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

Improved imaging of magnetically labeled cells using rotational magnetomotive optical coherence tomography

In this paper, we present a reliable and robust method for magnetomotive optical coherence tomography (MM-OCT) imaging of single cells labeled with iron oxide particles. This method employs modulated longitudinal and transverse magnetic fields to evoke alignment and rotation of anisotropic magnetic structures in the sample volume. Experimental evidence suggests that magnetic particles assemble themselves in elongated chains when exposed to a permanent magnetic field. Magnetomotion in the intracellular space was detected and visualized by means of 3D OCT as well as laser speckle reflectometry as a 2D reference imaging method. Our experiments on mesenchymal stem cells embedded in agar scaffolds show that the magnetomotive signal in rotational MM-OCT is significantly increased by a factor of ˜3 compared to previous pulsed MM-OCT, although the solenoid's power consumption was 16 times lower. Finally, we use our novel method to image ARPE-19 cells, a human retinal pigment epithelium cell line. Our results permit magnetomotive imaging with higher sensitivity and the use of low power magnetic fields or larger working distances for future three-dimensional cell tracking in target tissues and organs

A Step-by-step Guide to the Realisation of Advanced Optical Tweezers

Since the pioneering work of Arthur Ashkin, optical tweezers have become an indispensable tool for contactless manipulation of micro- and nanoparticles. Nowadays optical tweezers are employed in a myriad of applications demonstrating the importance of these tools. While the basic principle of optical tweezers is the use of a strongly focused laser beam to trap and manipulate particles, ever more complex experimental set-ups are required in order to perform novel and challenging experiments. With this article, we provide a detailed step- by-step guide for the construction of advanced optical manipulation systems. First, we explain how to build a single-beam optical tweezers on a home-made microscope and how to calibrate it. Improving on this design, we realize a holographic optical tweezers, which can manipulate independently multiple particles and generate more sophisticated wavefronts such as Laguerre-Gaussian beams. Finally, we explain how to implement a speckle optical tweezers, which permit one to employ random speckle light fields for deterministic optical manipulation.Comment: 29 pages, 7 figure

DYNAMICS OF LINEAGE RESTRICTION FORMATION IN THE VERTEBRATE MIDBRAIN-HINDBRAIN BOUNDARY

Proper formation of lineage restrictions is important in developing animals to ensure healthy development. While mechanisms that form these restrictions are understood in invertebrates, it is not yet known what contributes to lineage restriction in vertebrates. We have used Ultrashort Pulse Microscopy to obtain 3-dimensional multimodal images of the developing midbrain-hindbrain boundary, a known lineage restriction in the zebrafish. By using this form of imaging, we have visualized both morphological and genetic parameters in this area and have used these to make quantitative and qualitative models of MHB morphogenesis and lineage restriction formation. These models allow us to have a better understanding of what exactly is responsible for the formation of the midbrain-hindbrain boundary and its lineage restriction in zebrafish with implications for all vertebrates, including humans

DYNAMICS OF LINEAGE RESTRICTION FORMATION IN THE VERTEBRATE MIDBRAIN-HINDBRAIN BOUNDARY

Proper formation of lineage restrictions is important in developing animals to ensure healthy development. While mechanisms that form these restrictions are understood in invertebrates, it is not yet known what contributes to lineage restriction in vertebrates. We have used Ultrashort Pulse Microscopy to obtain 3-dimensional multimodal images of the developing midbrain-hindbrain boundary, a known lineage restriction in the zebrafish. By using this form of imaging, we have visualized both morphological and genetic parameters in this area and have used these to make quantitative and qualitative models of MHB morphogenesis and lineage restriction formation. These models allow us to have a better understanding of what exactly is responsible for the formation of the midbrain-hindbrain boundary and its lineage restriction in zebrafish with implications for all vertebrates, including humans

Physiological Role of Gap-Junctional Hemichannels: Extracellular Calcium-Dependent Isosmotic Volume Regulation

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to ≤1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and β-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation

Metal-Based Optical Probes for Live Cell Imaging of Nitroxyl (HNO)

Nitroxyl (HNO) is a biological signaling agent that displays distinctive reactivity compared to nitric oxide (NO). As a consequence, these two reactive nitrogen species trigger different physiological responses. Selective detection of HNO over NO has been a challenge for chemists, and several fluorogenic molecular probes have been recently developed with that goal in mind. Common constructs take advantage of the HNO-induced reduction of Cu(II) to Cu(I). The sensing mechanism of such probes relies on the ability of the unpaired electron in a d orbital of the Cu(II) center to quench the fluorescence of a photoemissive ligand by either an electron or energy transfer mechanism. Experimental and theoretical mechanistic studies suggest that proton-coupled electron transfer mediates this process, and careful tuning of the copper coordination environment has led to sensors with optimized selectivity and kinetics. The current optical probes cover the visible and near-infrared regions of the spectrum. This palette of sensors comprises structurally and functionally diverse fluorophores such as coumarin (blue/green emission), boron dipyrromethane (BODIPY, green emission), benzoresorufin (red emission), and dihydroxanthenes (near-infrared emission). Many of these sensors have been successfully applied to detect HNO production in live cells. For example, copper-based optical probes have been used to detect HNO production in live mammalian cells that have been treated with H2S and various nitrosating agents. These studies have established a link between HSNO, the smallest S-nitrosothiol, and HNO. In addition, a near-infrared HNO sensor has been used to perform multicolor/multianalyte microscopy, revealing that exogenously applied HNO elevates the concentration of intracellular mobile zinc. This mobilization of zinc ions is presumably a consequence of nitrosation of cysteine residues in zinc-chelating proteins such as metallothionein. Future challenges for the optical imaging of HNO include devising probes that can detect HNO reversibly, especially because ratiometric imaging can only report equilibrium concentrations when the sensing event is reversible. Another important aspect that needs to be addressed is the creation of probes that can sense HNO in specific subcellular locations. These tools would be useful to identify the organelles in which HNO is produced in mammalian cells and probe the intracellular signaling networks in which this reactive nitrogen species is involved. In addition, near-infrared emitting probes might be applied to detect HNO in thicker specimens, such as acute tissue slices and even live animals, enabling the investigation of the roles of HNO in physiological or pathological conditions in multicellular systems.National Science Foundation (U.S.)Swiss National Science Foundation (Postdoctoral Fellowship

A brief account of nanoparticle contrast agents for photoacoustic imaging

Photoacoustic imaging (PAI) is a hybrid, nonionizing modality offering excellent spatial resolution, deep penetration, and high soft tissue contrast. In PAI, signal is generated based on the absorption of laser-generated optical energy by endogenous tissues or exogenous contrast agents leading to acoustic emissions detected by an ultrasound transducer. Research in this area over the years has shown that PAI has the ability to provide both physiological and molecular imaging, which can be viewed alone or used in a hybrid modality fashion to extend the anatomic and hemodynamic sensitivities of clinical ultrasound. PAI may be performed using inherent contrast afforded by light absorbing molecules such as hemoglobin, myoglobin, and melanin or exogenous small molecule contrast agent such as near infrared dyes and porphyrins. However, this review summarizes the potential of exogenous nanoparticle-based agents for PAI applications including contrast based on gold particles, carbon nanotubes, and encapsulated copper compounds

조직재생 추적을 위한 세포 분자 영상 기법

학위논문(박사)--서울대학교 대학원 :공과대학 협동과정 바이오엔지니어링전공,2019. 8. Hwang , Nathaniel S..Regenerative medicine holds immense potential for treating a wide range of human disease, yet the mechanisms behind the therapeutic function of transplanted cells and biomaterials are not fully understood. Advances in molecular imaging have made it possible to quantitatively characterize damaged tissue, and they have allowed for the visualization of dynamic processes within a cell and even in the living body. Particularly, intraoperative optical imaging with monitoring the pathophysiological alteration of the target tissue/cell. However, a common set of challenges involving the development of targeted probes is the long-term detection of cell survival, differentiation, and integration with the host tissue for the early delineation of toxicity and function. In this study, we developed a novel fluorescent cell-labeling probe, CTNF126, in the near-infrared (NIR) window, which can be covalently fixed within the cells using formalin or paraformaldehyde, while retaining high stability. Exploiting this technique to the next step, we achieved simultaneously monitoring of brain tissue regeneration and scaffold degradation in animal models using brain-specific NIR fluorophores, Ox1, in the 700 nm channel and ZW800-3a conjugated NIR hydrogel in the 800 nm channel. Taken together, these results highlight that targeted imaging can be used as a promising platform for supporting therapeutic intervention and mechanistic insight in tissue regeneration.의학의 발전과 함께 세포를 이용한 재생의학 및 난치성 질병 치료의 연구가 활발히 이루어지고 있다. 일반적으로 질병이나 사고, 혹은 노령화 등으로 손상된 심장, 뇌, 척추신경 등의 조직과 장기는 자가 재생능력이 부족하여 일반적인 치료 및 수술을 통한 기능복원이 어려운 것이 현실이다. 즉, 한번 손상된 조직은 그 기능을 잃어버리게 되고, 이때에는 기존 약물치료나 수술을 통한 기능복원이 불가능하다. 최근에는 이를 극복하기 위해 다분화능을 가진 다양한 줄기세포 또는 스캐폴드(scaffold)를 이용, 재생이 어려운 장기를 복원시키거나 난치성 질병 치료에 관한 연구가 활발히 이루어지고 있다. 현재 다양한 치료 기술이 개발되어 그 효능평가 및 안전성 검증에 대한 연구가 이루어지고 있지만, 세포 치료제를 임상에 적용하려면 인체에 주입한 후 일어나는 일련의 현상들을 자세히 관찰할 수 있어야 한다. 특히, 분자 영상 기술을 이용하여 체내에 투여된 세포들과 주변 세포들의 거동을 영상화하고 정량화 할 수 있다면 치료제의 임상적용을 더욱 촉진시킬 수 있을 것이다. 앞서 기술한 효능평가 및 안전성 검증과 관련된 문제를 해결하기 위한 본 논문의 첫 번째 연구내용은 세포 라이소좀(lysosome)을 타깃 하는 근적외선 형광 조영제를 개발하여 체내에 투여된 세포를 장시간 관찰하는 것이며, 이에 성공하였다. 다음 연구내용으로는 뇌를 특이적으로 목표하여 실시간 이미징을 가능하게 하는 조영제를 개발함이다. 일반적으로 뇌는 자가재생이 어려운 장기로서 뇌 손상이 생기게 되면 영구적인 장애를 가질 수 있는 바, 본 연구에서는 뇌 특이적 조영제를 이용, 뇌 조직 손상/재생 및 스캐폴드(scaffold) 마모속도를 서로 다른 두 가지 파장을 내는 형광 물질을 활용하여 다중채널 이미징을 함으로써 그 치료과정을 육안으로 쉽게 구분이 가능하게 하였다. 이번 연구는 기존 저해상도 초음파나 MRI를 이용한 영상기법과 달리, 조직 특이적으로 시각화 하였고, 이는 특정 생체 조직들의 맞춤형 표적영상화 가능성을 높일 수 있을 것이라고 생각된다. 따라서, 본 논문에서는 제시한 조직재생 추적 기술 및 독립형 다중영상 시스템 은 조직재생 추적뿐만 아니라 정상/손상조직 모사체 개발, 진단의학을 위한 플랫폼 구축, 신약개발 등의 신의료기술 연구에 응용될 가능성이 높다고 판단된다.Abstract ii Table of Contents iv List of Tables vii List of Figures viii CHAPTER ONE: THE SCIENTIFIC BACKGROUND AND RESEARCH PROGRESS 1 1.1 Overview 1 1.2 Optical fluorescence imaging probes 5 1.3 Fluorescent organic dyes 8 1.4 Fluorescent proteins 8 1.5 Fluorescent nanoparticles 9 1.6 Intraoperative fluorescence imaging systems 10 1.7 Multispectral imaging systems 11 1.8 Optical imaging in cardiac stem cell therapy 14 1.9 Optical imaging in tissue regeneration 17 1.10 Summary 21 CHAPTER TWO: LYSOSOME-TARGETED BIOPROBES FOR SEQUENTIAL CELL TRACKING FROM MACROSCOPIC TO MICROSCOPIC SCALES 22 2.1 Introduction 22 2.2 Materials and methods 25 2.2.1 Chemicals and syntheses 25 2.2.2 Measurement of optical properties 26 2.2.3 Live cell labeling and in vitro imaging 26 2.2.4 In vivo cell tracking and histological analysis 27 2.2.5 Quantitation and statistical analysis 29 2.3 Results and discussion 30 2.3.1 Synthesis of cell tracking fluorophores 30 2.3.2 Physicochemical and optical properties of CTNFs 36 2.3.3 Lysosome-Targeted bioprobes for sequential cell tracking 38 2.3.4 In vitro stability test of CTNFs 40 2.3.5 In vivo stability test of CTNFs 43 2.3.6 Fixable properties of CTNF126 45 2.4 Summary 51 CHAPTER THREE: DUAL-CHANNEL FLUORESCENCE IMAGING OF HYDROGEL DEGRADATION AND TISSUE REGENERATION IN THE BRAIN 52 3.1 Introduction 52 3.2 Materials and methods 54 3.2.1 NIR fluorophores and hydrogels 54 3.2.2 Optical property measurements 55 3.2.3 Analysis of mechanical properties 55 3.2.4 Rheological analysis 56 3.2.5 Live cell labeling and tracking in the NIR hydrogel 56 3.2.6 In vitro cell viability test 57 3.2.7 Animal models for hydrogel implantation 58 3.2.8 Optical fluorescence imaging system and quantification 59 3.2.9 Intracranial transplantation of NIR hydrogel and brain tissue imaging 59 3.2.10 Histological analysis and NIR fluorescence microscopy 60 3.3 Results and discussion 61 3.3.1 Engineering of injectable NIR hydrogels 61 3.3.2 Mechanical and rheological property of NIR hydrogels 67 3.3.3 In vitro cell viability test of NIR hydrogels 70 3.3.4 In vivo physiological stability and degradation of NIR hydrogels 75 3.3.5 Multichannel imaging of hydrogel degradation and tissue growth 77 3.3.6 Quantitative annotation of brain tissue ingrowth 82 3.3.7 Histological analysis using NIR fluorescence microscopy 82 3.4 Summary 85 CHAPTER FOUR: CONCLUDING REMARKS 86 4.1 Summary 86 4.2 Future perspective 87 4.2.1 Multimodal imaging 87 4.2.2 Fluorescence lifetime imaging 88 4.2.3 Photoacoustic imaging 89 4.2.4 NIR-II fluorophores 90 Reference 92Docto