Correlative live-cell light and electron microscopy (live-cell CLEM) has revolutionized bioimaging, since it is the only approach that infers molecular and dynamic information to ultrastructural context. CLEM is used to identify rare or transient events for EM analysis, that are nearly impossible to detect by electron microscopy (EM) alone. Live-cell fluorescence data can also be used to register live-cell dynamics to an ultrastructural snapshot. Finding back regions of interest (ROI) between modalities in 3D can prove challenging however. In addition, accurate overlay of fluorescence and EM data is crucial to interpret the correlated datasets. These challenges must be overcome to retrace individual organelles between live-cell FM data and EM.
PhD Defence of J. Fermie MSc
PhD Defence: Bridging cellular dimensions by CLEM: novel tools for live-cell correlative imaging
This thesis explored novel approaches to reliably facilitate live-cell correlative imaging. We demonstrate how correlative live-cell imaging and EM link dynamic and functional properties to ultrastructural context. Furthermore, we demonstrate how CLEM is used to significantly reduce imaging time in the EM by providing a system of reference points for targeting in focused ion beam scanning electron microscopy (FIB-SEM). Finally, we show the potential of different bimodal fiducial particles for (live-cell) CLEM using both 2D and 3D imaging systems.
Overall, this thesis highlights the advances necessary to efficiently place live-cell dynamics and molecular composition into ultrastructural context, and demonstrates the benefits of the use of bimodal particles for more efficient CLEM applications. These, and other recent developments have made CLEM into an increasingly powerful tool in cell biology, by tightly interlinking the best that fluorescence and electron imaging have to offer.