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Calibrating excitation light fluxes for quantitative light microscopy in cell biology


Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.

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Figure 1: Principle setup and components.
Figure 2: Beam profile along the optical axis of the microscope.
Figure 3: Accounting for differences in the objective back aperture.
Figure 4: Lateral intensity profile of the excitation light.
Figure 5: Assembling of the refocusing unit.
Figure 6: Installation of refocusing unit and power meter on the microscope.
Figure 7: Repositioning of the microscope stage to center the detector head.
Figure 8: Visualization of the beam position relative to a detector.
Figure 9: Effect of excitation intensity variations on images.


  1. Model, M.A. & Blank, J.L. Intensity calibration of a laser scanning confocal microscope based on concentrated beads. Anal. Quant. Cytol. Histol. 28, 253–261 (2006).

    PubMed  Google Scholar 

  2. Zwier, J.M., Van Rooij, G.J., Hofstraat, J.W. & Brakenhoff, G.J. Image calibration in fluorescence microscopy. J. Microsc. 216, 15–24 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Murray, J.M., Appleton, P.L., Swedlow, J.R. & Waters, J.C. Evaluating performance in three-dimensional fluorescence microscopy. J. Microsc. 228, 390–405 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Beach, J.M. A LED calibration source for dual-wavelength microscopy. Cell Calcium 105, 55–63 (1997).

    Google Scholar 

  6. Cho, E.H. & Lockett, S.J. Calibration and standardization of the emission light path of confocal microscopes. J. Microsc. 223, 15–25 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Kubitscheck, U. et al. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168, 233–243 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Grünwald, D., Hoekstra, A., Dange, T., Buschmann, V. & Kubitscheck, U. Direct observation of single protein molecules in aqueous solution. ChemPhysChem 7, 812–815 (2006).

    Article  PubMed  Google Scholar 

  9. Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Burger, W. & Burge, M.J. Digital Image Processing: An Algorithmic Introduction using Java (Springer-Verlag, New York, 2008).

Download references


The authors thank Fedor Subach for help with photography, Amber Wells for providing cells and stainings, Christina Polumbo for testing the protocol for facility use and Saumil Gandhi for bringing the original problem back on our agenda. This work was supported by National Institutes of Health grants to R.H.S and a DFG postdoctoral fellowship (GR3388/1) to D.G. Photographs have been adjusted in size and for best display of features using Photoshop CS (Adobe). Images for Figure 9 have been adjusted to 8-bit using ImageJ.

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Correspondence to Robert H Singer.

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Grünwald, D., Shenoy, S., Burke, S. et al. Calibrating excitation light fluxes for quantitative light microscopy in cell biology. Nat Protoc 3, 1809–1814 (2008).

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