtubules, and the RhoGEF FARP1 regulate shape in ways dependent on the microenvironment. Thus, changes in cancer cell shape in response to 3D geometric and mechanical cues are modulated in both an environmentally dependent and independent fashion.

ULTRA-SR Challenge: Assessment of Ultrasound Localization and TRacking Algorithms for Super-Resolution Imaging.

With the widespread interest and uptake of super-resolution ultrasound (SRUS) through localization and tracking of microbubbles, also known as ultrasound localization microscopy (ULM), many localization and tracking algorithms have been developed. ULM can image many centimeters into tissue in-vivo and track microvascular flow non-invasively with sub-diffraction resolution. In a significant community effort, we organized a challenge, Ultrasound Localization and TRacking Algorithms for Super-Resolution (ULTRA-SR). The aims of this paper are threefold: to describe the challenge organization, data generation, and winning algorithms; to present the metrics and methods for evaluating challenge entrants; and to report results and findings of the evaluation. Realistic ultrasound datasets containing microvascular flow for different clinical ultrasound frequencies were simulated, using vascular flow physics, acoustic field simulation and nonlinear bubble dynamics simulation. Based on these datasets, 38 submissions from 24 research groups were evaluated against ground truth using an evaluation framework with six metrics, three for localization and three for tracking. In-vivo mouse brain and human lymph node data were also provided, and performance assessed by an expert panel. Winning algorithms are described and discussed. The publicly available data with ground truth and the defined metrics for both localization and tracking present a valuable resource for researchers to benchmark algorithms and software, identify optimized methods/software for their data, and provide insight into the current limits of the field. In conclusion, Ultra-SR challenge has provided benchmarking data and tools as well as direct comparison and insights for a number of the state-of-the art localization and tracking algorithms.

Alignment and characterization of remote-refocusing systems.

The technique of remote refocusing is used in optical microscopy to provide rapid axial scanning without mechanically perturbing the sample and in techniques such as oblique plane microscopy that build on remote refocusing to image a tilted plane within the sample. The magnification between the pupils of the primary (O1) and secondary (O2) microscope objectives of the remote-refocusing system has been shown previously by Mohanan and Corbett [J. Microsc.288, 95 (2022)JMICAR0022-272010.1111/jmi.12991] to be crucial in obtaining the broadest possible remote-refocusing range. In this work, we performed an initial alignment of a remote-refocusing system and then studied the effect of axial misalignments of O1 and O2, axial misalignment of the primary tube lens (TL1) relative to the secondary tube lens (TL2), lateral misalignments of TL2, and changes in the focal length of  TL2. For each instance of the setup, we measured the mean point spread function F W H M xy of 100 nm fluorescent beads and the normalized bead integrated fluorescence signal, and we calculated the axial and lateral distortion of the system; all of these quantities were mapped over the remote-refocusing range and as a function of lateral image position. This allowed us to estimate the volume over which diffraction-limited performance is achieved and how this changes with the alignment of the system.

Active mesh and neural network pipeline for cell aggregate segmentation.

Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.

High-speed 2D light-sheet fluorescence microscopy enables quantification of spatially varying calcium dynamics in ventricular cardiomyocytes.

Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity. Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 µm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell. Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules. Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure.

MultiFRET: A Detailed Protocol for High-Throughput Multiplexed Ratiometric FRET.

The newly generated software plugin MultiFRET allows for real-time measurements of multiplexed fluorescent biosensors in a near high-throughput fashion. Here we describe a detailed protocol for setup and use of this software for any purpose requiring instant feedback during fluorescence measurement experiments. We further describe its non-primary features including beam splitter misalignment correction, custom calculations through input of simple equations typed in a .txt format, customizable Excel output, and offline bulk analysis of image stacks. Finally, we supply a usage example of a cAMP measurement in cultured rat neonatal cardiomyocytes.

Automatic tube lens design from stock optics for microscope remote-refocusing systems.

The remote-refocusing approach of Botcherby et al. [Opt. Lett.32, 2007 (2007)10.1364/OL.32.002007] has been applied widely to 2D and 3D fluorescence microscopes to enable rapid refocusing of the optical system without mechanically perturbing the sample. In order for this approach to operate correctly, it requires that the overall magnification of the first two microscope systems matches the ratio of the refractive indices in sample and intermedia image spaces. However, commercially available tube lenses are not always suitable to produce the desired overall magnification. Therefore, a practical approach to produce tube lenses with low expense and diffraction-limited performance is required. Tube lenses can be formed using a pair of stock achromatic doublets, however, selecting appropriate pairs of achromatic doublets from stock optics is a time-consuming process, as many combinations can be considered. In this paper, we present two software packages (Catalogue Generator and Doublet Selector) developed in MATLAB that use the application programming interface (ZOS-API) to the Zemax OpticStudio optical design software to realise an automatic search of stock achromatic doublets to produce microscope tube lenses with a specified focal length, entrance pupil diameter and maximum design field angle. An algorithm to optimise principal plane positions in versions of OpticStudio before 20.2 was also introduced to enable the use of older software versions. To evaluate the performance of Catalogue Generator and Doublet Selector, we used them to generate ten tube lens designs. All of the software-produced tube lenses have a better optical performance than those using manually selected pairs of stock doublets lenses.

Video-rate remote refocusing through continuous oscillation of a membrane deformable mirror.

This paper presents the use of a deformable mirror (DM) configured to rapidly refocus a microscope employing a high numerical aperture (NA) objective lens. An Alpao DM97-15 membrane DM was used to refocus a 40×/0.80 NA water-immersion objective through a defocus range of -50-50 µm at 26.3 sweeps s-1. We achieved imaging with a mean Strehl metric of >0.6 over a field of view in the sample of 200 × 200 µm2 over a defocus range of 77 µm. We describe an optimisation procedure where the mirror is swept continuously in order to avoid known problems of hysteresis associated with the membrane DM employed. This work demonstrates that a DM-based refocusing system could in the future be used in light-sheet fluorescence microscopes to achieve video-rate volumetric imaging.

Characterization of NADH fluorescence properties under one-photon excitation with respect to temperature, pH, and binding to lactate dehydrogenase.

Reduced nicotinamide adenine dinucleotide (NADH) is the principal electron donor in glycolysis and oxidative metabolism and is thus recognized as a key biomarker for probing metabolic state. While the fluorescence characteristics of NADH have been investigated extensively, there are discrepancies in the published data due to diverse experimental conditions, instrumentation and microenvironmental parameters that can affect NADH fluorescence. Using a cuvette-based time-resolved spectrofluorimeter employing one-photon excitation at 375 nm, we characterized the fluorescence intensity, lifetime, spectral response, anisotropy and time-resolved anisotropy of NADH in aqueous solution under varying microenvironmental conditions, namely temperature, pH, and binding to lactate dehydrogenase (LDH). Our results demonstrate how temperature, pH, and binding partners each impact the fluorescence signature of NADH and highlight the complexity of the fluorescence data when different parameters produce competing effects. We hope that the data presented in this study will provide a reference for potential sources of variation in experiments measuring NADH fluorescence.

Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast.

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire four images from which phase contrast images can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be easily integrated with fluorescence microscopy.

Genetic and biased agonist-mediated reductions in ß-arrestin recruitment prolong cAMP signaling at glucagon family receptors.

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of ß-arrestins. Indeed, recently described GLP-1R agonists with reduced ß-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both ß-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in ß-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced ß-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

The Influence of Peptide Context on Signaling and Trafficking of Glucagon-like Peptide-1 Receptor Biased Agonists.

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated cyclic AMP signaling, recruitment of ß-arrestins, endocytosis, and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific midpeptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level.

Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure.

We report a flexible light-sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination-detection modes: the first uses angle-dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video-rate volumetric imaging; the second combines digitally-scanned light-sheet illumination with an axially-swept light-sheet waist and stage-scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination-detection mode achieves dual spectral-channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time-lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination-detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t-tubule network in a fixed cardiomyocyte cell.

Autofluorescence Lifetime Reports Cartilage Damage in Osteoarthritis.

Osteoarthritis (OA) is the most common arthritis and its hallmark is degradation of articular cartilage by proteolytic enzymes leading to loss of joint function. It is challenging to monitor the status of cartilage in vivo and this study explores the use of autofluorescence lifetime (AFL) measurements to provide a label-free optical readout of cartilage degradation that could enable earlier detection and evaluation of potential therapies. We previously reported that treatment of ex vivo porcine cartilage with proteolytic enzymes resulted in decreased AFL. Here we report changes in AFL of ex vivo mouse knee joints, porcine metacarpophalangeal joints, normal human metatarsophalangeal articular tissue and human OA tibial plateau tissues measured with or without treatment using a compact single-point time resolved spectrofluorometer. Our data show that proteolytically damaged areas in porcine metacarpophalangeal joints present a reduced AFL and that inducing aggrecanases in mouse and human joints also significantly reduces AFL. Further, human cartilage from OA patients presents a significantly lower AFL compared to normal human cartilage. Our data suggest that AFL can detect areas of cartilage erosion and may potentially be utilised as a minimally-invasive diagnostic readout for early stage OA in combination with arthroscopy devices.

Dual-view oblique plane microscopy (dOPM).

We present a new folded dual-view oblique plane microscopy (OPM) technique termed dOPM that enables two orthogonal views of the sample to be obtained by translating a pair of tilted mirrors in refocussing space. Using a water immersion 40× 1.15 NA primary objective, deconvolved image volumes of 200 nm beads were measured to have full width at half maxima (FWHM) of 0.35 ± 0.04 µm and 0.39 ± 0.02 µm laterally and 0.81 ± 0.07 µm axially. The measured z-sectioning value was 1.33 ± 0.45 µm using light-sheet FWHM in the frames of the two views of 4.99 ± 0.58 µm and 4.89 ± 0.63 µm. To qualitatively demonstrate that the system can reduce shadow artefacts while providing a more isotropic resolution, a multi-cellular spheroid approximately 100 µm in diameter was imaged.

3-D Super-Resolution Ultrasound Imaging With a 2-D Sparse Array.

High-frame-rate 3-D ultrasound imaging technology combined with super-resolution processing method can visualize 3-D microvascular structures by overcoming the diffraction-limited resolution in every spatial direction. However, 3-D super-resolution ultrasound imaging using a full 2-D array requires a system with a large number of independent channels, the design of which might be impractical due to the high cost, complexity, and volume of data produced. In this study, a 2-D sparse array was designed and fabricated with 512 elements chosen from a density-tapered 2-D spiral layout. High-frame-rate volumetric imaging was performed using two synchronized ULA-OP 256 research scanners. Volumetric images were constructed by coherently compounding nine-angle plane waves acquired at a pulse repetition frequency of 4500 Hz. Localization-based 3-D super-resolution images of two touching subwavelength tubes were generated from 6000 volumes acquired in 12 s. Finally, this work demonstrates the feasibility of 3-D super-resolution imaging and super-resolved velocity mapping using a customized 2-D sparse array transducer.

A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET.

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.

In vivo label-free optical monitoring of structural and metabolic remodeling of myocardium following infarction.

Cardiac remodeling following myocardial infarction (MI) involves structural and functional alterations in the infarcted and remote viable myocardium that can ultimately lead to heart failure. The underlying mechanisms are not fully understood and, following our previous study of the autofluorescence lifetime and diffuse reflectance signatures of the myocardium in vivo at 16 weeks post MI in rats [Biomed. Opt. Express6(2), 324 (2015)], we here present data obtained at 1, 2 and 4 weeks post myocardial infarction that help follow the temporal progression of these changes. Our results demonstrate that both structural and metabolic changes in the heart can be monitored from the earliest time points following MI using label-free optical readouts, not only in the region of infarction but also in the remote non-infarcted myocardium. Changes in the autofluorescence intensity and lifetime parameters associated with collagen type I autofluorescence were indicative of progressive collagen deposition in tissue that was most pronounced at earlier time points and in the region of infarction. In addition to significant collagen deposition in infarcted and non-infarcted myocardium, we also report changes in the autofluorescence parameters associated with reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD), which we associate with metabolic alterations throughout the heart. Parallel measurements of the diffuse reflectance spectra indicated an increased contribution of reduced cytochrome c. Our findings suggest that combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy could provide a useful means to monitor cardiac function in vivo at the time of surgery.

Characterization of NAD(P)H and FAD autofluorescence signatures in a Langendorff isolated-perfused rat heart model.

Autofluorescence spectroscopy is a promising label-free approach to characterize biological samples with demonstrated potential to report structural and biochemical alterations in tissues in a number of clinical applications. We report a characterization of the ex vivo autofluorescence fingerprint of cardiac tissue, exploiting a Langendorff-perfused isolated rat heart model to induce physiological insults to the heart, with a view to understanding how metabolic alterations affect the autofluorescence signals. Changes in the autofluorescence intensity and lifetime signatures associated with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized during oxygen- or glucose-depletion protocols. Results suggest that both NAD(P)H and FAD autofluorescence intensity and lifetime parameters are sensitive to changes in the metabolic state of the heart owing to oxygen deprivation. We also observed changes in NAD(P)H fluorescence intensity and FAD lifetime parameter on reperfusion of oxygen, which might provide information on reperfusion injury, and permanent tissue damage or changes to the tissue during recovery from oxygen deprivation. We found that changes in the autofluorescence signature following glucose-depletion are, in general, less pronounced, and most clearly visible in NAD(P)H related parameters. Overall, the results reported in this investigation can serve as baseline for future investigations of cardiac tissue involving autofluorescence measurements.