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

Stephen D. Carter, Shrawan K. Mageswaran, Zachary J. Farino, João I. Mamede, Catherine M. Oikonomou, Thomas J. Hope, Zachary Freyberg*, Grant J. Jensen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)15-25
Number of pages11
JournalJournal of Structural Biology
Volume201
Issue number1
DOIs
StatePublished - Jan 2018

Keywords

  • Autofluorescence
  • Cryo-CLEM
  • Electron cryo-tomography
  • Fluorescent proteins
  • Mammalian cells

ASJC Scopus subject areas

  • Structural Biology

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