From colors to grayscale: illuminating the way to the nanometer world

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Correlative Microscopy Across the Scales
Anna Steyer (1)
1. EMBL Heidelberg
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Correlative light and electron microscopy (CLEM) experiments uniquely provide a highly accurate link between the imaging of living cells and their 3D ultrastructure. However, CLEM generally suffers from a low throughput. The major hurdles include tracking the object throughout the different imaging modalities, the tedious procedures for sample preparation and the lack of automation in the data acquisition by electron microscopy. In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume acquisition at the ultrastructural level. We present a toolset for adherent cultured cells that enables tracking and finding cell regions previously identified in light microscopy, in the FIB-SEM along with automatic acquisition of high-resolution volume datasets. The automation of the workflow includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope’s field of view reducing or even eliminating operator supervision. The approach enables to target the regions of interest in EM within 5 μm accuracy, while iterating between different targets including unattended data acquisition. This allows to collect statistically significant data from a large number of cells in a heterogeneous population, demonstrating that executing high throughput volume acquisition in electron microscopy is feasible.