The technique offers an improvement on existing techniques. Cryogenic electron microscopy tomography offers subnanometer 3D resolution; however, it is limited to deposits of macromers or sections of submicrometer thickness. Alternatively, cryo-focused ion beam (FIB) imaging in combination with scanning electron microscopy (FIB-SEM) can achieve isotropic 3D sampling with greater precision.

All types of electron microscopy (EM) produce grayscale images, which makes it difficult to identify many subcellular structures. To address the concerns with existing EM methods, correlative light and electron microscopy (CLEM) techniques were developed that provide global control, high resolution, and molecular specificity. CLEM is a combination of fluorescence microscopy with high-resolution electron microscopy. This technique allows for the visualization of specific molecular components at the nanoscale in super-resolution (SR). Correlative microscopy can be achieved through a variety of fluorescence and electron microscopy techniques.

In the current study, the researchers utilized cryogenic structured illumination microscopy (SIM) and single-molecule localization microscopy (SMLM) to obtain superresolution images that were correlated and then contrasted with 3D FIB-SEM images. Together, superresolution whole-cell cryo-SR/FIB-SEM revealed compartmentalized proteins within subcellular components, assisted in the discovery of new subcellular components, and classified unknown morphologies and their roles in cell biology.

In this case, the pipeline for correlative microscopy included first freezing the cells under high pressure. Next, the researchers imaged cells in a cryogenic chamber with 3D superresolution fluorescence microscopy. The cells were removed and embedded in resin and then imaged with an electron microscope that milled away the sample during imaging to expose each new layer. Finally, the data from both imaging techniques were stitched together to create a 3D reconstruction.

Correlative cryo-SR/FIB-SEM enabled the researchers to combine two datasets that revealed protein location patterns and ultrastructural morphologies. The researchers identified a few unique details within cells.

For example, they found significant changes within the nucleus as DNA is repackaged when genes are turned on and off. In this case, FIB-SEM alone showed little difference in the absolute volume of compacted heterochromatin before and after differentiation, but correlative microscopy revealed that the developed neurons actually had 50% greater nuclear volume than the progenitor neurons.

"The technique provided an amazingly detailed snapshot of the state of the nucleus before and after differentiation," said study co-author David Solecki, PhD, from St. Jude Children's Research Hospital.

Neural adhesions are essential for brain development, but the researchers wanted to understand how neurons are connected during differentiation. They found that in developing neurons, neurons adhere to one another in a web-like structure composed of drebrin and specifically placed in relation to plasma membranes.

Lastly, correlative microscopy revealed that vesicles, including peroxisomes, lysosomes, and endosomes, are ubiquitous throughout cells. The analysis revealed some of the specific shapes and volume that characterize peroxisomes present in HeLa cells, which are responsible for transferring cargo between organelles. Moreover, FIB-SEM in combination with SIM was used to determine specific morphology of endosomes, including elongated tubules, a very specific subcellular characteristic of endosomes.

"This is a very powerful method," said Harald Hess, a senior group leader at the Howard Hughes Medical Institute's Janelia Research Campus.

The research group is already working on making improvements and additional applications based on the results of their current research.

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