Dialysis-assisted fiber optic spectroscopy for in situ biomedical sensing

A miniature fiber optic spectrometer enclosed within a semipermeable (dialysis) membrane is proposed for in vivo interstitial sensing applications. The semipermeable membrane acts as a molecular filter, allowing only small molecules to pass through to the sampling volume. This filtering, in principle, should enable continuous in vivo drug sensing, removing the necessity for complex microdialysis systems. We use a biological phantom to examine the reliable detection of a fluorescence signal from small dye molecules in the presence of large fluorophores and scatterers. We find that spectral artefacts arising from scatterers and large fluorophores are substantially suppressed, simplifying the spectral analysis. In addition, the measured sampling rate of 157 s is superior to existing in vivo tissue assaying techniques such as microdialysis, which can take tens of minutes. (c) 2006 Society of Photo- Optical Instrumentation Engineers

Novel contrasts in photoacoustic tomography

Photoacoustic tomography (PAT) combines rich optical contrast and high ultrasonic resolution in optically scattering tissue at depths. Taking advantage of its 100% sensitivity to optical absorption, PAT has been widely applied to structural, functional and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. This dissertation explores novel absorption contrast mechanisms of PAT based on optical/thermal patterns, endogenous cellular chromophores, nanoparticles, small-molecule dyes and genetically-encoded proteins. With these novel contrasts, the proof-of-concept applications of PAT have been extended to include homogenous flow measurements, targeted angiogenesis imaging and therapy, label-free white blood cell imaging, 3D-whole-organ cell nuclei imaging with a subcellular resolution, and in vivo neural activity imaging with voltage/calcium-sensitive indicators. Specifically, Chapter 1 introduces photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT) systems and discuss the motivation of the dissertation. Chapter 2 describes two photoacoustic (PA) flow measurement methods with optical and thermal patterns, which are applicable to homogenous flowing medium. In the first method, a Doppler frequency shift in PA signals of the flow was detected and used to calculate flow speeds. In the second method, unique features in an externally imposed thermal pattern of the flow, captured by repeated B-scans along the flow direction with a PAM system, revealed different flow speeds. Chapter 3 explores the unique PA contrast of macrophages, an important type of white blood cells. Macrophages were imaged by PAM without any label, and their measured PA spectrum was distinctive from the hemoglobin spectrum, so they can be potentially differentiated from red blood cells in the blood stream. Next, with a microtomy-assisted PAM system, cell nuclei distribution in whole organs, including mouse brain and mouse lung, were imaged with subcellular resolution. Chapter 4 introduces a type of target copper nanoparticles, which are less expensive and more biocompatible than its counterpart gold nanoparticles. The PA signals of neovasculature in the mouse flank were enhanced by the ___3-targeted copper nanoparticles. Moreover, the work shows the first example of a systemically targeted antiangiogenic drug delivery with a photoacoustic contrast nanoparticle in vivo. Chapter 5 demonstrates the voltage imaging capability of PA. A voltage sensitive dye with sufficient signal change was discovered and used as a PA voltage indicator for the first time. The mechanism was characterized through both PA imaging and spectroscopic methods. Its use was explored in a mouse epilepsy model and cortical electrical stimulation model in vivo. Finally, the deep imaging potential of PA was realized by imaging the voltage response of cells under 4.5 mm thick slice of rat brain tissue using a PACT system. Chapter 6 proves the neural calcium imaging capability of PA with a genetically encoded calcium indicator. In a fly model, I ambiguously demonstrated for the first time that PA can be used to imaging neural activities in the fly brain without the interference signals from hemoglobin. In the a live-mouse-brain-slice model, I successfully demonstrated the deep imaging capability of PA for calcium imaging by imaging through a 2-mm-thick scattering medium with a PACT system

Noninvasive optical estimation of CSF thickness for brain-atrophy monitoring

Dementia disorders are increasingly becoming sources of a broad range of problems, strongly interfering with normal daily tasks of a growing number of individuals. Such neurodegenerative diseases are often accompanied with progressive brain atrophy that, at late stages, leads to drastically reduced brain dimensions. At the moment, this structural involution can be followed with XCT or MRI measurements that share numerous disadvantages in terms of usability, invasiveness and costs. In this work, we aim to retrieve information concerning the brain atrophy stage and its evolution, proposing a novel approach based on non-invasive time-resolved Near Infra-Red (tr-NIR) measurements. For this purpose, we created a set of human-head atlases, in which we eroded the brain as it would happen in a clinical brain-atrophy progression. With these realistic meshes, we reproduced a longitudinal tr-NIR study exploiting a Monte-Carlo photon propagation algorithm to model the varying cerebral spinal fluid (CSF). The study of the time-resolved reflectance curve at late photon arrival times exhibited peculiar slope-changes upon CSF layer increase that were confirmed under several measurement conditions. The performance of the technique suggests good sensitivity to CSF variation, useful for a fast and non-invasive observation of the dementia progression.Comment: 32 pages, double spaced, 11 figure

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease.

Three-dimensional (3D) optical imaging techniques can expand our knowledge about physiological and pathological processes that cannot be fully understood with 2D approaches. Standard diagnostic tests frequently are not sufficient to unequivocally determine the presence of a pathological condition. Whole-organ optical imaging requires tissue transparency, which can be achieved by using tissue clearing procedures enabling deeper image acquisition and therefore making possible the analysis of large-scale biological tissue samples. Here, we review currently available clearing agents, methods, and their application in imaging of physiological or pathological conditions in different animal and human organs. We also compare different optical tissue clearing methods discussing their advantages and disadvantages and review the use of different 3D imaging techniques for the visualization and image acquisition of cleared tissues. The use of optical tissue clearing resources for large-scale biological tissues 3D imaging paves the way for future applications in translational and clinical research.This work was supported by Ministerio de Ciencia, Innovacion y Universidades, ISCIII-FIS grants PI18/00462 co-financed by ERDF, European Union (FEDER) Funds from the European Commission, European Union, ‘‘A way of making Europe’’; the CNIC is supported by theMinisterio de Ciencia, Innovacion y Universidades y the Pro CNIC Foundation, Severo Ochoa Center of Excellence (SEV-2015-0505), CIBER de Salud Mental (CIBERSAM), and COST-action CA16124. J.R. acknowledges funding from EU H2020 FET Open project SENSITIVE, ID 801347, and Ministerio de Ciencia, Innovacion y Universidades Grant FIS2016-77892-RS

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model

Monitoring of tumor response to Cisplatin using optical spectroscopy

INTRODUCTION Anatomic imaging alone is often inadequate for tuning systemic treatment for individual tumor response. Optically based techniques could potentially contribute to fast and objective response monitoring in personalized cancer therapy. In the present study, we evaluated the feasibility of dual-modality diffuse reflectance spectroscopy-autofluorescence spectroscopy (DRS-AFS) to monitor the effects of systemic treatment in a mouse model for hereditary breast cancer. METHODS Brca1(-/-); p53(-/-) mammary tumors were grown in 36 mice, half of which were treated with a single dose of cisplatin. Changes in the tumor physiology and morphology were measured for a period of 1 week using dual-modality DRS-AFS. Liver and muscle tissues were also measured to distinguish tumor-specific alterations from systemic changes. Model-based analyses were used to derive different optical parameters like the scattering and absorption coefficients, as well as sources of intrinsic fluorescence. Histopathologic analysis was performed for cross-validation with trends in optically based parameters. RESULTS Treated tumors showed a significant decrease in Mie-scattering slope and Mie-to-total scattering fraction and an increase in both fat volume fraction and tissue oxygenation after 2 days of follow-up. Additionally, significant tumor-specific changes in the fluorescence spectra were seen. These longitudinal trends were consistent with changes observed in the histopathologic analysis, such as vital tumor content and formation of fibrosis. CONCLUSIONS This study demonstrates that dual-modality DRS-AFS provides quantitative functional information that corresponds well with the degree of pathologic response. DRS-AFS, in conjunction with other imaging modalities, could be used to optimize systemic cancer treatment on the basis of early individual tumor response

PDT in the Thoracic Cavity: Spectroscopic Methods and Fluence Modeling for Treatment Planning

PDT for the thoracic cavity provides a promising cancer treatment modality, but improvements in treatment planning, particularly in PDT dosimetry, can be made to improve uniformity of light delivery. When a cavity of arbitrary geometry is illuminated, the fluence increases due to multiple-scattered photons, referred to as the Integrating Sphere Effect (ISE). Current pleural PDT treatment protocol at the University of Pennsylvania monitors light fluence (hereafter simply fluence, measured in W/cm2) via seven isotropic detectors sutured at different locations in thoracic cavity of a patient. This protocol monitors light at discrete locations, but does not provide a measurement of fluence for the thoracic cavity as a whole. Current calculation of light fluence includes direct light only and thus does not account for the unique optical properties of each tissue type present, which in turn affects the accuracy of the calculated light distribution in the surrounding tissue and, in turn, the overall cell death and treatment efficacy. Treatment planning for pleural PDT can be improved, in part, by considering the contribution of scattered light, which is affected by the two factors of geometry and in vivo optical properties. We expanded the work by Willem Star in regards to the ISE in a spherical cavity. A series of Monte Carlo (MC) simulations were run for semi-infinite planar, spherical, and ellipsoidal geometries for a range of optical properties. The results of these simulations are compared to theory and numerical solutions for fluence in the cavity and at the cavity-medium boundary. The development via MC simulations offers a general method of calculating the required light fluence specialized to each patient, based on the treatment surface area. The scattered fluence calculation is dependent on in vivo optical properties (μa and μs\u27) of the tissues treated. Diffuse reflectance and fluorescence spectroscopy methods are used to determine the optical properties and oxygenation (reflectance measurements) and drug concentration (fluorescence measurements) of different tissues in vivo, before and after treatment, in patients enrolled the Phase I HPPH study ongoing at the University of Pennsylvania. This work aims to provide the building blocks essential to pleural PDT treatment planning by more accurately calculating the required fluence using a model that accounts for the effects of treatment geometry and optical properties measured in vivo

Targeted Photodynamic Therapy and Photochemical Internalization of Human Head and Neck Cancer:a preclinical study in vitro and in vivo

Photodynamic therapy (PDT) is a treatment modality based on a tumour-localising photosensitizer and light exposure to induce necrosis and apoptosis of tumour cells. It is used to treat head and neck cancer, but its inadequate selectivity and specificity lead to phototoxicity of normal tissues. Targeted PDT employs a conjugate, a dye and an antibody (against a tumour-overexpressing molecule), to enhance selectivity and specificity of PDT. Photochemical internalisation (PCI) uses the principle of PDT for light-enhanced cytosolic release of anti-cancer drugs that are entrapped in the endo/lysosomal vesicles of cancer cells. The aims of this thesis were to improve selectivity and specificity of PDT and PCI with cetuximab-IR700DX conjugate. The thesis started with studying killing effects of targeted PDT in human head and neck tumour cell lines. Such therapeutic effects were then confirmed in a xenografted human head and neck tumour in a mouse skin-fold window-chamber model in vivo. A low light fluence rate enhanced such targeted PDT effects. The thesis was ended with investigating bleomycin-based PCI with temoporfin and gelonin-based PCI with targeted PDT in the human tumour cell lines in vitro