Detecting, segmenting and tracking bio-medical objects

Studying the behavior patterns of biomedical objects helps scientists understand the underlying mechanisms. With computer vision techniques, automated monitoring can be implemented for efficient and effective analysis in biomedical studies. Promising applications have been carried out in various research topics, including insect group monitoring, malignant cell detection and segmentation, human organ segmentation and nano-particle tracking. In general, applications of computer vision techniques in monitoring biomedical objects include the following stages: detection, segmentation and tracking. Challenges in each stage will potentially lead to unsatisfactory results of automated monitoring. These challenges include different foreground-background contrast, fast motion blur, clutter, object overlap and etc. In this thesis, we investigate the challenges in each stage, and we propose novel solutions with computer vision methods to overcome these challenges and help automatically monitor biomedical objects with high accuracy in different cases --Abstract, page iii

Locating Particles Accurately in Microscope Images Requires Image-Processing Kernels to be Rotationally Symmetric

Computerized image-analysis routines deployed widely to locate and track the positions of particles in microscope images include several steps where images are convolved with kernels to remove noise. In many common implementations, some kernels are rotationally asymmetric. Here we show that the use of these asymmetric kernels creates significant artifacts, distorting apparent particle positions in a way that gives the artificial appearance of orientational crystalline order, even in such fully-disordered isotropic systems as simple fluids of hard-sphere-like colloids. We rectify this problem by replacing all asymmetric kernels with rotationally-symmetric kernels, which does not impact code performance. We show that these corrected codes locate particle positions properly, restoring measured isotropy to colloidal fluids. We also investigate rapidly-formed colloidal sediments, and with the corrected codes show that these sediments, often thought to be amorphous, may exhibit strong orientational correlations among bonds between neighboring colloidal particles.Engineering and Applied Science

Electromechanical Lifting Actuation of a MEMS Cantilever and Nano-Scale Analysis of Diffusion in Semiconductor Device Dielectrics

This dissertation presents experimental and theoretical studies of physical phenomena in micro- and nano-electronic devices. Firstly, a novel and unproven means of electromechanical actuation in a micro-electro-mechanical system (MEMS) cantilever was investigated. In nearly all MEMS devices, electric forces cause suspended components to move toward the substrate. I demonstrated a design with the unusual and potentially very useful property of having a suspended MEMS cantilever lift away from the substrate. The effect was observed by optical micro-videography, by electrical sensing, and it was quantified by optical interferometry. The results agree with predictions of analytic and numerical calculations. One potential application is infrared sensing in which absorbed radiation changes the temperature of the cantilever, changing the duty cycle of an electrically-driven, repetitively closing micro-relay. Secondly, ultra-thin high-k gate dielectric layers in two 22 nm technology node semiconductor devices were studied. The purpose of the investigation was to characterize the morphology and composition of these layers as a means to verify whether the transmission electron microscope (TEM) with energy dispersive spectroscopy (EDS) could sufficiently resolve the atomic diffusion at such small length scales. Results of analytic and Monte-Carlo numerical calculations were compared to empirical data to validate the ongoing viability of TEM EDS as a tool for nanoscale characterization of semiconductor devices in an era where transistor dimensions will soon be less than 10 nm

Laser-assisted single-molecule refolding

Non-coding RNAs must fold into precise secondary and tertiary structures in order to perform the biological functions. Due to the flexibility of RNA, the RNA folding energy landscape can be rugged and full of local minimum (kinetic trap). To provide a means to study kinetically trapped RNAs, we have developed a new technique combining single-molecule FRET detection with laser induced temperature jump. We have calibrated the magnitude of the temperature jump with 1˚C accuracy using gold micro-size sensor. The accuracy of temperature calibration was confirmed by close agreement between single-molecule and bulk DNA duplex melting experiments. HIV 1 DIS RNAs form a kissing complex in the loop region and proceed to the extended duplex structure with the help of enzymes or other cofactors in the later stage of viral replication. The kissing complex itself is very stable, which makes it a unique optimal model system to study kinetically trapped RNAs. The application of LASR to kissing hairpins has allowed us, for the first time, to drive a molecular reaction and monitor the process at the single molecule level. The melting curve for the dissociation and dimerization was used to estimate the thermodynamic properties of the reaction, such as melting temperature, cooperativity, and the enthalpy change. Mutational studies have allowed dissection of the contribution of base pairs to kissing complex stability. And the ratio of the competing reaction pathways was determined. LASR experiments designed to the study of the origin of the memory effect in the hairpin ribozyme folding introduced inter-conversions between the subpopulations of hairpin ribozyme with distinct undocking kinetic rates. This provides strong evidence for the hypothesis that the memory effect is an intrinsic property of hairpin ribozyme folding. Eyring analysis was adopted to fit the enthalpic and entropic height of the inter-conversion barrier. Our results suggest that for inter-conversion to occur, surprisingly, a small number of interactions are broken. However inter-conversion is rare due to a large negative entropic term. Negative entropy indicates a rigid transition state for inter-conversion and an increased free energy barrier height with increased temperature. This explains why there are few inter-conversions even at high temperatures such as 78 ˚C. The entropic barrier may primarily arise from the stretching of the S-turn

Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000 m off Hawaii

© International Society for Microbial Ecology, 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in The ISME Journal 5 (2011): 1748–1758, doi:10.1038/ismej.2011.48.A novel hydrothermal field has been discovered at the base of Lōihi Seamount, Hawaii, at 5000 mbsl. Geochemical analyses demonstrate that ‘FeMO Deep’, while only 0.2 °C above ambient seawater temperature, derives from a distal, ultra-diffuse hydrothermal source. FeMO Deep is expressed as regional seafloor seepage of gelatinous iron- and silica-rich deposits, pooling between and over basalt pillows, in places over a meter thick. The system is capped by mm to cm thick hydrothermally derived iron-oxyhydroxide- and manganese-oxide-layered crusts. We use molecular analyses (16S rDNA-based) of extant communities combined with fluorescent in situ hybridizations to demonstrate that FeMO Deep deposits contain living iron-oxidizing Zetaproteobacteria related to the recently isolated strain Mariprofundus ferroxydans. Bioenergetic calculations, based on in-situ electrochemical measurements and cell counts, indicate that reactions between iron and oxygen are important in supporting chemosynthesis in the mats, which we infer forms a trophic base of the mat ecosystem. We suggest that the biogenic FeMO Deep hydrothermal deposit represents a modern analog for one class of geological iron deposits known as ‘umbers’ (for example, Troodos ophilolites, Cyprus) because of striking similarities in size, setting and internal structures.Funding has been provided by the NSF Microbial Observatories Program (KJE, DE, BT, HS and CM), by the Gordon and Betty Moore Foundation (KJE), the College of Letters, Arts, and Sciences at the University of Southern California (KJE) and by the NASA Astrobiology Institute (KJE, DE)

Magnetic Resonance Imaging of a Natural Killer Cell Therapy in a Mouse Model of Prostate Cancer

Purpose: This thesis uses magnetic resonance imaging (MRI) techniques to study natural killer (NK) cell therapy in a nude mouse model of prostate cancer. Cellular MRI, anatomical MRI and hyperpolarized 13C spectroscopy were used to study various aspects of the model. Methods: The cells used were KHYG-1 NK cells and PC-3M prostate cancer cells. Imaging was performed on a clinical 3T GE MR 750 scanner, using a high-performance gradient insert for acquisition with the balanced steady state free precession (bSSFP) sequence, and using the built-in gradients for 13C pyruvate spectroscopy. Pyruvate was hyperpolarized by dynamic nuclear polarization. Results: KHYG-1 were toxic to PC-3M in vitro and were successfully labeled with MoldayRhodamine, a superparamagnetic iron oxide nanoparticle (SPIO). A subcutaneous PC-3M tumour model was used to investigate tracking of KHYG-1 in vivo using the bSSFP sequence. Four days after administration, KHYG-1 accumulation in the tumours was detected by histology but not by MRI, although labeled KHYG-1 at high density were visible in MR images. The bSSFP sequence was then optimized for imaging the mouse prostate and the whole mouse body. Tumour development in an orthotopic prostate cancer model was characterized by MRI and histology for tumour growth, metastasis and tumour metabolism. Tumours were visible by MRI day 9 after injection. Using histology, metastasis was detected in the lymph nodes and spleen of the mice. Necrotic regions in the tumours were detected on day 22 by both anatomical imaging and pyruvate spectroscopy and were confirmed by histology. Conclusions: KHYG-1 cell therapy shows promise as a treatment of prostate cancer. A mouse model that developed lymph node metastases was characterized. Based on the accumulation of KHYG-1 in SC tumours 4 days after administration, and the consistent presence of MRI-visible tumours on day 9-13, a treatment time point of 9-13 days is proposed for future NK cell tracking experiments

Optical mapping and optogenetics in cardiac electrophysiology research and therapy:a state-of-the-art review

State-of-the-art innovations in optical cardiac electrophysiology are significantly enhancing cardiac research. A potential leap into patient care is now on the horizon. Optical mapping, using fluorescent probes and high-speed cameras, offers detailed insights into cardiac activity and arrhythmias by analysing electrical signals, calcium dynamics, and metabolism. Optogenetics utilizes light-sensitive ion channels and pumps to realize contactless, cell-selective cardiac actuation for modelling arrhythmia, restoring sinus rhythm, and probing complex cell–cell interactions. The merging of optogenetics and optical mapping techniques for ‘all-optical’ electrophysiology marks a significant step forward. This combination allows for the contactless actuation and sensing of cardiac electrophysiology, offering unprecedented spatial–temporal resolution and control. Recent studies have performed all-optical imaging ex vivo and achieved reliable optogenetic pacing in vivo, narrowing the gap for clinical use. Progress in optical electrophysiology continues at pace. Advances in motion tracking methods are removing the necessity of motion uncoupling, a key limitation of optical mapping. Innovations in optoelectronics, including miniaturized, biocompatible illumination and circuitry, are enabling the creation of implantable cardiac pacemakers and defibrillators with optoelectrical closed-loop systems. Computational modelling and machine learning are emerging as pivotal tools in enhancing optical techniques, offering new avenues for analysing complex data and optimizing therapeutic strategies. However, key challenges remain including opsin delivery, real-time data processing, longevity, and chronic effects of optoelectronic devices. This review provides a comprehensive overview of recent advances in optical mapping and optogenetics and outlines the promising future of optics in reshaping cardiac electrophysiology and therapeutic strategies

Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanu-cleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocy-toplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models