Volume correlative light and electron microscopy
Figure 1: Correlated Light and Electron Microscopy allows the combination of 3D confocal data taken on living cells with 3D ultrastructural analysis. In this example taken from Spiegelhalter et al 2010, the volume analysis of the GFP tagged protein BIN1 is used to focus the serial Immuno EM (@GFP) to a chosen region of interest.
Figure 2: CLEM techniques, and especially targeted ultramicrotomy, allow the fine selection of objects of interest by light microscopy and a detailed 3D ultrastructural analysis by electron microscopy (taken from Kolotuev et al., 2013)
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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 through the changes in imaging modalities. On adherent cultured cells, we have developed specific substrates with coordinates to precisely record the position of cells (Spiegelhalter et al., 2009; Gibbings et al., 2011). To make the correlation easy, one key feature of the technique is the preservation of the coordinates in the EM sample. The 3D mapping of the cell of interest by confocal microscopy is also used to focus the 3D analysis to a given subset of serial sections.
On more complex specimen, such as multicellular organisms, this targeting is even more critical as systematic EM acquisition of their entire volume would otherwise involve tedious and extremely long processes. We took advantage of the 3D mapping by confocal microscopy to characterise the position of an object of interest (cell or organ) that was then tracked at the EM level by targeted ultramicrotomy (Kolotuev et al. 2009; 2012). With sub-micrometric precision, this technique has greatly improved the yield of data targeted to a given region of interest.
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 transmission electron microscopy (TEM) and scanning electron microscopy (SEM) tomography. Our team, in collaboration with other research teams at EMBL, will now combine these advanced techniques to perform CLEM in the 3D space of complex model specimen for cell and developmental biology. By automating the targeting and acquisition, we also intend to improve the throughput of data collection.