Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

Carter, S. D.; Mageswaran, S. K.; Farino, Z. J.; Mamede, J. I.; Oikonomou, C. M.; Hope, T. J.; Freyberg, Z.; Jensen, G. J.

doi:10.1101/120642

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bioRxiv · 10.1101/120642

Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells

Carter, S. D.·Mageswaran, S. K.·Farino, Z. J.·Mamede, J. I.·Oikonomou, C. M.·Hope, T. J.·Freyberg, Z.·Jensen, G. J.

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Abstract

Cryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

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Carter, S. D., Mageswaran, S. K., Farino, Z. J., Mamede, J. I., Oikonomou, C. M., Hope, T. J., Freyberg, Z., Jensen, G. J.. 2017-03-26. Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells. https://doi.org/10.1101/120642

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