Professor Simon Watkins' lab at University of Pittsburgh, in collaboration with JEOL USA, has been developing novel ways to analyze biological structures in 3D, utilizing correlative FESEM and fluorescence microscopy. The paper “Correlative Fluorescence and Electron Microscopy in 3D – Scanning Electron Microscope Perspective” (Current Protocols in Cytrometry) describes how the ability to correlate fluorescence microscopy and electron microscopy data obtained on biological specimens bridges the resolution gap between the data obtained by these different imaging techniques. From the abstract: “In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three-dimensional EM tomogram of the sample can be directly correlated to the FM data.”
Early in the COVID-19 outbreak in 2020, Jonathan Franks, Lab Manager in the Watkin’s lab, applied this methodology to achieve a stunning image elucidating the interaction of COVID-19 particles with cells. We look forward to more published results on this study using correlative EM/FM.
Meanwhile they continue to use the method and, Franks says, “This technique of using the Backscatter Electron Detector to take TEM-like images has a wide range of uses. We have continued to develop this CLEM technique to help collaborators look at a variety of tissues like neurons in a mouse brain as an example. We also see the potential for this technique to be able to look at a much larger area of tissue than traditional TEM. With TEM we are limited to ~1mm x 1mm x 70nm section. The SEM equipped with the BED, we can scan up to ~1cm squared piece of tissue and can stitch together a montage of multiple images at EM resolution.”
Sample: Plaque Assays using vero-E3 cells. Cells are on either side of the image and in the center is space between the cells where the virus is being released.
Credit: Jonathan Franks, with additional credit to William Klimstra and Alan Watson for providing the samples, Donna Stolz and Mara Sullivan for doing the sample processing.