1 research outputs found
Automated Correlative Light and Electron Microscopy using FIB-SEM as a tool to screen for ultrastructural phenotypes
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