Nanometric depth resolution from multi-focal images in microscopy

Heather 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

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)942-951
Number of pages10
JournalJournal of the Royal Society Interface
Volume8
Issue number60
DOIs
Publication statusPublished - 6 Jul 2011

Keywords

  • particle tracking
  • three-dimensional imaging
  • photo-activated localization microscopy
  • micro-fluid flow
  • image sharpness
  • maximum likelihood
  • FLUORESCENCE MICROSCOPY
  • PARTICLE-TRACKING
  • 3 DIMENSIONS
  • SUPERRESOLUTION MICROSCOPY
  • PLANE MICROSCOPY
  • LIVING CELLS
  • DYNAMICS
  • LOCALIZATION
  • ACCURACY
  • NETWORK

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