Photoacoustic Imaging of the Eye

Photoacoustic imaging (PAI) is a novel, hybrid, non-ionizing, and non-invasive imaging technology with high-resolution, high sensitivity, high-contrast, and high depth of penetration. Hence, it has particularly useful applications in eye investigations. It can provide both anatomic and functional ocular characterizations. Many eye diseases, including macular degeneration and diabetic retinopathy, involve abnormalities in the vasculature, and thus the ability of PAI to affectively visualize the vasculature can be incredibly helpful to evaluate normal and disease states of the eye. In future research, PAI of the eye can be dramatically improved in terms of its resolution, use of contrast agents for molecular imaging, safety evaluations to develop a clinically approved system, and integration with existing fundus imaging modalities. Multimodality ocular imaging platforms have also been successfully developed by a combination of photoacoustic microscopy (PAM) with other optical imaging such as optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), and fluorescence microscopy (FM). The multimodal images can accurately be acquired from a single imaging system and co-registered on the same image plane, enabling improved evaluation of eye disease states. In this book chapter, the potential application of photoacoustic imaging of the eye in both research and clinical diagnosis are comprehensively discussed as a powerful medical screening technique for visualization of various ocular diseases

Optical Coherence Photoacoustic Microscopy (OC-PAM) for Multimodal Imaging

Optical coherence tomography (OCT) and Photoacoustic microscopy (PAM) are two noninvasive, high-resolution, three-dimensional, biomedical imaging modalities based on different contrast mechanisms. OCT detects the light backscattered from a biological sample either in the time or spectral domain using an interferometer to form an image. PAM is sensitive to optical absorption by detecting the light-induced acoustic waves to form an image. Due to their complementary contrast mechanisms, OCT and PAM are suitable for being combined to achieve multimodal imaging. In this dissertation, an optical coherence photoacoustic microscopy (OC-PAM) system was developed for in vivo multimodal retinal imaging with a pulsed broadband NIR light source. To test the capabilities of the system on multimodal ophthalmic imaging, the retina of pigmented rats was imaged. The OCT images showed the retinal structures with quality similar to conventional OCT, while the PAM images revealed the distribution of melanin in the retina since the NIR PAM signals are generated mainly from melanin in the posterior segment of the eye. By using the pulsed broadband light source, the OCT image quality highly depends on the pulse-to-pulse stability of the light source without averaging. In addition, laser safety is always a concern for in vivo applications, especially for eye imaging with a pulsed light source. Therefore, a continuous wave (CW) light source is desired for OC-PAM applications. An OC-PAM system using an intensity-modulated CW superluminescent diode was then developed. The system was tested for multimodal imaging the vasculature of a mouse ear in vivo by using Gold Nanorods (GNRs) as contrast agent for PAM, as well as excised porcine eyes ex vivo. Since the quantitative information of the optical properties extracted from the proposed NIR OC-PAM system is potentially able to provide a unique technique to evaluate the existence of melanin and lipofuscin specifically, a phantom study has been conducted and the relationship between image intensity of OCT and PAM was interpreted to represent the relationship between the optical scattering property and optical absorption property. It will be strong evidence for practical application of the proposed NIR OC-PAM system

Penta-Modal Imaging Platform with OCT- Guided Dynamic Focusing for Simultaneous Multimodal Imaging

Complex diseases, such as Alzheimer’s disease, are associated with sequences of changes in multiple disease-specific biomarkers. These biomarkers may show dynamic changes at specific stages of disease progression. Thus, testing/monitoring each biomarker may provide insight into specific disease-related processes, which can result in early diagnosis or even development of preventive measures. Obtaining a comprehensive information of biological tissues requires imaging of multiple optical contrasts, which is not typically offered by a single imaging modality. Thus, combining different contrast mechanisms to achieve simultaneous multimodal imaging is desirable. However, this process is highly challenging due to specific optical and hardware requirements for each optical imaging system. The objective of this dissertation is to develop a novel Penta-modal optical imaging system integrating photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT), OCT angiography (OCTA) and confocal fluorescence microscopy (CFM) in one platform providing comprehensive structural, functional, and molecular information of living biological tissues. The system can simultaneously image different biomarkers with a large field-of-view (FOV) and high-speed imaging. The large FOV and the high imaging speed is achieved by combining optical and mechanical scanning mechanisms. To compensate for an uneven surface of biological samples, which result in images with non-uniform resolution and low signal to noise ratio (SNR), we further develop a novel OCT-guided surface contour scanning methodology, a technique for adjusting objective lens focus to follow the contour of the sample surface, to provide a uniform spatial resolution and SNR across the region of interest (ROI). The imaging system was tested by imaging phantoms, ex vivo biological samples, and in vivo. The OCT-guided surface contour scanning methodology was utilized for imaging a leaf of purple queen plant, which resulted in a significant contrast improvement of 41% and 38% across a large imaging area for CFM and PAM, respectively. The nuclei and cells walls were also clearly observed in both images. In an in vivo imaging of the Swiss Webster mouse ear, our multimodal imaging system was able to provide images with uniform resolution in an FOV of 10 mm x 10 mm with an imaging time of around 5 minutes. In addition to measuring the blood flow in the mouse ear, the system also successfully imaged mouse ear blood vessels, sebaceous glands, as well as several tissue structures. We further conducted a comparative study of OCTA for rodent retinal imaging by evaluating the performance of three OCTA algorithms, namely the phase variance (PV), improved speckle contrast (ISC), and optical microangiography (OMAG). It was concluded that the OMAG algorithm provided statistically significant higher mean values of BVD and VPI compared to the ISC algorithm (0.27±0.07 vs. 0.24±0.05 for BVD; 0.09±0.04 and 0.08±0.04 for VPI), while no statistically significant difference was observed for VDI and VCI among the algorithms. Results showed that both the ISC and OMAG algorithms are more robust than PV, and they can reveal similar vasculature features. Lastly, we utilized the proposed imaging system to monitor, for the first time, the invasion process of malaria parasites in the mosquito midgut. The system shows a promising potential to detect parasite motion as well as structural changes inside the mosquito midgut. The multimodal imaging system outlined in this dissertation can be useful in a variety of applications thanks to the specific optical contrast offered by each employed modality, including retinal and brain imaging

Comprehensive in vivo micro-vascular imaging of the human eye by dual-beam-scan Doppler optical coherence angiography

Comprehensive angiography provides insight into the diagnosis of vascular-related diseases. However, complex microvascular networks of unstable in vivo organs such as the eye require micron-scale resolution in three dimensions and a high sampling rate to access a wide area as maintaining the high resolution. Here, we introduce dual-beam-scan Doppler optical coherence angiography (OCA) as a label-free comprehensive ophthalmic angiography that satisfies theses requirements. In addition to high resolution and high imaging speed, high sensitivity to motion for detecting tiny blood flow of microvessels is achieved by detecting two time-delayed signals with scanning of two probing beams separated on a sample. We present in vivo three-dimensional imaging of the microvasculature of the posterior part of the human eye. The demonstrated results show that this technique may be used for comprehensive ophthalmic angiography to evaluate the vasculature of the posterior human eye and to diagnose variety of vascular diseases.This paper was published in Optics Express and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/abstract.cfm?uri=oe-19-2-1271. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law

Multimodal photoacoustic remote sensing (PARS) microscopy combined with swept-source optical coherence tomography (SS-OCT) for in-vivo, non-contact, functional and structural ophthalmic imaging applications

Ophthalmic imaging has long played an important role in the understanding, diagnosis, and treatment of a wide variety of ocular disorders. Currently, available clinical ophthalmic imaging instruments are primarily optical-based, including slit-lamp microscopy, fundus photography, confocal microscopy, scanning laser ophthalmoscopy, and optical coherence tomography (OCT). The development of these imaging instruments has greatly extended our ability to evaluate the ocular environment. Studies have shown that at least 40% of blinding disorders in the United States are either preventable or treatable with timely diagnosis and intervention. OCT is a state-of-the-art imaging technique extensively used in preclinical and clinical applications for imaging both anterior and posterior parts of the eye. OCT has become a standard of care for the assessment and treatment of most ocular conditions. The technology enables non-contact, high-speed, cross-sectional imaging over a large field of view with submicron resolutions. In eye imaging applications, functional extensions of OCT such as spectroscopic OCT and Doppler OCT have been applied to provide a better understanding of tissue activity. Spectroscopic OCT is usually achieved through OCT systems in the visible spectral range, and it enables the amount of light absorption inside the ocular environment to be measured. This indirect optical absorption measurement is used to estimate the amount of ocular oxygen saturation (SO2) which is a well-known biomarker in prevalent eye diseases including diabetic retinopathy, glaucoma, and retinal vein occlusions. Despite all the advancements in functional spectroscopic OCT methods, they still rely primarily on measuring the backscattered photons to quantify the absorption of chromophores inside the tissue. Therefore, they are sensitive to local geometrical parameters, such as retinal thickness, vessel diameters, and retinal pigmentation, and may result in biased estimations. Of the various optical imaging modalities, photoacoustic imaging (PAI) offers unique imaging contrast of optical absorption because PAI can image any target that absorbs light energy. This unique imaging ability makes PAI a favorable candidate for various functional and molecular imaging applications as well as for measuring chromophore concentration. Over the past decade, photoacoustic ophthalmoscopy has been applied for visualizing hemoglobin and melanin content in ocular tissue, quantifying ocular SO2, and measuring the metabolic rate of oxygen consumption (MRO2). Despite all these advantages offered by PAI devices, a major limitation arises from their need to be in contact with the ocular tissues. This physical contact may increase the risk of infection and cause patient discomfort. Furthermore, this contact-based imaging approach applies pressure to the eye and introduces barriers to oxygen diffusion. Thus, it has a crucial influence on the physiological and pathophysiological balance of ocular vasculature function, and it is not capable of studying dynamic processes under normal conditions. To overcome these limitations and to benefit from the numerous advantages offered by photoacoustic ophthalmoscopy, non-contact detection of photoacoustic signals has been a long-lasting goal in the field of ocular imaging. In 2017 Haji Reza et al. developed photoacoustic remote sensing (PARS) for non-contact, non-interferometric detection of photoacoustic signals. PARS is the non-contact, all-optical version of optical-resolution photoacoustic microscopy (OR-PAM), where the acoustically coupled ultrasound transducer is replaced with a co-focused probe beam. This all-optical detection scheme allows the system to measure the photoacoustic pressure waves at the subsurface origin where the pressure is at a maximum. In a very short time, PARS technology has proven its potential for various biomedical applications, including label-free histological imaging, SO2 mapping, and angiogenesis imaging. PARS is an ideal companion for OCT in ophthalmic applications, where the depth-resolved, detailed scattering information of OCT is well complemented by rich absorption information of PARS. This combined multimodal imaging technology has the potential to provide chromophore selective absorption contrast in concert with depth-resolved scattering contrast in the ocular environment. The main goals of this PhD project are to: • Develop a photoacoustic remote sensing microscopy system for in-vivo, non-contact ophthalmic imaging. This is the first time a non-contact photoacoustic imaging has been used for in-vivo imaging of the eye. • Develop a robust and temporally stable multiwavelength light source for functional photoacoustic imaging applications. • Develop a multimodal PARS-OCT imaging system that can image in-vivo and record, simultaneously, functional, and structural information in the anterior segment of a rodent eye. This is the first time a multiwavelength non-contact photoacoustic system is used for in-vivo measurement of oxygen saturation in the ocular environment. • Develop and modify the multimodal PARS-OCT imaging system for non-contact, in-vivo, functional, and structural imaging of the posterior part of the rodent eye

Imaging Sensors and Applications

In past decades, various sensor technologies have been used in all areas of our lives, thus improving our quality of life. In particular, imaging sensors have been widely applied in the development of various imaging approaches such as optical imaging, ultrasound imaging, X-ray imaging, and nuclear imaging, and contributed to achieve high sensitivity, miniaturization, and real-time imaging. These advanced image sensing technologies play an important role not only in the medical field but also in the industrial field. This Special Issue covers broad topics on imaging sensors and applications. The scope range of imaging sensors can be extended to novel imaging sensors and diverse imaging systems, including hardware and software advancements. Additionally, biomedical and nondestructive sensing applications are welcome

Optical imaging in vivo with a focus on paediatric disease: technical progress, current preclinical and clinical applications and future perspectives

To obtain information on the occurrence and location of molecular events as well as to track target-specific probes such as antibodies or peptides, drugs or even cells non-invasively over time, optical imaging (OI) technologies are increasingly applied. Although OI strongly contributes to the advances made in preclinical research, it is so far, with the exception of optical coherence tomography (OCT), only very sparingly applied in clinical settings. Nevertheless, as OI technologies evolve and improve continuously and represent relatively inexpensive and harmful methods, their implementation as clinical tools for the assessment of children disease is increasing. This review focuses on the current preclinical and clinical applications as well as on the future potential of OI in the clinical routine. Herein, we summarize the development of different fluorescence and bioluminescence imaging techniques for microscopic and macroscopic visualization of microstructures and biological processes. In addition, we discuss advantages and limitations of optical probes with distinct mechanisms of target-detection as well as of different bioluminescent reporter systems. Particular attention has been given to the use of near-infrared (NIR) fluorescent probes enabling observation of molecular events in deeper tissue