Multiple-view microscopy with light-sheet based fluorescence microscope

The axial resolution of any standard single-lens light microscope is lower than its lateral resolution. The ratio is approximately 3-4 when high numerical aperture objective lenses are used (NA 1.2 -1.4) and more than 10 with low numerical apertures (NA 0.2 and below). In biological imaging, the axial resolution is normally insufficient to resolve subcellular phenomena. Furthermore, parts of the images of opaque specimens are often highly degraded or obscured. Multiple-view fluorescence microscopy overcomes both problems simultaneously by recording multiple images of the same specimen along different directions. The images are digitally fused into a single high-quality image. Multiple-view imaging was developed as an extension to the light-sheet based fluorescence microscope (LSFM), a novel technique that seems to be better suited for multiple-view imaging than any other fluorescence microscopy method to date. In this contribution, the LSFM properties, which are important for multiple-view imaging, are characterized and the implementation of LSFM based multiple-view microscopy is described. The important aspects of multiple-view image alignment and fusion are discussed, the published algorithms are reviewed and original solutions are proposed. The advantages and limitations of multiple-view imaging with LSFM are demonstrated using a number of specimens, which range in size from a single yeast cell to an adult fruit fly and to Medaka fish

Recent Progress in Image Deblurring

This paper comprehensively reviews the recent development of image deblurring, including non-blind/blind, spatially invariant/variant deblurring techniques. Indeed, these techniques share the same objective of inferring a latent sharp image from one or several corresponding blurry images, while the blind deblurring techniques are also required to derive an accurate blur kernel. Considering the critical role of image restoration in modern imaging systems to provide high-quality images under complex environments such as motion, undesirable lighting conditions, and imperfect system components, image deblurring has attracted growing attention in recent years. From the viewpoint of how to handle the ill-posedness which is a crucial issue in deblurring tasks, existing methods can be grouped into five categories: Bayesian inference framework, variational methods, sparse representation-based methods, homography-based modeling, and region-based methods. In spite of achieving a certain level of development, image deblurring, especially the blind case, is limited in its success by complex application conditions which make the blur kernel hard to obtain and be spatially variant. We provide a holistic understanding and deep insight into image deblurring in this review. An analysis of the empirical evidence for representative methods, practical issues, as well as a discussion of promising future directions are also presented.Comment: 53 pages, 17 figure

Super-resolution imaging of cell-surface Sonic hedgehog multimolecular signalling complexes

Sonic hedgehog is a fascinating protein with great responsibility over the formation and upkeep of our bodies. It is widely studied, not least because dysregulation of the Shh signalling pathway leads to repercussions on human health, such as contraction of cancer. Gaining an understanding of its signalling mechanism is central to inventing preventative measures and treatments against this disease. This thesis focuses on the study of the spatial organisation of Shh multimolecular signalling complexes on the surface of producing cells, and those dispatched in the vicinity of those cells, using high-resolution optical imaging beyond the diffraction limit. With un-precedented resolution, the differences in organisation of Shh pre- and post-release from the surface were characterised, and the influence of the lipid modifications of Shh, namely choles-terol and palmitate, investigated. The main findings were that both lipid adducts are necessary for large-scale multimerisation, but not for the formation of small, sub-diffraction limit oligomers. Together with data I collected about the profile of the clusters’ size distributions, I find that electrostatic interactions between the molecules may be the engine driving the multimerisation process. Furthermore, the role of lipid modifications may, at least in part, be to retain Shh on the surface while multimerisation proceeding according to the law of mass action builds upon the small oligomer nucleation sites prepared presumably by the electrostatic interactions in the first place. Other, more indirect lines of evidence again based on the profile of the multimer size distribution insinuated that Shh complexes may not undergo any proteolytic modifications prior to release – contrary to some reports in the literature. The results presented in this thesis are the fruits of a completely fresh and innovative approach to examining Shh, which for the first time delivers concrete dimensional details about the elusive structure of the Shh multimer.Open Acces

Fast, Three-Dimensional Fluorescence Imaging of Living Cells

This thesis focuses on multi-plane fluorescence microscopy for fast live-cell imaging. To improve the performance of multi-plane microscopy, I developed new image analysis methods. I used these methods to measure and analyze the movements of cardiomyocytesand Dictyostelium discoideum cells.The multi-plane setup is based on a conventional wide-field microscope using a custom multiple beam-splitter in the detection path. This prism creates separate images of eight distinct focal planes in the sample. Since 3D volume is imaged without scanning, three-dimensional imaging at a very high speed becomes possible. However, as in conventional wide-field microscopy, the "missing cone" of spatial frequencies along the optical axis in the optical transfer function (OTF) prevents optical sectioning in such a microscope. This is in stark contrast to other truly three-dimensional imaging modalities like confocal and light-sheet microscopy. In order to overcome the lack of optical sectioning, I developed a new deconvolution method. Deconvolution describes methods that restore or sharpen an image based on physical assumptions and knowledge of the imaging process. Deconvolution methods have been widely used to sharpen images of microscopes and telescopes. The recently developed SUPPOSe algorithm is a deconvolution algorithm that uses a set of numerous virtual point sources. It tries to reconstruct an image by distributing these point sources in space and optimizing their positions so that the resulting image reproduces as good as possible the measured data. SUPPOSe has never been used for 3D images. Compared to other algorithms, this method has superior performance when the number of pixels is increased by interpolation. In this work, I extended the method to work also with 3D image data. The 3D-SUPPOSe program is suitable for analyzing data of our multi-plane setup. The multi-plane setup has only eight vertically aligned image planes. Furthermore, for accurate reconstruction of 3D images, I studied a method of correcting each image plane's relative brightness constituting an image, and I also developed a method of measuring the movement of point emitters in 3D space. Using these methods, I measured and analyzed the beating motion of cardiomyocytes and the chemotaxis of Dicyosteilium discoidem. Cardiomyocytes are the cells of the heart muscle and consist of repetitive sarcomeres. These cells are characterized by fast and periodic movements, and so far the dynamics of these cells was studied only with two-dimensional imaging. In this thesis, the beating motion was analyzed by tracing the spatial distribution of the so-called z-discs, one of the constituent components of cardiomyocytes. I found that the vertical distribution of $\alpha$-actinine-2 in a single z-disc changed very rapidly, which may serve as a starting point for a better understanding the motion of cardiomyocytes. \textit{Dictyostelium discoideum} is a well established single cell model organism that migrates along the gradient of a chemoattractant. One has conducted much research to understand the mechanism of chemotaxis, and many efforts have been made to understand the role of actin in the chemotactic motion. By suppressing the motor protein, myosin, a cell line was created that prevented the formation of normal actin filaments. In these myosin null cells, F-actin moves in a flow-like behaviour and induces cell movement. In this study, I imaged the actin dynamics, and I analyzed the flow using the newly created deconvolution and flow estimation methods. As a result of the analysis, the spatio-temporal correlation between pseudo-pod formation and dynamics and actin flow was investigated.2022-01-2

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