@article {Carter120642, author = {Stephen D. Carter and Shrawan K. Mageswaran and Zachary J. Farino and Jo{\~a}o I. Mamede and Catherine M. Oikonomou and Thomas J. Hope and Zachary Freyberg and Grant J. Jensen}, title = {Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells}, elocation-id = {120642}, year = {2017}, doi = {10.1101/120642}, publisher = {Cold Spring Harbor Laboratory}, 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.}, URL = {https://www.biorxiv.org/content/early/2017/03/26/120642}, eprint = {https://www.biorxiv.org/content/early/2017/03/26/120642.full.pdf}, journal = {bioRxiv} }