The CryoChem Method - optimal morphology meets correlative imaging (CLEM)

The CryoChem Method (CCM) is a new hybrid approach to the processing of cryofixed cells and tissues that enables preservation of ultrastructural morphology while being compatible with genetic EM tags, fluorescent proteins and counterstaining, and onwards to SBEM and CLEM.

The research, led by Professor Mark Ellisman, UCSD, benchmarks this new approach in a variety of samples to test its broad applicability.  The work, firstly shows that, following rehydration, a genetically-encoded EM tag (peroxidase APEX2) is able to accumulate electron-dense DAB.  Next, the quantitative measurement of two structurally different fluorescent proteins GFP and tdTomato is demonstrated.

To explore the opportunities presented further in correlated light and electron microscopy (CLEM), a mouse brain slice expressing tdTomato in specific neurons is processed by CCM and the ability to counterstain the nuclei tested, using the far-red DNA probe DRAQ5™.  This is established successfully at a typical concentration (5 µM), on ice for 60 minutes.  Thereafter the tissue slice is subjected to en bloc staining with heavy metals and embedded in readiness for serial block-face electron microscopy (SBEM).  The block is then analyzed by micro-CT to correlate this X-ray volume to the DRAQ5™-stained nuclei as fiducial markers of the confocal imaging data to determine a region of interest for SBEM imaging of tdTomato neurons.

It is also possible to correlate confocal and SBEM data using the bright chromatin labelling by DRAQ5™ to the matching finer SBEM features and the tdTomato fluorescence to similarly delineate cell bodies and the neuronal processes.

The authors suggest that this method should also be compatible with the earlier ChromEMT method (see separate blog) which uses DRAQ5™ to photo-catalyze the accumulation of DAB to chromatin and thereby enabling OsO4 labelling for EM tomography (EMT).

This new work further extends the breadth of use for DRAQ5™ as a nuclear counterstain.

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Reference:
Tsang, T. K., Bushong, E. A., Boassa, D., Hu, J., Romoli, B., Phan, S., ... & Ellisman, M. H. (2018). High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues. eLife. 2018; 7: e35524

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

• Simplified CLEM - from in vivo imaging to FIB/SEM
• ChromEMT for EM-Tomography of chromatin - direct labelling of chromatin