Multi-plane remote refocussing epifluorescence microscopy to image dynamic Ca2+ events

Penelope F. Lawton, Charlotte Buckley, Christopher D. Saunter, Calum Wilson, Alexander D. Corbett, Patrick S. Slater, John G. McCarron, John M. Girkin

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)
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Rapid imaging of multiple focal planes without sample movement may be achieved through remote refocussing, where imaging is carried out in a plane conjugate to the sample plane. The technique is ideally suited to studying the endothelial and smooth muscle cell layers of blood vessels. These are intrinsically linked through rapid communication and must be separately imaged at a sufficiently high frame rate in order to understand this biologically crucial interaction. We have designed and implemented an epifluoresence-based remote refocussing imaging system that can image each layer at up to 20fps using different dyes and excitation light for each layer, without the requirement for optically sectioning microscopy. A novel triggering system is used to activate the appropriate laser and image acquisition at each plane of interest. Using this method, we are able to achieve axial plane separations down to 15 m, with a mean lateral stability of 0.32 um displacement using a 60x, 1.4NA imaging objective and a 60x, 0.7NA reimaging objective. The system allows us to image and quantify endothelial cell activity and smooth muscle cell activity at a high frame rate with excellent lateral and good axial resolution without requiring complex beam scanning confocal microscopes, delivering a cost effective solution for imaging two planes rapidly. We have successfully imaged and analysed Ca2+ activity of the endothelial cell layer independently of the smooth muscle layer for several minutes.
Original languageEnglish
Pages (from-to)5611-5624
Number of pages14
JournalBiomedical Optics Express
Issue number11
Early online date10 Oct 2019
Publication statusPublished - 1 Nov 2019


  • multiple focal planes
  • imaging
  • blood vessels
  • epifluoresence
  • endothelial cell activity
  • smooth muscle cell activity


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