Advanced Image Acquisition, Processing Techniques and Applications

"Advanced Image Acquisition, Processing Techniques and Applications" is the first book of a series that provides image processing principles and practical software implementation on a broad range of applications. The book integrates material from leading researchers on Applied Digital Image Acquisition and Processing. An important feature of the book is its emphasis on software tools and scientific computing in order to enhance results and arrive at problem solution

STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

Single atom imaging with time-resolved electron microscopy

Developments in scanning transmission electron microscopy (STEM) have opened up new possibilities for time-resolved imaging at the atomic scale. However, rapid imaging of single atom dynamics brings with it a new set of challenges, particularly regarding noise and the interaction between the electron beam and the specimen. This thesis develops a set of analytical tools for capturing atomic motion and analyzing the dynamic behaviour of materials at the atomic scale. Machine learning is increasingly playing an important role in the analysis of electron microscopy data. In this light, new unsupervised learning tools are developed here for noise removal under low-dose imaging conditions and for identifying the motion of surface atoms. The scope for real-time processing and analysis is also explored, which is of rising importance as electron microscopy datasets grow in size and complexity. These advances in image processing and analysis are combined with computational modelling to uncover new chemical and physical insights into the motion of atoms adsorbed onto surfaces. Of particular interest are systems for heterogeneous catalysis, where the catalytic activity can depend intimately on the atomic environment. The study of Cu atoms on a graphene oxide support reveals that the atoms undergo anomalous diffusion as a result of spatial and energetic disorder present in the substrate. The investigation is extended to examine the structure and stability of small Cu clusters on graphene oxide, with atomistic modelling used to understand the significant role played by the substrate. Finally, the analytical methods are used to study the surface reconstruction of silicon alongside the electron beam-induced motion of adatoms on the surface. Taken together, these studies demonstrate the materials insights that can be obtained with time-resolved STEM imaging, and highlight the importance of combining state-ofthe- art imaging with computational analysis and atomistic modelling to quantitatively characterize the behaviour of materials with atomic resolution.The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement 291522–3DIMAGE, as well as from the European Union Seventh Framework Programme under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative -I3)

AI for time-resolved imaging: from fluorescence lifetime to single-pixel time of flight

Time-resolved imaging is a field of optics which measures the arrival time of light on the camera. This thesis looks at two time-resolved imaging modalities: fluorescence lifetime imaging and time-of-flight measurement for depth imaging and ranging. Both of these applications require temporal accuracy on the order of pico- or nanosecond (10−12 − 10−9s) scales. This demands special camera technology and optics that can sample light-intensity extremely quickly, much faster than an ordinary video camera. However, such detectors can be very expensive compared to regular cameras while offering lower image quality. Further, information of interest is often hidden (encoded) in the raw temporal data. Therefore, computational imaging algorithms are used to enhance, analyse and extract information from time-resolved images. "A picture is worth a thousand words". This describes a fundamental blessing and curse of image analysis: images contain extreme amounts of data. Consequently, it is very difficult to design algorithms that encompass all the possible pixel permutations and combinations that can encode this information. Fortunately, the rise of AI and machine learning (ML) allow us to instead create algorithms in a data-driven way. This thesis demonstrates the application of ML to time-resolved imaging tasks, ranging from parameter estimation in noisy data and decoding of overlapping information, through super-resolution, to inferring 3D information from 1D (temporal) data

Optical Coherence Tomography and Its Non-medical Applications

Optical coherence tomography (OCT) is a promising non-invasive non-contact 3D imaging technique that can be used to evaluate and inspect material surfaces, multilayer polymer films, fiber coils, and coatings. OCT can be used for the examination of cultural heritage objects and 3D imaging of microstructures. With subsurface 3D fingerprint imaging capability, OCT could be a valuable tool for enhancing security in biometric applications. OCT can also be used for the evaluation of fastener flushness for improving aerodynamic performance of high-speed aircraft. More and more OCT non-medical applications are emerging. In this book, we present some recent advancements in OCT technology and non-medical applications

Image Restoration

This book represents a sample of recent contributions of researchers all around the world in the field of image restoration. The book consists of 15 chapters organized in three main sections (Theory, Applications, Interdisciplinarity). Topics cover some different aspects of the theory of image restoration, but this book is also an occasion to highlight some new topics of research related to the emergence of some original imaging devices. From this arise some real challenging problems related to image reconstruction/restoration that open the way to some new fundamental scientific questions closely related with the world we interact with

Physical Properties of a Tardigrade Desiccation-Tolerance Protein Gel

Protein based drugs are gaining power in lockstep with our understanding of their physical properties. Unfortunately, due to the requirement for constant refrigeration this usefulness is hard to convey to those in need. Lyophilization of proteins produces more stable powders, but usually require special excipients. Cytosolically abundant heat soluble (CAHS) proteins from tardigrades show promise as excipients and have rich phase behaviors forming cold setting gels. My dissertation attempts to quantify the physical properties of a representative CAHS protein with regards to gelation and lyophilization. I first discuss the history of protein structure and how this informs the methodology to deconvolute CAHS D’s behavior. I then examine a CAHS hydrogel through the rheological, thermodynamic, statistical, and structural methods. The gels have fine stranded fibril microstructures comprised of coiled-coil helical oligomers and disordered end terminal domains that drive gelation through intermolecular β-sheet formation. These gels have strengths like that of human skin and melt around human body temperature. I then move to the lyophilized state where I observe bulk structural changes through electron micrographs and secondary structure rearrangement through vibrational spectroscopy. This revealed a strong pre-lyophilization concentration dependence on the structure of the final sample. The fibril size dependence and increase in large intermolecular β-sheets imply that after a certain size fibrils transition to slabs that form the pore walls. Together this gives a model of gelation that involves the formation of coiled-coil fibers that repeatedly oligomerize, increasing their thickness, until intermolecular β-sheets knit together pore walls and junctions. The physical properties of CAHS D hydrogels show promise in medical applications, while the aerogel highlights the importance of structure in formulation as an excipient.Doctor of Philosoph

Methods for analyzing the influence of molecular dynamics on neuronal activity

Magdeburg, Univ., Fak. für Informatik, Diss., 2015von Stefan Sokol

Single-pixel, single-photon three-dimensional imaging

The 3D recovery of a scene is a crucial task with many real-life applications such as self-driving vehicles, X-ray tomography and virtual reality. The recent development of time-resolving detectors sensible to single photons allowed the recovery of the 3D information at high frame rate with unprecedented capabilities. Combined with a timing system, single-photon sensitive detectors allow the 3D image recovery by measuring the Time-of-Flight (ToF) of the photons scattered back by the scene with a millimetre depth resolution. Current ToF 3D imaging techniques rely on scanning detection systems or multi-pixel sensor. Here, we discuss an approach to simplify the hardware complexity of the current 3D imaging ToF techniques using a single-pixel, single-photon sensitive detector and computational imaging algorithms. The 3D imaging approaches discussed in this thesis do not require mechanical moving parts as in standard Lidar systems. The single-pixel detector allows to reduce the pixel complexity to a single unit and offers several advantages in terms of size, flexibility, wavelength range and cost. The experimental results demonstrate the 3D image recovery of hidden scenes with a subsecond acquisition time, allowing also non-line-of-sight scenes 3D recovery in real-time. We also introduce the concept of intelligent Lidar, a 3D imaging paradigm based uniquely on the temporal trace of the return photons and a data-driven 3D retrieval algorithm