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

Carter, Stephen D. and Mageswaran, Shrawan K. and Farino, Zachary J. and Mamede, João I. and Oikonomou, Catherine M. and Hope, Thomas J. and Freyberg, Zachary and Jensen, Grant J. (2018) Distinguishing Signal From Autofluorescence In Cryogenic Correlated Light And Electron Microscopy Of Mammalian Cells. Journal of Structural Biology, 201 (1). pp. 15-25. ISSN 1047-8477. PMCID PMC5771259.

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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 (80K). 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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle Paper
Carter, Stephen D.0000-0002-4237-4276
Mamede, João I.0000-0002-6048-1876
Oikonomou, Catherine M.0000-0003-2312-4746
Hope, Thomas J.0000-0001-7183-8319
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© 2017 Elsevier Inc. Received 12 April 2017, Revised 21 October 2017, Accepted 23 October 2017, Available online 25 October 2017. This work was supported by the NIH (grant GM082545 to G.J.J., grant GM082545-6935 to T.J.H., grant K08 DA031241 to Z.F.), the Department of Defense (grant PR141292 to Z.F.), and the John F. and Nancy A. Emmerling Fund of The Pittsburgh Foundation (to Z.F.). We thank Dr. Joachim Frank, Dr. Maïté Courel, Dr. Hans Breunig, Dr. Estela Area-Gomez, Robert Grassucci and Stephanie Siegmund for guidance, suggestions and reagents. We thank Dr. Pierre Maechler for generously providing INS-1E cells for our studies. We would like to thank Steven Wilbert for technical assistance. Confocal imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation.
Funding AgencyGrant Number
NIHK08 DA031241
Department of DefensePR141292
Pittsburgh FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Subject Keywords:Cryo-clem; Autofluorescence; Mammalian cells; Electron cryotomography; Fluorescent proteins
Issue or Number:1
PubMed Central ID:PMC5771259
Record Number:CaltechAUTHORS:20170705-091308246
Persistent URL:
Official Citation:Stephen D. Carter, Shrawan K. Mageswaran, Zachary J. Farino, João I. Mamede, Catherine M. Oikonomou, Thomas J. Hope, Zachary Freyberg, Grant J. Jensen, Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells, Journal of Structural Biology, Volume 201, Issue 1, 2018, Pages 15-25, ISSN 1047-8477,
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:78754
Deposited By: Tony Diaz
Deposited On:05 Jul 2017 22:23
Last Modified:21 Mar 2022 17:25

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