Development of Multi-modal and Super-resolved Retinal Imaging Systems

Advancements in retinal imaging technologies have drastically improved the quality of eye care in the past couple decades. Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) are two examples of critical imaging modalities for the diagnosis of retinal pathologies. However current-generation SLO and OCT systems have limitations in diagnostic capability due to the following factors: the use of bulky tabletop systems, monochromatic imaging, and resolution degradation due to ocular aberrations and diffraction.

Bulky tabletop SLO and OCT systems are incapable of imaging patients that are supine, under anesthesia, or otherwise unable to maintain the required posture and fixation. Monochromatic SLO and OCT imaging prevents the identification of various color-specific diagnostic markers visible with color fundus photography like those of neovascular age-related macular degeneration. Resolution degradation due to ocular aberrations and diffraction has prevented the imaging of photoreceptors close to the fovea without the use of adaptive optics (AO), which require bulky and expensive components that limit the potential for widespread clinical use.

In this dissertation, techniques for extending the diagnostic capability of SLO and OCT systems are developed. These techniques include design strategies for miniaturizing and combining SLO and OCT to permit multi-modal, lightweight handheld probes to extend high quality retinal imaging to pediatric eye care. In addition, a method for extending true color retinal imaging to SLO to enable high-contrast, depth-resolved, high-fidelity color fundus imaging is demonstrated using a supercontinuum light source. Finally, the development and combination of SLO with a super-resolution confocal microscopy technique known as optical photon reassignment (OPRA) is demonstrated to enable high-resolution imaging of retinal photoreceptors without the use of adaptive optics.

Development of Swept Source Optical Coherence Tomography and Adaptive Optics Scanning Laser Ophthalmoscopy: Improved Imaging Speed and Handheld Applications

Optical coherence tomography (OCT) is a noninvasive three-dimensional interferometric imaging technique capable of achieving micrometer scale resolution. It is now a standard of care in ophthalmology, where it is used to improve the accuracy of early diagnosis, to better understand the source of pathophysiology, and to monitor disease progression and response to therapy. In particular, retinal imaging has been the most prevalent clinical application of OCT, but researchers and companies alike are developing OCT systems for cardiology, dermatology, dentistry, and many other medical and industrial applications.

Adaptive optics (AO) is a technique used to reduce monochromatic aberrations in optical instruments. It is used in astronomical telescopes, laser communications, high-power lasers, retinal imaging, optical fabrication and microscopy to improve system performance. Scanning laser ophthalmoscopy (SLO) is a noninvasive confocal imaging technique that produces high contrast two-dimensional retinal images. AO is combined with SLO (AOSLO) to compensate for the wavefront distortions caused by the optics of the eye, providing the ability to visualize the living retina with cellular resolution. AOSLO has shown great promise to advance the understanding of the etiology of retinal diseases on a cellular level.

Broadly, we endeavor to enhance the vision outcome of ophthalmic patients through improved diagnostics and personalized therapy. Toward this end, the objective of the work presented herein was the development of advanced techniques for increasing the imaging speed, reducing the form factor, and broadening the versatility of OCT and AOSLO. Despite our focus on applications in ophthalmology, the techniques developed could be applied to other medical and industrial applications. In this dissertation, a technique to quadruple the imaging speed of OCT was developed. This technique was demonstrated by imaging the retinas of healthy human subjects. A handheld, dual depth OCT system was developed. This system enabled sequential imaging of the anterior segment and retina of human eyes. Finally, handheld SLO/OCT systems were developed, culminating in the design of a handheld AOSLO system. This system has the potential to provide cellular level imaging of the human retina, resolving even the most densely packed foveal cones.

Development and Optimization of Four-dimensional Magnetic Resonance Imaging (4D-MRI) for Radiation Therapy

A tenet of modern radiotherapy (RT) is to identify the treatment target accurately, following which the high-dose treatment volume may be expanded into the surrounding tissues in order to create the clinical and planning target volumes. Respiratory motion can induce errors in target volume delineation and dose delivery in radiation therapy for thoracic and abdominal cancers. Historically, radiotherapy treatment planning in the thoracic and abdominal regions has used 2D or 3D images acquired under uncoached free-breathing conditions, irrespective of whether the target tumor is moving or not. Once the gross target volume has been delineated, standard margins are commonly added in order to account for motion. However, the generic margins do not usually take the target motion trajectory into consideration. That may lead to under- or over-estimate motion with subsequent risk of missing the target during treatment or irradiating excessive normal tissue. That introduces systematic errors into treatment planning and delivery. In clinical practice, four-dimensional (4D) imaging has been popular in For RT motion management. It provides temporal information about tumor and organ at risk motion, and it permits patient-specific treatment planning. The most common contemporary imaging technique for identifying tumor motion is 4D computed tomography (4D-CT). However, CT has poor soft tissue contrast and it induce ionizing radiation hazard. In the last decade, 4D magnetic resonance imaging (4D-MRI) has become an emerging tool to image respiratory motion, especially in the abdomen, because of the superior soft-tissue contrast. Recently, several 4D-MRI techniques have been proposed, including prospective and retrospective approaches. Nevertheless, 4D-MRI techniques are faced with several challenges: 1) suboptimal and inconsistent tumor contrast with large inter-patient variation; 2) relatively low temporal-spatial resolution; 3) it lacks a reliable respiratory surrogate. In this research work, novel 4D-MRI techniques applying MRI weightings that was not used in existing 4D-MRI techniques, including T2/T1-weighted, T2-weighted and Diffusion-weighted MRI were investigated. A result-driven phase retrospective sorting method was proposed, and it was applied to image space as well as k-space of MR imaging. Novel image-based respiratory surrogates were developed, improved and evaluated.

CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.