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 language | English (US) |
|---|---|
| Pages (from-to) | 15-25 |
| Number of pages | 11 |
| Journal | Journal of Structural Biology |
| Volume | 201 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 2018 |
Funding
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. Karsten König, 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.
Keywords
- Autofluorescence
- Cryo-CLEM
- Electron cryo-tomography
- Fluorescent proteins
- Mammalian cells
ASJC Scopus subject areas
- Structural Biology