CORRELATIVE LIGHT ELECTRON MICROSCOPY (CLEM)
CLEM combines the capabilities of two typically separate microscopy platforms: light (or fluorescence) microscopy (LM) and electron microscopy (EM). The advantage of LM is that it can provide wide field images of whole, often living cells, but its resolution is limited. The advantage of EM is that it can provide much higher resolution images, up to molecular dimensions, but only over specific regions of a cell at a time and in fixed cells. CLEM combines the advantages of both techniques, allowing scientists to spot cellular structures and processes of interest in whole cell images with LM and then zoom in for a closer look with EM
CLEM from live cells to plastic sections ("live" CLEM)
Cells are imaged live by light microscopy (e.g. wide field, confocal or light sheet). Following the light microscopy image acquisition, specific CLEM protocols enable the scientist to retrace the position of the same cells in order to acquire high resolution EM images. This method allows one to image a labelled molecule of interest within the cell and combine it with the high-resolution information of the cellular landscape surrounding it. After live imaging, cells undergo chemical fixation or high pressure freezing and further processing for EM. The cells/regions of interest are retrieved by making use of a coordinate system present on the cell growing substrate, which is imprinted on the surface of the resulting resin block. Sections (or serial sections) acquired through the cell of interest are inspected at the EM.
Correlative light-electron microscopy from live cells to 3D plastic embedded samples
A recently developed workflow allows one to link dynamic live imaging of subcellular compartments to 3D EM for providing ultrastructural context. Following this workflow, one can image live cells expressing fluorescent markers, then fix cells in-situ and process them for 3D EM imaging using FIB-SEM (see Cellular EM). This approach is very suitable to study live-cell organelle dynamics in relation to their high-resolution morphology in 3D.
This is one specific application of the above CLEM, where full organisms such as zebrafish or mouse embryos are imaged in vivo. By 3D targeting, the same region of interest is extracted and imaged by EM (any of the 3D EM techniques listed here).
High accuracy on section CLEM
Here fluorescently-tagged molecules within the sample are preserved during the sample preparation for EM (high pressure freezing and freeze substitution). Plastic sections are collected and screened with a fluorescence microscope. Thanks to fiducial markers which are fluorescent and electron dense, the precise location of the fluorescent spots is retrieved in the EM enabling the ultrastructural assignment to the fluorescent marker. The precision of such CLEM is in the range of 50 to 100 nm. Very often, TEM tomography is performed on such samples.
CLEM on Tokuyasu sections
CLEM on Tokuyasu sections belongs to the “on-section” CLEM techniques, similar to the “high-accuracy" described above. The difference is on the sample preparation. Here, specimens are chemically fixed, cryo-protected and frozen. The sample is then hard enough to be sectioned by cryo-ultramicrotomy. Next, the cryo-sections are thawed and exposed to probes or antibodies. If fluorescent probes are used, their signal can be correlated to the ultrastructure by CLEM.
The principle is the same as for the high accuracy CLEM, but the full workflow is performed on vitrified samples (cryo-ultramicrotomy, cryo-fluorescence microscopy, cryo-EM). cryoCLEM is currently not offered as open access technology in the EuBI technology portfolio.