Simplified CLEM method - from in vivo imaging to FIB/SEM

The lab of Prof. Jochen Herms (LMU-Munich) has developed a simplified CLEM sample-processing workflow to allow study of a single region of interest (ROI) - the tripartite synapse - from in vivo 2-P microscopy through to FIB/SEM.  The alignment and registration of the tissue section through the various stages was achieved by slide mounting and natural landmarks without the need for complex EM labelling techniques known to be associated with deterioration or obscuration of target and proximal ultrastructure.

Instead, natural landmarks - blood vessels, nuclei and myelinated axons - were utilised, and identified by a variety of light microscopy techniques, combined with a simplified "flat embedding" which preserved the various landmarks for alignment between the LM and EM images.

For high resolution nuclear imaging by confocal laser scanning microscopy DRAQ5™ was chosen as nuclear counterstain, as a proven vital (cell permeant) DNA dye that therefore eliminates the need for tissue permeabilization.

In some cases the three landmarks could be identified in a label-free manner utilising DIC and SCoRe (spectral confocal reflectance) microscopy, albeit at lower resolution. In all cases registration of the various LM images could be achieved with SEM of the carbon-coated sample.

In the example on mouse brain tissue, 50 µm vibratome sections were slide mounted under coverslip and spacer for LM and then re-exposed on-slide for FIB/SEM sample processing.  Thereby, sample orientation remained the same throughout and the various landmarks used as fiducial points for overlaying the LM and EM images to enable localisation of the region of interest (ROI), in this case the tripartite synapse.

Here, far-red DRAQ5™ again demonstrates its utility to reliably mark nuclei without resort to unwanted sample permeabilization and processing in already challenging and complex workflows.  Based on the evidence of a number of papers from leading laboratories DRAQ5™ appears to be the nuclear counterstain of choice for CLEM!

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Reference: Luckner, M., Burgold, S., Filser, S., Scheungrab, M., Niyaz, Y., Hummel, E., ... & Herms, J. (2018). Label-free 3D-CLEM using endogenous tissue landmarks. iScience 6:92-101.

Read more articles on DRAQ5™ in exciting new EM techniques: 

• CryoChem for CLEM - preservation of high definition in fluorescence & EM dimensions
• ChromEMT for EM-Tomography of chromatin - direct labelling of chromatin