Figure 1: On adherent cultured cells, CLEM relies on coordinates that are visible by both light and electron microscopy. Precise targeting enables the acquisition of selected subcellular volumes with a FIB-SEM.
Figure 2: On multicellular specimens (here, mouse brain), our targeting methods enable studying identified cells (tumour cells) by multimodal correlative microscopy (intravital microscopy, microCT and FIB-SEM; from Karreman et al. 2016).
The Schwab team is interested in developing tools for the 3D correlation of data generated by fluorescent imaging and by electron microscopy.
Previous and current research
Correlative light and electron microscopy (CLEM) is a set of techniques that allow data acquisition with both imaging modalities on a single object. It is a growing field that now includes a large variety of strategies, and one that reaches a high degree of precision, even in complex biological models. Before joining EMBL, we were developing tools and protocols to track rare objects or dynamic phenomena on cultured cells and bulk specimen such as nematodes and murine tissues.
One common challenge when trying to combine imaging modalities on the same sample is to identify space cues (external or internal) to track single objects when switching from light microscopy (LM) to electron microscopy (EM). On adherent cultured cells, we have previously developed specific substrates with coordinates to precisely record the position of cells (Spiegelhalter et al., 2009). Currently, we are exploiting these approaches to develop new workflows allowing the study of a higher number of cells.
On more complex specimens, such as multicellular organisms, this targeting is even more critical, as systematic EM acquisition of their entire volume is close to impossible. For this reason, we are developing new methods to map the region of interest (ROI) within large living specimens, taking advantage of structural hallmarks in the sample that are visible with both LM and EM. The position of the ROI is mapped in 3D by confocal or multiphoton microscopy and then tracked at the EM level by targeted ultramicrotomy (Kolotuev et al. 2009; 2012; Goetz et al. 2014). Relying on structural features of the sample as anchor points, the cell or structure of interest can then be retrieved with sub-micrometric precision (Durdu et al. 2014, Karreman et al. 2014, 2015).
Future projects and goals
In parallel to the fast evolution of CLEM techniques over the past decade, acquisition methods in electron microscopes have significantly evolved with special breakthroughs in the volume analysis of cells by TEM tomography and automated serial imaging in scanning electron microscopy (ASI-SEM). Our team, in collaboration with other scientists and our industrial partners, combines these advanced techniques to perform CLEM in the 3D space on complex model specimens for cell and developmental biology. We aim to develop new techniques and software to facilitate and automate the correlation and acquisition of large amounts and volumes of sample. By automating these tedious procedures, we intend to improve the throughput of data collection.