title: Automated Correlative Light and  Electron Microscopy using FIB-SEM as  a tool to screen for ultrastructural  phenotypes
creator: Serra Lleti, Jose Miguel
subject: ddc-004
subject: 004 Data processing Computer science
subject: ddc-570
subject: 570 Life sciences
description: In Correlative Light and Electron Microscopy (CLEM), two imaging modalities are combined  to take advantage of the localization capabilities of light microscopy (LM) to guide the capture  of high-resolution details in the electron microscope (EM). However, traditional approaches  have proven to be very laborious, thus yielding a too low throughput for quantitative or  exploratory studies of populations.  Recently, in the electron microscopy field, FIB-SEM  (Focused Ion Beam -Scanning Electron Microscope) tomography has emerged as a flexible  method that enables semi-automated 3D volume acquisitions. During my thesis, I developed  CLEMSite, a tool that takes advantage of the semi-automation and scanning capabilities  of the FIB-SEM to automatically acquire volumes of adherent cultured cells.  CLEMSite is a combination of computer vision and machine learning applications with a library for  controlling the microscope ( product from a collaboration with Carl Zeiss GmbH and Fibics Inc.). Thanks to this, the microscope was able to automatically track, find and acquire cell regions previously identified in the light microscope. More specifically, two main modules  were implemented. First, a correlation module was designed to detect and record reference  points from a grid pattern present on the culture substrate in both modalities (LM and EM).  Second, I designed a module that retrieves the regions of interest in the FIB-SEM and that  drives the acquisition of image stacks between different targets in an unattended fashion. The  automated CLEM approach is demonstrated on a project where 3D EM volumes are examined  upon multiple siRNA treatments for knocking down genes involved in the morphogenesis  of the Golgi apparatus.  Additionally, the power of CLEM approaches using FIB-SEM is  demonstrated with the detailed structural analysis of two events: the breakage of the nuclear  envelope within constricted cells and an intriguing catastrophic DNA Damage Response in  binucleated cells. Our results demonstrate that executing high throughput volume acquisition  in electron microscopy is possible and that EM can provide incredible insights to guide new  biological discoveries.
date: 2020
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/26154/1/Thesis_JoseMiguelSerraLleti.pdf
identifier: DOI:10.11588/heidok.00026154
identifier: urn:nbn:de:bsz:16-heidok-261544
identifier:   Serra Lleti, Jose Miguel  (2020) Automated Correlative Light and Electron Microscopy using FIB-SEM as a tool to screen for ultrastructural phenotypes.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/26154/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng