BUbble Flow Field: a Simulation Framework for Evaluating Ultrasound Localization Microscopy Algorithms

Ultrasound contrast enhanced imaging has seen widespread uptake in research and clinical diagnostic imaging. This includes applications such as vector flow imaging, functional ultrasound and super-resolution Ultrasound Localization Microscopy (ULM). All of these require testing and validation during development of new algorithms with ground truth data. In this work we present a comprehensive simulation platform BUbble Flow Field (BUFF) that generates contrast enhanced ultrasound images in vascular tree geometries with realistic flow characteristics and validation algorithms for ULM. BUFF allows complex micro-vascular network generation of random and user-defined vascular networks. Blood flow is simulated with a fast Computational Fluid Dynamics (CFD) solver and allows arbitrary input and output positions and custom pressures. The acoustic field simulation is combined with non-linear Microbubble (MB) dynamics and simulates a range of point spread functions based on user-defined MB characteristics. The validation combines both binary and quantitative metrics. BFF's capacity to generate and validate user-defined networks is demonstrated through its implementation in the Ultrasound Localisation and TRacking Algorithms for Super Resolution (ULTRA-SR) Challenge at the International Ultrasonics Symposium (IUS) 2022 of the Institute of Electrical and Electronics Engineers (IEEE). The ability to produce ULM images, and the availability of a ground truth in localisation and tracking enables objective and quantitative evaluation of the large number of localisation and tracking algorithms developed in the field. BUFF can also benefit deep learning based methods by automatically generating datasets for training. BUFF is a fully comprehensive simulation platform for testing and validation of novel ULM techniques and is open source.Comment: 10 Pages, 9 Figure

Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy

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3D acoustic wave sparsely activated localization microscopy with phase change contrast agents

Objective The aim of this study is to demonstrate 3-dimensional (3D) acoustic wave sparsely activated localization microscopy (AWSALM) of microvascular flow in vivo using phase change contrast agents (PCCAs). Materials and Methods Three-dimensional AWSALM using acoustically activable PCCAs was evaluated on a crossed tube microflow phantom, the kidney of New Zealand White rabbits, and the brain of C57BL/6J mice through intact skull. A mixture of C3F8 and C4F10 low-boiling-point fluorocarbon gas was used to generate PCCAs with an appropriate activation pressure. A multiplexed 8-MHz matrix array connected to a 256-channel ultrasound research platform was used for transmitting activation and imaging ultrasound pulses and recording echoes. The in vitro and in vivo echo data were subsequently beamformed and processed using a set of customized algorithms for generating 3D super-resolution ultrasound images through localizing and tracking activated contrast agents. Results With 3D AWSALM, the acoustic activation of PCCAs can be controlled both spatially and temporally, enabling contrast on demand and capable of revealing 3D microvascular connectivity. The spatial resolution of the 3D AWSALM images measured using Fourier shell correlation is 64 μm, presenting a 9-time improvement compared with the point spread function and 1.5 times compared with half the wavelength. Compared with the microbubble-based approach, more signals were localized in the microvasculature at similar concentrations while retaining sparsity and longer tracks in larger vessels. Transcranial imaging was demonstrated as a proof of principle of PCCA activation in the mouse brain with 3D AWSALM. Conclusions Three-dimensional AWSALM generates volumetric ultrasound super-resolution microvascular images in vivo with spatiotemporal selectivity and enhanced microvascular penetration

Application of ultrafast gold luminescence to measuring the instrument response function for multispectral multiphoton fluorescence lifetime imaging

When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo

FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ**

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated

Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway

Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules

Wide-field coherence-gated imaging techniques including photorefractive holography

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