Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.

Assessment of physical and hydrological properties of biological soil crusts using x-ray microtomography and modelling

Biological soil crusts (BSCs) are formed by aggregates of soil particles and communities of microbial organisms and are common in all drylands. The role of BSCs on infiltration remains uncertain due to the lack of data on their role in affecting soil physical properties such as porosity and structure. Quantitative assessment of these properties is primarily hindered by the fragile nature of the crusts. Here we show how the use of a combination of non-destructive imaging X-ray microtomography (XMT) and Lattice Boltzmann method (LBM) enables quantification of key soil physical parameters and the modeling of water flow through BSCs samples from Kalahari Sands, Botswana. We quantify porosity and flow changes as a result of mechanical disturbance of such a fragile cyanobacteria-dominated crust. Results show significant variations in porosity between different types of crusts and how they affect the flow and that disturbance of a cyanobacteria-dominated crust results in the breakdown of larger pore spaces and reduces flow rates through the surface layer. We conclude that the XMT–LBM approach is well suited for study of fragile surface crust samples where physical and hydraulic properties cannot be easily quantified using conventional methods.

Physical and biological properties of a novel siloxane adhesive for soft tissue applications

The aim of this study was to investigate the adhesive properties of an in-house amino-propyltrimethoxysilane-methylenebisacrylamide (APTMS-MBA) siloxane system and compare them with a commercially available adhesive, n-butyl cyanoacrylate (nBCA). The ability of the material to perform as a soft tissue adhesive was established by measuring the physical (bond strength, curing time) and biological (cytotoxicity) properties of the adhesives on cartilage. Complementary physical techniques, X-ray photoelectron spectroscopy, Raman and infrared imaging, enabled the mode of action of the adhesive to the cartilage surface to be determined. Adhesion strength to cartilage was measured using a simple butt joint test after storage in phosphate-buffered saline solution at 37°C for periods up to 1 month. The adhesives were also characterised using two in vitro biological techniques. A live/dead stain assay enabled a measure of the viability of chondrocytes attached to the two adhesives to be made. A water-soluble tetrazolium assay was carried out using two different cell types, human dermal fibroblasts and ovine meniscal chondrocytes, in order to measure material cytotoxicity as a function of both supernatant concentration and time. IR imaging of the surface of cartilage treated with APTMS-MBA siloxane adhesive indicated that the adhesive penetrated the tissue surface marginally compared to nBCA which showed a greater depth of penetration. The curing time and adhesion strength values for APTMS-MBA siloxane and nBCA adhesives were measured to be 60 s/0.23 MPa and 38 min/0.62 MPa, respectively. These materials were found to be significantly stronger than either commercially available fibrin (0.02 MPa) or gelatin resorcinol formaldehyde (GRF) adhesives (0.1 MPa) (P <0.01). Cell culture experiments revealed that APTMS-MBA siloxane adhesive induced 2% cell death compared to 95% for the nBCA adhesive, which extended to a depth of approximately 100-150 μm into the cartilage surface. The WST-1 assay demonstrated that APTMS-MBA siloxane was significantly less cytotoxic than nBCA adhesive as an undiluted conditioned supernatant (P <0.001). These results suggest that the APTMS-MBA siloxane may be a useful adhesive for medical applications. © VSP 2005.

Bottleneck Problem Solution using Biological Models of Attention in High Resolution Tracking Sensors

Every high resolution imaging system suffers from the bottleneck problem. This problem relates to the huge amount of data transmission from the sensor array to a digital signal processing (DSP) and to bottleneck in performance, caused by the requirement to process a large amount of information in parallel. The same problem exists in biological vision systems, where the information, sensed by many millions of receptors should be transmitted and processed in real time. Models, describing the bottleneck problem solutions in biological systems fall in the field of visual attention. This paper presents the bottleneck problem existing in imagers used for real time salient target tracking and proposes a simple solution by employing models of attention, found in biological systems. The bottleneck problem in imaging systems is presented, the existing models of visual attention are discussed and the architecture of the proposed imager is shown.

Longitudinal diffusion tensor imaging in dementia with Lewy bodies and Alzheimer's disease

Copyright © 2016 Elsevier Ltd. All rights reserved. Acknowledgements The study was supported by the NIHR Biomedical Research Unit in Dementia and the Biomedical Research Centre awarded to Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge, and the NIHR Biomedical Research Unit in Dementia and the Biomedical Research Centre awarded to Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University. Elijah Mak was in receipt of a Gates Cambridge PhD studentship.

Whole-brain patterns of 1H-magnetic resonance spectroscopy imaging in Alzheimer's disease and dementia with Lewy bodies

Acknowledgments We thank Craig Lambert for his help in processing the MRS data. The study was funded by the Sir Jules Thorn Charitable Trust (grant ref: 05/JTA) and was supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre and the Biomedical Research Unit in Lewy Body Dementia based at Newcastle upon Tyne Hospitals National Health Service (NHS) Foundation Trust and Newcastle University and the NIHR Biomedical Research Centre and Biomedical Research Unit in Dementia based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

Probing Tissue Microstructure Using Susceptibility Contrast Magnetic Resonance Imaging

Magnetic resonance imaging is a research and clinical tool that has been applied in a wide variety of sciences. One area of magnetic resonance imaging that has exhibited terrific promise and growth in the past decade is magnetic susceptibility imaging. Imaging tissue susceptibility provides insight into the microstructural organization and chemical properties of biological tissues, but this image contrast is not well understood. The purpose of this work is to develop effective approaches to image, assess, and model the mechanisms that generate both isotropic and anisotropic magnetic susceptibility contrast in biological tissues, including myocardium and central nervous system white matter.

This document contains the first report of MRI-measured susceptibility anisotropy in myocardium. Intact mouse heart specimens were scanned using MRI at 9.4 T to ascertain both the magnetic susceptibility and myofiber orientation of the tissue. The susceptibility anisotropy of myocardium was observed and measured by relating the apparent tissue susceptibility as a function of the myofiber angle with respect to the applied magnetic field. A multi-filament model of myocardial tissue revealed that the diamagnetically anisotropy α-helix peptide bonds in myofilament proteins are capable of producing bulk susceptibility anisotropy on a scale measurable by MRI, and are potentially the chief sources of the experimentally observed anisotropy.

The growing use of paramagnetic contrast agents in magnetic susceptibility imaging motivated a series of investigations regarding the effect of these exogenous agents on susceptibility imaging in the brain, heart, and kidney. In each of these organs, gadolinium increases susceptibility contrast and anisotropy, though the enhancements depend on the tissue type, compartmentalization of contrast agent, and complex multi-pool relaxation. In the brain, the introduction of paramagnetic contrast agents actually makes white matter tissue regions appear more diamagnetic relative to the reference susceptibility. Gadolinium-enhanced MRI yields tensor-valued susceptibility images with eigenvectors that more accurately reflect the underlying tissue orientation.

Despite the boost gadolinium provides, tensor-valued susceptibility image reconstruction is prone to image artifacts. A novel algorithm was developed to mitigate these artifacts by incorporating orientation-dependent tissue relaxation information into susceptibility tensor estimation. The technique was verified using a numerical phantom simulation, and improves susceptibility-based tractography in the brain, kidney, and heart. This work represents the first successful application of susceptibility-based tractography to a whole, intact heart.

The knowledge and tools developed throughout the course of this research were then applied to studying mouse models of Alzheimer’s disease in vivo, and studying hypertrophic human myocardium specimens ex vivo. Though a preliminary study using contrast-enhanced quantitative susceptibility mapping has revealed diamagnetic amyloid plaques associated with Alzheimer’s disease in the mouse brain ex vivo, non-contrast susceptibility imaging was unable to precisely identify these plaques in vivo. Susceptibility tensor imaging of human myocardium specimens at 9.4 T shows that susceptibility anisotropy is larger and mean susceptibility is more diamagnetic in hypertrophic tissue than in normal tissue. These findings support the hypothesis that myofilament proteins are a source of susceptibility contrast and anisotropy in myocardium. This collection of preclinical studies provides new tools and context for analyzing tissue structure, chemistry, and health in a variety of organs throughout the body.

Tissue Equivalent Phantom Design for Optimization of a Coherent Scatter Imaging System

Scatter in medical imaging is typically cast off as image-related noise that detracts from meaningful diagnosis. It is therefore typically rejected or removed from medical images. However, it has been found that every material, including cancerous tissue, has a unique X-ray coherent scatter signature that can be used to identify the material or tissue. Such scatter-based tissue-identification provides the advantage of locating and identifying particular materials over conventional anatomical imaging through X-ray radiography. A coded aperture X-ray coherent scatter spectral imaging system has been developed in our group to classify different tissue types based on their unique scatter signatures. Previous experiments using our prototype have demonstrated that the depth-resolved coherent scatter spectral imaging system (CACSSI) can discriminate healthy and cancerous tissue present in the path of a non-destructive x-ray beam. A key to the successful optimization of CACSSI as a clinical imaging method is to obtain anatomically accurate phantoms of the human body. This thesis describes the development and fabrication of 3D printed anatomical scatter phantoms of the breast and lung.

The purpose of this work is to accurately model different breast geometries using a tissue equivalent phantom, and to classify these tissues in a coherent x-ray scatter imaging system. Tissue-equivalent anatomical phantoms were designed to assess the capability of the CACSSI system to classify different types of breast tissue (adipose, fibroglandular, malignant). These phantoms were 3D printed based on DICOM data obtained from CT scans of prone breasts. The phantoms were tested through comparison of measured scatter signatures with those of adipose and fibroglandular tissue from literature. Tumors in the phantom were modeled using a variety of biological tissue including actual surgically excised benign and malignant tissue specimens. Lung based phantoms have also been printed for future testing. Our imaging system has been able to define the location and composition of the various materials in the phantom. These phantoms were used to characterize the CACSSI system in terms of beam width and imaging technique. The result of this work showed accurate modeling and characterization of the phantoms through comparison of the tissue-equivalent form factors to those from literature. The physical construction of the phantoms, based on actual patient anatomy, was validated using mammography and computed tomography to visually compare the clinical images to those of actual patient anatomy.

Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.

Sparse Signal Representation in Digital and Biological Systems

Theories of sparse signal representation, wherein a signal is decomposed as the sum of a small number of constituent elements, play increasing roles in both mathematical signal processing and neuroscience. This happens despite the differences between signal models in the two domains. After reviewing preliminary material on sparse signal models, I use work on compressed sensing for the electron tomography of biological structures as a target for exploring the efficacy of sparse signal reconstruction in a challenging application domain. My research in this area addresses a topic of keen interest to the biological microscopy community, and has resulted in the development of tomographic reconstruction software which is competitive with the state of the art in its field. Moving from the linear signal domain into the nonlinear dynamics of neural encoding, I explain the sparse coding hypothesis in neuroscience and its relationship with olfaction in locusts. I implement a numerical ODE model of the activity of neural populations responsible for sparse odor coding in locusts as part of a project involving offset spiking in the Kenyon cells. I also explain the validation procedures we have devised to help assess the model's similarity to the biology. The thesis concludes with the development of a new, simplified model of locust olfactory network activity, which seeks with some success to explain statistical properties of the sparse coding processes carried out in the network.

New bodipy dyads for two-photons fluorescence imaging in cells

Fluorescent probes are essential tools for studying biological systems. The last decade has witnessed particular interest in the development of two-photon excitable probes, due to their advantageous features in tissue imaging compared to the corresponding one-photon probes [1]. Recently, we have designed and synthetized an aminonaphthalimide–BODIPY derivative as energy transfer cassettes and were found to show very fast and efficient BODIPY fluorescence sensitization [2]. This was observed upon one- and two-photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In order to increase the two-photon absorption of the system aminonaphthalimide fluorophore was replace with a Prodan analog (BODIPY dyad 1), which presents found a variety of applications as probes and labels in biology [3]. The two-photon absorption cross-section  of the dyads is significantly incremented by the presence of the 6-acetyl-2-naphthylamine donor group. The emission maximum of a BODIPY fluorophore can significantly be red-shifted in comparison to their precursors by conjugation with aromatic aldehydes. [4] We use a synthetic strategy to obtain BODIPY dyad 2 that incorporates an imidazole ring. This molecule can be used in biological media as a near-neutral pH indicator based on one- and two-photon excitable BODIPY acceptor.

Fiber Pathways for Language in the Developing Brain: A Diffusion Tensor Imaging (DTI) Study

The present study characterized two fiber pathways important for language, the superior longitudinal fasciculus/arcuate fasciculus (SLF/AF) and the frontal aslant tract (FAT), and related these tracts to speech, language, and literacy skill in children five to eight years old. We used Diffusion Tensor Imaging (DTI) to characterize the fiber pathways and administered several language assessments. The FAT was identified for the first time in children. Results showed no age-related change in integrity of the FAT, but did show age-related change in the left (but not right) SLF/AF. Moreover, only the integrity of the right FAT was related to phonology but not audiovisual speech perception, articulation, language, or literacy. Both the left and right SLF/AF related to language measures, specifically receptive and expressive language, and language content. These findings are important for understanding the neurobiology of language in the developing brain, and can be incorporated within contemporary dorsal-ventral-motor models for language.

Challenges in imaging and predictive modeling of rhizosphere processes

Background Plant-soil interaction is central to human food production and ecosystem function. Thus, it is essential to not only understand, but also to develop predictive mathematical models which can be used to assess how climate and soil management practices will affect these interactions. Scope In this paper we review the current developments in structural and chemical imaging of rhizosphere processes within the context of multiscale mathematical image based modeling. We outline areas that need more research and areas which would benefit from more detailed understanding. Conclusions We conclude that the combination of structural and chemical imaging with modeling is an incredibly powerful tool which is fundamental for understanding how plant roots interact with soil. We emphasize the need for more researchers to be attracted to this area that is so fertile for future discoveries. Finally, model building must go hand in hand with experiments. In particular, there is a real need to integrate rhizosphere structural and chemical imaging with modeling for better understanding of the rhizosphere processes leading to models which explicitly account for pore scale processes.