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Research On Inverted Light Sheet Fluorescence Microscopy And Image Analysis

Posted on:2017-03-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:M GuoFull Text:PDF
GTID:1108330491462866Subject:Measuring and Testing Technology and Instruments
Abstract/Summary:PDF Full Text Request
It is crucial to dynamically imaging 3D biological activities with high spatial-temporal resolution in modern biological research, especially in immune proteins tracking, observing nematode activities in its late embryogenesis and other developmental processes. Featured for its capacity on fast 3D imaging with high spatial resolution, low phototoxicity and low bleaching, Light Sheet Fluorescence Microscopy (LSFM), or Selective Plane Illumination Microscopy (SPIM), has filled this niche and quickly proofed useful for a variety of applications.Focused on inverted type of LSFM and its applictions, this paper extends work including developing new microscopes, image reconstruction and LSFM acquired C. elegans embryo detwitching techniques:1. In the work on microscopes developing, we first developed an asymmetrical dual-view inverted Selective Plane Illumination Microscopy (adiSPIM) system by employing a high NA objective and a relative low NA objective. Both light-sheet scanning mode and stage scanning mode for data acquisition are realized. Performance test and biological imaging experiments demonstrate its improved spatial resolution in one view compared to our previous system. To analyze fluorescence anisotropy, we then developed a microscope for fluorescence anisotropy imaging based on inverted Selective Plane Illumination Microscopy (iSPIM) system. We used it to track protein fluorescence anisotropy dynamics in nematode during its embryogenesis and investigated the phosphorylation of the myosin â…¡ in 3D.2. For image reconstruction, we implemented joint deconvolution within Graphics Processing Unit (GPU) to accelerating the image reconstruction, making it more powerful for processing large scale of time-lapse dataset. Meanwhile we formulated a general system model for Tomographic Phase Microscopy (TPM) and proposed a image reconstruction framework based on Total Variation (TV) regularization. The proposed reconstruction method is able to retrieve the refractive index map from the measurement data with reduced angular sample frequency and limited angular coverage of illumination. In this way, the missing-angle problem is alleviated and better isotropic resolution is achieved. On the other hand, only sparse angular illuminations are required, making the imaging process faster.3. We also paid efforts on analyzing images acquired by LSFM microscope. We presented an algorithm for semi-automated untwisting nematode embryos which allows the investigation of neurodevelopmental events in late embryogenesis, and applied it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos.
Keywords/Search Tags:microscopy, light sheet microscopy, selective plane illumination microscopy, image reconstruction, deconvolution, nematode
PDF Full Text Request
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