Modelling the application of adaptive optics to wide-field microscope live imaging
ABSTRACT Wide-field fluorescence microscopy is an essential tool in modern cell biology. Unfortunately the image quality of fluorescence microscopes is often significantly degraded due to aberrations that occur under normal imaging conditions. In this article, we examine the use of adaptive optics technology to dynamically correct these problems to achieve close to ideal diffraction limited performance. Simultaneously, this technology also allows ultra-rapid focusing without having to move either the stage or the objective lens. We perform optical simulations to demonstrate the degree of correction that can be achieved.
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ABSTRACT: Depth aberrations are a major source of image degradation in three-dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of an inevitable mismatch between the sample refractive index and the immersion medium index. We have built a wide-field fluorescence microscope that incorporates a large-throw deformable mirror to correct for depth aberrations in 3D imaging. We demonstrate a corrected point spread function imaging beads in water with an oil immersion lens and a twofold improvement in peak signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.Proceedings of SPIE - The International Society for Optical Engineering 01/2008; DOI:10.1117/12.773731 · 0.20 Impact Factor
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ABSTRACT: Although many of the techniques of live cell imaging in Drosophila melanogaster are also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging of Drosophila tissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research using Drosophila.Cold Spring Harbor Protocols 04/2010; 2010(4):pdb.top75. DOI:10.1101/pdb.top75 · 4.63 Impact Factor
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ABSTRACT: We present a novel approach for three-dimensional localization of single molecules using adaptive optics. A 52-actuator deformable mirror is used to both correct aberrations and induce two-dimensional astigmatism in the point-spread-function. The dependence of the z-localization precision on the degree of astigmatism is discussed. We achieve a z-localization precision of 40 nm for fluorescent proteins and 20 nm for fluorescent dyes, over an axial depth of ~800 nm. We illustrate the capabilities of our approach for three-dimensional high-resolution microscopy with super-resolution images of actin filaments in fixed cells and single-molecule tracking of quantum-dot labeled transmembrane proteins in live HeLa cells.