Carter, S. D. , Mageswaran, S. K., Farino, Z. J., Mamede, J. I., Oikonomou, C. M., Hope, T. J., Freyberg, Z. and Jensen, G. J. (2018) Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells. Journal of Structural Biology, 201(1), pp. 15-26. (doi: 10.1016/j.jsb.2017.10.009) (PMID:29078993) (PMCID:PMC5771259)
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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.
| Item Type: | Articles |
|---|---|
| Additional Information: | 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.). |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Carter, Dr Stephen |
| Authors: | Carter, S. D., Mageswaran, S. K., Farino, Z. J., Mamede, J. I., Oikonomou, C. M., Hope, T. J., Freyberg, Z., and Jensen, G. J. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research |
| Journal Name: | Journal of Structural Biology |
| Publisher: | Elsevier |
| ISSN: | 1047-8477 |
| ISSN (Online): | 1095-8657 |
| Published Online: | 25 October 2017 |
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