Correlative light and electron microscopy: new strategies for improved throughput and targeting precision

The need for quantitative analysis is crucial when studying fundamental mechanisms in cell biology. Common assays consist of interfering with a system via protein knockdowns or drug treatments. These very often lead to important response variability that is generally addressed by analyzing large populations. Whilst the imaging throughput in light microscopy (LM) is high enough for such large screens, electron microscopy (EM) still lags behind and is not adapted to collect large amounts of data from highly heterogeneous cell populations. Nevertheless, EM is the only technique that offers high-resolution imaging of the entire subcellular context. Correlative light and electron microscopy (CLEM) has made it possible to look at rare events or addressing heterogeneous populations. Our goal is to develop new strategies in CLEM. More specifically, we aim at automatizing the processes of screening large cell populations (living cells or pre-fixed), identifying the sub-populations of interest by LM, targeting these by EM and measuring the key components of the subcellular organization. New 3D-EM techniques like focused ion beam - scanning electron microscopy (FIB-SEM) enable a high degree of automation for the acquisition of high-resolution, full cell datasets. So far, this has only been applied to individual target volumes, often isotropic and has not been designed to acquire multiple regions of interest. The ability to acquire full cells with up to 5 nm x 5 nm x 5 nm voxel size (x, y referring to pixel size, z referring to slice thickness), leads to the accumulation of large datasets. Their analysis involves tedious manual segmentation or so far not well established automated segmentation algorithms. To enable the analysis and quantification of an extensive amount of data, we decided to explore the potential of stereology protocols in combination with automated acquisition in the FIB-SEM. Instead of isotropic datasets, a few evenly spaced sections are used to quantify subcellular structures. Our strategy therefore combines CLEM, 3D-EM and stereology to collect and analyze large amounts of cells selected based on their phenotype as visible by fluorescence microscopy. We demonstrate the power of the approach in a systematic screen of the Golgi apparatus morphology upon alteration of the expression of 10 proteins, plus negative and positive control. In parallel to this core project, we demonstrate the power of combining correlative approaches with 3D-EM for the detailed structural analysis of fundamental cell biology events during cell division and also for the understanding on complex physiological transitions in a multicellular model organism