Date of Award

May 2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Engineering

First Advisor

Mahsa Ranji

Committee Members

Amadou KS Camara, Brian Armstrong, Mahsa Dabagh, Yi Hu

Keywords

Intrinsic fluorescence, Label-free, NADH, Optical metabolic imaging, Vascular imaging, Whole organ

Abstract

The assessment of organ metabolic function using optical imaging techniques is an overgrowing field of disease diagnosis. The broad research objective of my PhD thesis is to detect quantitative biomarkers by developing and applying optical imaging and image processing tools to animal models of human diseases. To achieve this goal, I have designed and implemented an optical imaging instrument called in vivo fluorescence imager to study wound healing progress. I have also developed a 3-dimensional (3D) vascular segmentation technique that uses intrinsic fluorescence images of whole organs.

Intrinsic fluorophores (autofluorescence signals) provide information about the status of cellular bioenergetics in different tissue types. Reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) are two key Krebs cycle coenzymes in mitochondria, which are autofluorescent. The ratio of these two fluorophores (NADH/FAD) is used as an optical biomarker for mitochondrial redox state of the tissues. The custom-designed optical tools have enabled me to probe the metabolic state of diseases as well as structural information of the organs at different regimes (in vivo, at cryogenic temperature, and in vitro). Here are the main projects that I have conducted and significantly contributed to:

1) Fluorescent metabolic imaging. I have designed and implemented an in vivo fluorescence imaging device to study diabetic wounds in small animals. This device can monitor the dynamics of the metabolism of the skin by capturing the images of the surface fluorescence of NADH and FAD. The area of the wounds can also be monitored simultaneously. The spatiotemporal mitochondrial redox ratio changes can give information on the status of wound healing online. This device was utilized to study diabetic wounds and the effect of photo-biomodulation on the wound healing progress.

I have also utilized the optical cryo-imaging system to study the three-dimensional (3D) mitochondrial redox state of kidneys, hearts, livers, and wound biopsies of the small animal models of various injuries. For example, cryo-imaging was conducted on irradiated rat hearts during ischemia-reperfusion (IR) to investigate the role of mitochondrial metabolism in the differential susceptibility to IR injury. Also, I developed a 3D image processing tool that can segment and quantify the medullary versus the cortical redox state in the kidneys of animal injury models.

2) 3D Vascular-Metabolic Imaging (VMI). I have designed VMI, an image processing algorithm that segments vascular networks from intrinsic fluorescence. VMI allows the simultaneous acquisition of vasculature and metabolism in multiple organs. I demonstrate that this technique provides the vascular network of the whole organ without the need for a contrast agent. A proof validation has performed using TdTomato fluorescence expressing endothelium. The VMI also showed convincing evidence for the “minimum work” hypothesis in the vascular network by following Murray’s law. For a proof-of-concept, I have also utilized a partial body irradiation model that VMI can provide information on radiation-induced vascular regression.

3) Time-lapse fluorescence microscopy. I have utilized fluorescence microscopy to quantify the dynamics of cellular reactive oxygen species (ROS) concentration. ROS is imaged and quantified under oxygen or metabolic stress conditions in cells in vitro. This approach enabled me to study the sensitivity of retinal endothelial cells and pericytes to stress under high glucose conditions.

In short, I developed and utilized optical bio-instrumentation and image processing tools to be able to detect metabolic and vascular information about different diseases.

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