Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit...

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Bibliographic Details
Published in:	Journal of structural biology Vol. 201; no. 1; pp. 15 - 25
Main Authors:	Carter, Stephen D., Mageswaran, Shrawan K., Farino, Zachary J., Mamede, João I., Oikonomou, Catherine M., Hope, Thomas J., Freyberg, Zachary, Jensen, Grant J.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-01-2018
Subjects:	Autofluorescence Cryo-CLEM Electron cryo-tomography Fluorescent proteins Mammalian cells Cryo-CLEM Autofluorescence Fluorescent proteins Electron cryo-tomography Mammalian cells
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Summary:	In cryogenic correlated light and electron microscopy (cryo-CLEM), frozen targets of interest are identified and located on EM grids by fluorescence microscopy and then imaged at higher resolution by cryo-EM. Whilst working with these methods, we discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80 K). Autofluorescence originated from multilamellar bodies (MLBs) and secretory granules. Here we describe a method to distinguish fluorescent protein tags from these autofluorescent sources based on the narrower emission spectrum of the former. The method is first tested on mitochondria and then applied to examine the ultrastructural variability of secretory granules within insulin-secreting pancreatic beta-cell-derived INS-1E cells.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to the work.
ISSN:	1047-8477 1095-8657
DOI:	10.1016/j.jsb.2017.10.009