Fluorescence-enhanced optical imaging on three-dimensional phantoms using a hand-held probe based frequency-domain intensified charge coupled device (ICCD) optical imager

Fluorescence-enhanced optical imaging is an emerging non-invasive and non-ionizing modality towards breast cancer diagnosis. Various optical imaging systems are currently available, although most of them are limited by bulky instrumentation, or their inability to flexibly image different tissue volumes and shapes. Hand-held based optical imaging systems are a recent development for its improved portability, but are currently limited only to surface mapping. Herein, a novel optical imager, consisting primarily of a hand-held probe and a gain-modulated intensified charge coupled device (ICCD) detector, is developed towards both surface and tomographic breast imaging. The unique features of this hand-held probe based optical imager are its ability to; (i) image large tissue areas (5×10 sq. cm) in a single scan, (ii) reduce overall imaging time using a unique measurement geometry, and (iii) perform tomographic imaging for tumor three-dimensional (3-D) localization. Frequency-domain based experimental phantom studies have been performed on slab geometries (650 ml) under different target depths (1-2.5 cm), target volumes (0.45, 0.23 and 0.10 cc), fluorescence absorption contrast ratios (1:0, 1000:1 to 5:1), and number of targets (up to 3), using Indocyanine Green (ICG) as fluorescence contrast agents. An approximate extended Kalman filter based inverse algorithm has been adapted towards 3-D tomographic reconstructions. Single fluorescence target(s) was reconstructed when located: (i) up to 2.5 cm deep (at 1:0 contrast ratio) and 1.5 cm deep (up to 10:1 contrast ratio) for 0.45 cc-target; and (ii) 1.5 cm deep for target as small as 0.10 cc at 1:0 contrast ratio. In the case of multiple targets, two targets as close as 0.7 cm were tomographically resolved when located 1.5 cm deep. It was observed that performing multi-projection (here dual) based tomographic imaging using a priori target information from surface images, improved the target depth recovery over using single projection based imaging. From a total of 98 experimental phantom studies, the sensitivity and specificity of the imager was estimated as 81-86% and 43-50%, respectively. With 3-D tomographic imaging successfully demonstrated for the first time using a hand-held based optical imager, the clinical translation of this technology is promising upon further experimental validation from in-vitro and in-vivo studies.

Motion correction algorithm of lung tumors for respiratory gated PET images

Respiratory gating in lung PET imaging to compensate for respiratory motion artifacts is a current research issue with broad potential impact on quantitation, diagnosis and clinical management of lung tumors. However, PET images collected at discrete bins can be significantly affected by noise as there are lower activity counts in each gated bin unless the total PET acquisition time is prolonged, so that gating methods should be combined with imaging-based motion correction and registration methods. The aim of this study was to develop and validate a fast and practical solution to the problem of respiratory motion for the detection and accurate quantitation of lung tumors in PET images. This included: (1) developing a computer-assisted algorithm for PET/CT images that automatically segments lung regions in CT images, identifies and localizes lung tumors of PET images; (2) developing and comparing different registration algorithms which processes all the information within the entire respiratory cycle and integrate all the tumor in different gated bins into a single reference bin. Four registration/integration algorithms: Centroid Based, Intensity Based, Rigid Body and Optical Flow registration were compared as well as two registration schemes: Direct Scheme and Successive Scheme. Validation was demonstrated by conducting experiments with the computerized 4D NCAT phantom and with a dynamic lung-chest phantom imaged using a GE PET/CT System. Iterations were conducted on different size simulated tumors and different noise levels. Static tumors without respiratory motion were used as gold standard; quantitative results were compared with respect to tumor activity concentration, cross-correlation coefficient, relative noise level and computation time. Comparing the results of the tumors before and after correction, the tumor activity values and tumor volumes were closer to the static tumors (gold standard). Higher correlation values and lower noise were also achieved after applying the correction algorithms. With this method the compromise between short PET scan time and reduced image noise can be achieved, while quantification and clinical analysis become fast and precise.

Clinical translation of a hand-held optical imager for breast cancer diagnostics: In vitro and in vivo tomography studies

Optical imaging is an emerging technology towards non-invasive breast cancer diagnostics. In recent years, portable and patient comfortable hand-held optical imagers are developed towards two-dimensional (2D) tumor detections. However, these imagers are not capable of three-dimensional (3D) tomography because they cannot register the positional information of the hand-held probe onto the imaged tissue. A hand-held optical imager has been developed in our Optical Imaging Laboratory with 3D tomography capabilities, as demonstrated from tissue phantom studies. The overall goal of my dissertation is towards the translation of our imager to the clinical setting for 3D tomographic imaging in human breast tissues. A systematic experimental approach was designed and executed as follows: (i) fast 2D imaging, (ii) coregistered imaging, and (iii) 3D tomographic imaging studies. (i) Fast 2D imaging was initially demonstrated in tissue phantoms (1% Liposyn solution) and in vitro (minced chicken breast and 1% Liposyn). A 0.45 cm3 fluorescent target at 1:0 contrast ratio was detectable up to 2.5 cm deep. Fast 2D imaging experiments performed in vivo with healthy female subjects also detected a 0.45 cm3 fluorescent target superficially placed ∼2.5 cm under the breast tissue. (ii) Coregistered imaging was automated and validated in phantoms with ∼0.19 cm error in the probe’s positional information. Coregistration also improved the target depth detection to 3.5 cm, from multi-location imaging approach. Coregistered imaging was further validated in-vivo , although the error in probe’s positional information increased to ∼0.9 cm (subject to soft tissue deformation and movement). (iii) Three-dimensional tomography studies were successfully demonstrated in vitro using 0.45 cm3 fluorescence targets. The feasibility of 3D tomography was demonstrated for the first time in breast tissues using the hand-held optical imager, wherein a 0.45 cm3 fluorescent target (superficially placed) was recovered along with artifacts. Diffuse optical imaging studies were performed in two breast cancer patients with invasive ductal carcinoma. The images showed greater absorption at the tumor cites (as observed from x-ray mammography, ultrasound, and/or MRI). In summary, my dissertation demonstrated the potential of a hand-held optical imager towards 2D breast tumor detection and 3D breast tomography, holding a promise for extensive clinical translational efforts.

Motion Correction Algorithm of Lung Tumors for Respiratory Gated PET Images

Respiratory gating in lung PET imaging to compensate for respiratory motion artifacts is a current research issue with broad potential impact on quantitation, diagnosis and clinical management of lung tumors. However, PET images collected at discrete bins can be significantly affected by noise as there are lower activity counts in each gated bin unless the total PET acquisition time is prolonged, so that gating methods should be combined with imaging-based motion correction and registration methods. The aim of this study was to develop and validate a fast and practical solution to the problem of respiratory motion for the detection and accurate quantitation of lung tumors in PET images. This included: (1) developing a computer-assisted algorithm for PET/CT images that automatically segments lung regions in CT images, identifies and localizes lung tumors of PET images; (2) developing and comparing different registration algorithms which processes all the information within the entire respiratory cycle and integrate all the tumor in different gated bins into a single reference bin. Four registration/integration algorithms: Centroid Based, Intensity Based, Rigid Body and Optical Flow registration were compared as well as two registration schemes: Direct Scheme and Successive Scheme. Validation was demonstrated by conducting experiments with the computerized 4D NCAT phantom and with a dynamic lung-chest phantom imaged using a GE PET/CT System. Iterations were conducted on different size simulated tumors and different noise levels. Static tumors without respiratory motion were used as gold standard; quantitative results were compared with respect to tumor activity concentration, cross-correlation coefficient, relative noise level and computation time. Comparing the results of the tumors before and after correction, the tumor activity values and tumor volumes were closer to the static tumors (gold standard). Higher correlation values and lower noise were also achieved after applying the correction algorithms. With this method the compromise between short PET scan time and reduced image noise can be achieved, while quantification and clinical analysis become fast and precise.

On-screen pre-deblurring of digital images using the wavefront aberration function of the human eye to improve computer access for the visually impaired

Traditional Optics has provided ways to compensate some common visual limitations (up to second order visual impairments) through spectacles or contact lenses. Recent developments in wavefront science make it possible to obtain an accurate model of the Point Spread Function (PSF) of the human eye. Through what is known as the "Wavefront Aberration Function" of the human eye, exact knowledge of the optical aberration of the human eye is possible, allowing a mathematical model of the PSF to be obtained. This model could be used to pre-compensate (inverse-filter) the images displayed on computer screens in order to counter the distortion in the user's eye. This project takes advantage of the fact that the wavefront aberration function, commonly expressed as a Zernike polynomial, can be generated from the ophthalmic prescription used to fit spectacles to a person. This allows the pre-compensation, or onscreen deblurring, to be done for various visual impairments, up to second order (commonly known as myopia, hyperopia, or astigmatism). The technique proposed towards that goal and results obtained using a lens, for which the PSF is known, that is introduced into the visual path of subjects without visual impairment will be presented. In addition to substituting the effect of spectacles or contact lenses in correcting the loworder visual limitations of the viewer, the significance of this approach is that it has the potential to address higher-order abnormalities in the eye, currently not correctable by simple means.

Systematic Monitoring of Wound Healing Using a Hand-held Near-Infrared Optical Scanner

According to the American Podiatric Medical Association, about 15 percent of the patients with diabetes would develop a diabetic foot ulcer. Furthermore, foot ulcerations leads to 85 percent of the diabetes-related amputations. Foot ulcers are caused due to a combination of factors, such as lack of feeling in the foot, poor circulation, foot deformities and the duration of the diabetes. To date, the wounds are inspected visually to monitor the wound healing, without any objective imaging approach to look before the wound’s surface. Herein, a non-contact, portable handheld optical device was developed at the Optical Imaging Laboratory as an objective approach to monitor wound healing in foot ulcer. This near-infrared optical technology is non-radiative, safe and fast in imaging large wounds on patients. The FIU IRB-approved study will involve subjects that have been diagnosed with diabetes by a physician and who have developed foot ulcers. Currently, in-vivo imaging studies are carried out every week on diabetic patients with foot ulcers at two clinical sites in Miami. Near-infrared images of the wound are captured on subjects every week and the data is processed using customdeveloped Matlab-based image processing tools. The optical contrast of the wound to its peripheries and the wound size are analyzed and compared from the NIR and white light images during the weekly systematic imaging of wound healing.

Sea-floor images during POLARSTERN cruise ANT-XIII/3 to the Weddell Sea, Antarctica

Transects of a Remotely Operated Vehicle (ROV) providing sea-bed videos and photographs were carried out during POLARSTERN expedition ANT-XIII/3 focussing on the ecology of benthic assemblages on the Antarctic shelf in the South-Eastern Weddell Sea. The ROV-system sprint 103 was equiped with two video- and one still camera, lights, flash-lights, compass, and parallel lasers providing a scale in the images, a tether-management system (TMS), a winch, and the board units. All cameras used the same main lense and could be tilted. Videos were recorded in Betacam-format and (film-)slides were made by decision of the scientific pilot. The latter were mainly made under the aspect to improve the identification of organisms depicted in the videos because the still photographs have a much higher optical resolution than the videos. In the photographs species larger than 3 mm, in the videos larger than 1 cm are recognisable and countable. Under optimum conditions the transects were strait; the speed and direction of the ROV were determined by the drift of the ship in the coastal current, since both, the ship and the ROV were used as a drifting system; the option to operate the vehicle actively was only used to avoide obstacles and to reach at best a distance of only approximately 30 cm to the sea-floor. As a consequence the width of the photographs in the foreground is approximately 50 cm. Deviations from this strategy resulted mainly from difficult ice- and weather conditions but also from high current velocity and local up-welling close to the sea-bed. The sea-bed images provide insights into the general composition of key species, higher systematic groups and ecological guilds. Within interdisciplinary approaches distributions of assemblages can be attributed to environmental conditions such as bathymetry, sediment characteristics, water masses and current regimes. The images also contain valuable information on how benthic species are associated to each other. Along the transects, small- to intermediate-scaled disturbances, e.g. by grounding icebergs were analysed and further impact to the entire benthic system by local succession of recolonisation was studied. This information can be used for models predicting the impact of climate change to benthic life in the Southern Ocean. All these approaches contribute to a better understanding of the fiunctioning of the benthic system and related components of the entire Antarctic marine ecosystem. Despite their scientific value the imaging methods meet concerns about the protection of sensitive Antarctic benthic systems since they are non-invasive and they also provide valuable material for education and outreach purposes.

Sea-floor images from ROV transects during POLARSTERN cruise ANT-XVII/3 (EASIZ III) to the Weddell Sea, Antarctica

Transects of a Remotely Operated Vehicle (ROV) providing sea-bed videos and photographs were carried out during POLARSTERN expedition ANT-XVII/3 focussing on the ecology of benthic assemblages on the Antarctic shelf in the South-Eastern Weddell Sea. The ROV-system sprint 103 was equiped with two video- and one still camera, lights, flash-lights, compass, and parallel lasers providing a scale in the images, a tether-management system (TMS), a winch, and the board units. All cameras used the same main lense and could be tilted. Videos were recorded in Betacam-format and (film-)slides were made by decision of the scientific pilot. The latter were mainly made under the aspect to improve the identification of organisms depicted in the videos because the still photographs have a much higher optical resolution than the videos. In the photographs species larger than 3 mm, in the videos larger than 1 cm are recognisable and countable. Under optimum conditions the transects were strait; the speed and direction of the ROV were determined by the drift of the ship in the coastal current, since both, the ship and the ROV were used as a drifting system; the option to operate the vehicle actively was only used to avoide obstacles and to reach at best a distance of only approximately 30 cm to the sea-floor. As a consequence the width of the photographs in the foreground is approximately 50 cm. Deviations from this strategy resulted mainly from difficult ice- and weather conditions but also from high current velocity and local up-welling close to the sea-bed. The sea-bed images provide insights into the general composition of key species, higher systematic groups and ecological guilds. Within interdisciplinary approaches distributions of assemblages can be attributed to environmental conditions such as bathymetry, sediment characteristics, water masses and current regimes. The images also contain valuable information on how benthic species are associated to each other. Along the transects, small- to intermediate-scaled disturbances, e.g. by grounding icebergs were analysed and further impact to the entire benthic system by local succession of recolonisation was studied. This information can be used for models predicting the impact of climate change to benthic life in the Southern Ocean. All these approaches contribute to a better understanding of the fiunctioning of the benthic system and related components of the entire Antarctic marine ecosystem. Despite their scientific value the imaging methods meet concerns about the protection of sensitive Antarctic benthic systems since they are non-invasive and they also provide valuable material for education and outreach purposes.

Enhancing the Visualization of the Peripheral Retina with Wide Field-of-View Optical Coherence Tomography

The goal of my Ph.D. thesis is to enhance the visualization of the peripheral retina using wide-field optical coherence tomography (OCT) in a clinical setting.

OCT has gain widespread adoption in clinical ophthalmology due to its ability to visualize the diseases of the macula and central retina in three-dimensions, however, clinical OCT has a limited field-of-view of 300. There has been increasing interest to obtain high-resolution images outside of this narrow field-of-view, because three-dimensional imaging of the peripheral retina may prove to be important in the early detection of neurodegenerative diseases, such as Alzheimer's and dementia, and the monitoring of known ocular diseases, such as diabetic retinopathy, retinal vein occlusions, and choroid masses.

Before attempting to build a wide-field OCT system, we need to better understand the peripheral optics of the human eye. Shack-Hartmann wavefront sensors are commonly used tools for measuring the optical imperfections of the eye, but their acquisition speed is limited by their underlying camera hardware. The first aim of my thesis research is to create a fast method of ocular wavefront sensing such that we can measure the wavefront aberrations at numerous points across a wide visual field. In order to address aim one, we will develop a sparse Zernike reconstruction technique (SPARZER) that will enable Shack-Hartmann wavefront sensors to use as little as 1/10th of the data that would normally be required for an accurate wavefront reading. If less data needs to be acquired, then we can increase the speed at which wavefronts can be recorded.

For my second aim, we will create a sophisticated optical model that reproduces the measured aberrations of the human eye. If we know how the average eye's optics distort light, then we can engineer ophthalmic imaging systems that preemptively cancel inherent ocular aberrations. This invention will help the retinal imaging community to design systems that are capable of acquiring high resolution images across a wide visual field. The proposed model eye is also of interest to the field of vision science as it aids in the study of how anatomy affects visual performance in the peripheral retina.

Using the optical model from aim two, we will design and reduce to practice a clinical OCT system that is capable of imaging a large (800) field-of-view with enhanced visualization of the peripheral retina. A key aspect of this third and final aim is to make the imaging system compatible with standard clinical practices. To this end, we will incorporate sensorless adaptive optics in order to correct the inter- and intra- patient variability in ophthalmic aberrations. Sensorless adaptive optics will improve both the brightness (signal) and clarity (resolution) of features in the peripheral retina without affecting the size of the imaging system.

The proposed work should not only be a noteworthy contribution to the ophthalmic and engineering communities, but it should strengthen our existing collaborations with the Duke Eye Center by advancing their capability to diagnose pathologies of the peripheral retinal.

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.

Optical flow using color information: preliminary results

AIRES, Kelson R. T.; SANTANA, André M.; MEDEIROS, Adelardo A. D. Optical flow using color information: preliminary results. In: ANNUAL ACM SYMPOSIUM ON APPLIED COMPUTING, 23., 2008, Fortaleza. Proceedings... Fortaleza: SAC, 2008.

Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Background: Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology: We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions: This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.

Optical flow using color information: preliminary results

AIRES, Kelson R. T.; SANTANA, André M.; MEDEIROS, Adelardo A. D. Optical flow using color information: preliminary results. In: ANNUAL ACM SYMPOSIUM ON APPLIED COMPUTING, 23., 2008, Fortaleza. Proceedings... Fortaleza: SAC, 2008.

Topographic optical profilometry of steep slope micro-optical transparent surfaces

Optical profilometers based on light reflection may fail at surfaces presenting steep slopes and highly curved features. Missed light, interference and diffraction at steps, peaks and valleys are some of the reasons. Consequently, blind areas or profile artifacts may be observed when using common reflection micro-optical profilometers (confocal, scanning interferometers, etc…). The Topographic Optical Profilometry by Absorption in Fluids (TOPAF) essentially avoids these limitations. In this technique an absorbing fluid fills the gap between a reference surface and the surface to profile. By comparing transmission images at two different spectral bands we obtain a reliable topographic map of the surface. In this contribution we develop a model to obtain the profile under micro-optical observation, where high numerical aperture (NA) objectives are mandatory. We present several analytical and experimental results, validating the technique’s capabilities for profiling steep slopes and highly curved micro-optical surfaces with nanometric height resolution.