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Department of Biomedical EngineeringThe optical imaging has a critical role in biomedical research to analyze functional and morphological variation of an organ, tissue and even a single cell of animal models. Since the optical imaging modality has features of indirect access, volumetric analysis and high resolution, it has been used for biomedical analysis. Especially, as a low coherence interferometric imaging technique, optical coherence tomography (OCT) has been applied in scientific and medical fields from few decades ago. Since OCT can provide endogenous contrast of biological tissue using the infrared light source, it has high potential to be applied in practical medical diagnosis. However, it is hard to acquire uneven or thick sample due to the limited imaging window and penetration depth. To overcome those limitations, lots of optical, mathematical and chemical solutions comes within a decade such as adaptive optics, full-range method and tissue clearing. Despite the existence of suggested solutions, practical application of OCT is limitation due to the cost of time and effort. Here, we present practical methods to enhance acquirable endogenous information of sample through versatile scanning optical coherence tomography(VS-OCT). Conventional OCT utilizes dual-axis based flat focal plane scanning method providing limited depth information of curved samples. Thus, we developed advanced OCT, called VS-OCT, which can fully optimize imaging window by changing focal plane to dual plane and cylindrical plane. The VS-OCT is demonstrated for 1) quantification of engineered skin, 2) monitoring of tadpole development, 3) screening phenotype of zebrafish and 4) quantification of spinal cord injury (SCI) of mouse.ope

Toolbox for in vivo imaging of host-parasite interactions at multiple scales

Animal models have for long been pivotal for parasitology research. Over the last few years, techniques such as intravital, optoacoustic and magnetic resonance imaging, optical projection tomography, and selective plane illumination microscopy developed promising potential for gaining insights into host-pathogen interactions by allowing different visualization forms in vivo and ex vivo. Advances including increased resolution, penetration depth, and acquisition speed, together with more complex image analysis methods, facilitate tackling biological problems previously impossible to study and/or quantify. Here we discuss advances and challenges in the in vivo imaging toolbox, which hold promising potential for the field of parasitology

Photoacoustic tomography: fundamentals, advances and prospects

Optical microscopy has been contributing to the development of life science for more than three centuries. However, due to strong optical scattering in tissue, its in vivo imaging ability has been restricted to studies at superficial depths. Advances in photoacoustic tomography (PAT) now allow multiscale imaging at depths from sub-millimeter to several centimeters, with spatial resolutions from sub-micrometer to sub-millimeter. Because of this high scalability and its unique optical absorption contrast, PAT is capable of performing anatomical, functional, molecular and fluid-dynamic imaging at various system levels, and is playing an increasingly important role in fundamental biological research and clinical practice. This Review discusses recent technical progress in PAT and presents corresponding applications. It ends with a discussion of several prospects and their technical challenges

Luminescence lifetime imaging of three-dimensional biological objects

ABSTRACT A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.</jats:p

Challenges and advances in optical 3D mesoscale imaging

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence

Adaptive optics: principles and applications in ophthalmology

This is a comprehensive review of the principles and applications of adaptive optics (AO) in ophthalmology. It has been combined with flood illumination ophthalmoscopy, scanning laser ophthalmoscopy, as well as optical coherence tomography to image photoreceptors, retinal pigment epithelium (RPE), retinal ganglion cells, lamina cribrosa and the retinal vasculature. In this review, we highlight the clinical studies that have utilised AO to understand disease mechanisms. However, there are some limitations to using AO in a clinical setting including the cost of running an AO imaging service, the time needed to scan patients, the lack of normative databases and the very small size of area imaged. However, it is undoubtedly an exceptional research tool that enables visualisation of the retina at a cellular level

Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

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

Development of High-speed Optical Coherence Tomography for Time-lapse Non-destructive Characterization of Samples

Optical coherence tomography (OCT) is an established optical imaging modality which can obtain label-free, non-destructive 3D images of samples with micron-scale resolution and millimeter penetration. OCT has been widely adopted for biomedical researches