Measurement in the Confocal Microscope

Part of the Methods in Molecular Biology book series (MIMB, volume 1075)


All measurements require that the microscope must be aligned as accurately as possible, and the gain (or PMT voltage) and black level must be set to avoid any overflow or underflow. Measuring surface profiles and relative depths is straightforward and can be carried out to a higher accuracy than the depth resolution of the microscopes, even though the actual images may look poor. Measuring the thickness of objects which are labeled throughout is less accurate. Length and 2D area measurements are common image analysis problems and easily carried out with image analysis software. Volume measurements are conceptually equally simple but require manual techniques or 3D analysis software. 3D surface area measurements require specialist software, or can be carried out with stereological techniques. Fluorescence intensity measurements require careful calibration. For ratiometric measurements filters and/or laser lines should be chosen to optimize the response and calibration should be done in conditions as close as possible to the experimental ones. FLIM allows exploration of the chemical environment, and multiple labelling even where spectra overlap. When the hardware is available it is also usually the method of choice for measuring FRET, which can measure molecular interactions in the nanometer range. Without FLIM hardware, either intensity measurements with correction for bleed-through and cross talk or acceptor bleaching are the most popular methods of measuring FRET.

Key words

Alignment Contrast Depth measurement Thickness measurement Length measurement Area measurement Volume measurement Fluorescence measurement Stereology Ratiometry FLIM FRET 


  1. 1.
    Cox GC, Sheppard CJR (2001) Measurement of thin coatings in the confocal microscope. Micron 32:701–705PubMedCrossRefGoogle Scholar
  2. 2.
    Sheppard CJR, Török P (1997) Effects of specimen refractive index on confocal imaging. J Microsc 185:366–374CrossRefGoogle Scholar
  3. 3.
    Cox GC, Sheppard CJR (1998) Appropriate image processing for confocal microscopy. In: Cheng PC, Hwang PP, Wu JL, Wang G, Kim H (eds) Focus on multidimensional microscopy, vol 2. World Scientific Publishing, Singapore, pp 42–54. ISBN ISBN 981-02-3992-0Google Scholar
  4. 4.
    Underwood EE (1970) Quantitative stereology. Addison-Wesley, New YorkGoogle Scholar
  5. 5.
    Pawley J (1995) Fundamental limits in confocal microscopy. In: Pawley JB (ed) Handbook of biological confocal microscopy. Plenum Press, New York, pp 19–38CrossRefGoogle Scholar
  6. 6.
    Haughland RP (1996) Handbook of fluorescent probes and research chemicals. Molecular Probes Inc., Eugene, ORGoogle Scholar
  7. 7.
    Agronskaia AV, Tertoolen L, Gerritsen HC (2003) High frame rate fluorescence lifetime imaging. J Phys D: Appl Phys 36:1655–1662CrossRefGoogle Scholar
  8. 8.
    Vereb G, Matko J, Szollosi J (2004) Cytometry of fluorescence resonance energy transfer. Methods Cell Biol 75:105–152PubMedCrossRefGoogle Scholar
  9. 9.
    Elangovan M, Wallrabe H, Chen Y, Day R, Barroso M, Periasamy A (2003) Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29:58–73PubMedCrossRefGoogle Scholar
  10. 10.
    Gadella TWJ Jr, Jovin TM (1995) Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J Cell Biol 129:1543–1558PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Guy Cox
    • 1
  1. 1.Australian Centre for Microscopy & MicroanalysisUniversity of SydneySydneyAustralia

Personalised recommendations