Correlative light and electron microscopy (CLEM) combines the benefits of fluorescence and electron imaging, revealing protein localisation against the backdrop of cellular architecture. CLEM is usually performed by growing cells on gridded coverslips, imaging the cells (live or fixed) using confocal microscopy, preparing the cells for electron microscopy, relocating the cell position and plane for electron imaging of the fluorescent structure, selecting and modeling the 3D data in both modalities, and overlaying the two datasets to identify the structure of interest. This process usually requires collaboration with expert electron microscopists, and has sufficient steps and complexity to deter many researchers. Nevertheless, for those who commit to the process, there are rich rewards in the understanding of biological processes. Our recent work has focused on improving the speed, accuracy and accessibility of CLEM.
During this development work, it became clear that the technical challenges associated with CLEM are exaggerated when working in 3D. To increase protein localisation precision, we developed an ‘In-Resin Fluorescence’ (IRF) protocol that preserves the activity of GFP and related fluorophores in resin-embedded cells and tissues. The sample preparation is relatively fast, and also introduces electron contrast so that cell structure can be visualised in the electron microscope. Once the resin blocks have been cut into ultrathin sections, out-of-plane fluorescence is removed resulting in physical ‘super-resolution’ light microscopy in the axial direction, which increases the accuracy of the LM-EM overlays. Localisation precision is further increased by imaging the IRF sections in vacuo in the next generation of commercial integrated light and electron microscopes (ILEM). We were able to further improve accuracy by developing integrated super-resolution light and electron microscopy, using the novel and remarkable blinking property of GFP and YFP in-resin in vacuo, and implementing automated 3D imaging for 3D functional and structural analysis of whole cells and tissues at the nanoscale.
With the advent of dual fluorescence-electron samples comes the challenge of locating and following fluorescent cells during sample preparation and automated 3D EM image acquisition. We present a new locator tool – the miniature light microscope (miniLM), designed to integrate with the ultramicrotome to locate cells during trimming and sectioning, and an even smaller version that fits into the extremely tight space of the 3D SEM vacuum chamber for on-the-fly tracking of fluorescent cells during long automated imaging runs.
To cite this abstract:Lucy Collinson; Next Gen CLEM: super-accurate correlation and intelligent image acquisition. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/next-gen-clem-super-accurate-correlation-and-intelligent-image-acquisition/. Accessed: September 23, 2019
EMC Abstracts - https://emc-proceedings.com/abstract/next-gen-clem-super-accurate-correlation-and-intelligent-image-acquisition/