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Methods Mol Biol. 2017;1663:65-78. doi: 10.1007/978-1-4939-7265-4_6.

STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.

Author information

1
Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06520, USA.
2
Department of Chemistry, Yale University, New Haven, CT, 06520, USA.
3
Department of Chemistry, Yale University, New Haven, CT, 06520, USA. alanna.schepartz@yale.edu.
4
Department of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208107, New Haven, CT, 06520, USA. alanna.schepartz@yale.edu.

Abstract

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

KEYWORDS:

Bioorthogonal chemistry; Click chemistry; Fluorophores; Inverse electron demand Diels-Alder reaction; Membranes; Super-resolution microscopy

PMID:
28924659
PMCID:
PMC6146391
DOI:
10.1007/978-1-4939-7265-4_6
[Indexed for MEDLINE]
Free PMC Article

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