PLoS ONE (Jan 2011)

4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.

  • David Baddeley,
  • David Crossman,
  • Sabrina Rossberger,
  • Juliette E Cheyne,
  • Johanna M Montgomery,
  • Isuru D Jayasinghe,
  • Christoph Cremer,
  • Mark B Cannell,
  • Christian Soeller

DOI
https://doi.org/10.1371/journal.pone.0020645
Journal volume & issue
Vol. 6, no. 5
p. e20645

Abstract

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BackgroundOptical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.Methodology/principal findingsWe show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to Conclusions/significanceOur approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.