I am a Postdoc, employed at the University Hospital of Cologne, working in the aging institute CECAD
The microscopic visualization of biomolecules, their activity and distribution within cells and organisms, is the most compelling evidence to describe any biological phenomenon. For example, organismal development depends on the production and release of hormones that spread systemically and influence the shaping of organs and the whole organism. Any imbalance in this well-coordinated hormonal process can lead to developmental failure or the formation of cancer. To record such events in model organisms or tissue culture, scientists desire microscopes that produce three-dimensional (3D) images, with a resolution high enough to record subcellular events in whole-animal context, as they unfold over time.
Our interdisciplinary research team consisting of biologists, physicists and informaticians faces this challenge by combining two novel imaging technologies called Light Sheet Microscopy (LSM) and Optical Projection Tomography (OPT) in an experimental microscope platform. In LSM, a laser light sheet is produced that scans slice by slice through the specimen and the recorded data can be digitally stacked to produce a 3D image. LSM is rapid and less invasive for the specimen than other microscopy methods. OPT is the optical version of X-ray computed tomography (CT) and produces 3D pictures of anatomic structures. We combine the 3D datasets of LSM and OPT and analyze them in silico to reveal the tiniest subcellular processes and put them into the anatomic context of a living whole-organism.
We specifically adopt the environmental conditions of our microscope platform for development studies in various model organisms, including the roundworm Caenorhabditis elegans or the fruitfly Drosophila melanogaster, and constantly refine the computational methods for 3D data-reconstruction. Further, we follow the growth of cultivated 3D tumors for breast cancer cells. With our technology we are able to distinguish proliferating cells that define the progression of a malignant tumors and expose the cancer tissue to chemotherapeutics to find novel ways of treatment. Our microscopy setup combines the advantages of two novel 3D imaging techniques that enable us to visualize molecular events as they unfold over time in the context of a whole organism. This improves our understanding of basic biological processes, from organismal development to the growth of tumors, and enables us to test novel biomedical interventions.
Abstract: Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.
Pub.: 20 Apr '02, Pinned: 28 Aug '17
Abstract: Large, living biological specimens present challenges to existing optical imaging techniques because of their absorptive and scattering properties. We developed selective plane illumination microscopy (SPIM) to generate multidimensional images of samples up to a few millimeters in size. The system combines two-dimensional illumination with orthogonal camera-based detection to achieve high-resolution, optically sectioned imaging throughout the sample, with minimal photodamage and at speeds capable of capturing transient biological phenomena. We used SPIM to visualize all muscles in vivo in the transgenic Medaka line Arnie, which expresses green fluorescent protein in muscle tissue. We also demonstrate that SPIM can be applied to visualize the embryogenesis of the relatively opaque Drosophila melanogaster in vivo.
Pub.: 18 Aug '04, Pinned: 28 Aug '17
Abstract: The application of optical projection tomography to in-vivo experiments is limited by specimen movement during the acquisition. We present a set of mathematical correction methods applied to the acquired data stacks to correct for movement in both directions of the image plane. These methods have been applied to correct experimental data taken from in-vivo optical projection tomography experiments in Caenorhabditis elegans. Successful reconstructions for both fluorescence and white light (absorption) measurements are shown. Since no difference between movement of the animal and movement of the rotation axis is made, this approach at the same time removes artifacts due to mechanical drifts and errors in the assumed center of rotation.
Pub.: 25 Jan '11, Pinned: 28 Aug '17
Abstract: In in vivo optical projection tomography (OPT), object motion will significantly reduce the quality and resolution of the reconstructed image. Based on the well-known Helgason-Ludwig consistency condition (HLCC), we propose a novel method for motion correction in OPT under parallel beam illumination. The method estimates object motion from projection data directly and does not require any other additional information, which results in a straightforward implementation. We decompose object movement into translation and rotation, and discuss how to correct for both translation and general motion simultaneously. Since finding the center of rotation accurately is critical in OPT, we also point out that the system's geometrical offset can be considered as object translation and therefore also calibrated through the translation estimation method. In order to verify the algorithm effectiveness, both simulated and in vivo OPT experiments are performed. Our results demonstrate that the proposed approach is capable of decreasing movement artifacts significantly thus providing high quality reconstructed images in the presence of object motion.
Pub.: 01 Mar '12, Pinned: 28 Aug '17
Abstract: We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms.
Pub.: 12 May '11, Pinned: 28 Aug '17
Abstract: We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.
Pub.: 23 May '15, Pinned: 28 Aug '17
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