Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Hoffman, D. P.; Shtengel, G.; Xu, C. S.; Campbell, K. R.; Freeman, M.; Wang, L.; Milkie, D. E.; Pasolli, H. A.; Iyer, N.; Bogovic, J. A.; Stabley, D. R.; Shirinifard, A.; Pang, S.; Peale, D.; Schaefer, K.; Pomp, W.; Chang, C.-L.; Lippincott-Schwartz, J.; Kirchhausen, T.; Solecki, D. J.; Betzig, E.; Hess, H. F.

doi:10.1101/773986

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bioRxiv · 10.1101/773986

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

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Abstract

Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

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Hoffman, D. P., Shtengel, G., Xu, C. S., Campbell, K. R., Freeman, M., Wang, L., Milkie, D. E., Pasolli, H. A., Iyer, N., Bogovic, J. A., Stabley, D. R., Shirinifard, A., Pang, S., Peale, D., Schaefer, K., Pomp, W., Chang, C.-L., Lippincott-Schwartz, J., Kirchhausen, T., Solecki, D. J., Betzig, E., Hess, H. F.. 2019-09-18. Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells. https://doi.org/10.1101/773986

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