Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy

Hong Qian, Elliot L. Elson

Research output: Contribution to journalArticlepeer-review

158 Scopus citations


Quantitative fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR) measurements in bulk solution require a well characterized confocal laser microscope optical system. The introduction of a characteristic function, the collection efficiency function (CEF), provides a quantitative theoretical analysis of this system, which yields an interpretation of the FCS and FPR measurements in three dimensions. We demonstrate that when the proper field diaphragm is introduced, the 3-D FCS measurements can be mimicked by a 2-D theory with only minor error. The FPR characteristic recovery time for diffusion is expected to be slightly longer than the corresponding time measured by FCS in the same, conditions. This is because the profile of the laser beam used for photobleaching is not affected by the field diaphragm. The CEF is also important for quantitative analysis of standard scanning confocal microscopy when it is carried out using a finite detection pinhole.

Original languageEnglish
Pages (from-to)1185-1195
Number of pages11
JournalApplied Optics
Issue number10
StatePublished - Apr 1991


  • And field diaphragm
  • Collection efficiency function
  • Confocal microscopy
  • Laser focus
  • Point spread function


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