Cross-talk-free multi-color STORM imaging using a single fluorophore

PLoS One. 2014 Jul 7;9(7):e101772. doi: 10.1371/journal.pone.0101772. eCollection 2014.

Abstract

Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / metabolism
  • Cell Line
  • Color
  • Fluorescent Dyes / metabolism*
  • Microscopy, Fluorescence / methods*

Substances

  • Antibodies
  • Fluorescent Dyes

Grants and funding

This work was financially supported by Fundació CELLEX Barcelona (M.L.), the Systems Microscopy Network of Excellence consortium (call identifier number FP-7- HEALTH.2010.2.1.2.2) (M.L.), ERC-Starting grant (grant agreement number 337191-MOTORS) (M.L.), and the Whitaker International Fellows and Scholars Program (J.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.