Nanometric depth resolution from multi-focal images in microscopy

H. I C Dalgarno, Paul A. Dalgarno, Adetunmise C Dada, Catherine E. Towers, Gavin J. Gibson, Richard M. Parton, Ilan Davis, Richard J. Warburton, Alan H. Greenaway

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)


We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 mm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels. © 2011 The Royal Society.

Original languageEnglish
Pages (from-to)942-951
Number of pages10
JournalJournal of the Royal Society. Interface
Issue number60
Publication statusPublished - 6 Jul 2011


  • Image sharpness
  • Maximum likelihood
  • Micro-fluid flow
  • Particle tracking
  • Photo-activated localization microscopy
  • Three-dimensional imaging


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