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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ceramides / chemical synthesis
  • Ceramides / metabolism*
  • Drug Stability
  • Fluorescent Dyes / metabolism*
  • Golgi Apparatus / metabolism*
  • HeLa Cells
  • Humans
  • Imaging, Three-Dimensional
  • Microscopy, Fluorescence / methods
  • Rhodamines / metabolism*

Substances

  • Ceramides
  • Fluorescent Dyes
  • Rhodamines