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Calibrating excitation light fluxes for quantitative light microscopy in cell biology
Biochemistry and Molecular Pharmacology Publications and Presentations
  • David Grunwald, University of Massachusetts Medical School
  • Shailesh M. Shenoy, Albert Einstein College of Medicine
  • Sean Burke, Albert Einstein College of Medicine
  • Robert H. Singer, Albert Einstein College of Medicine
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Publication Date
Document Type
Calibration; *Light; Microscopy

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.

DOI of Published Version

Grünwald D, Shenoy SM, Burke S, Singer RH. Calibrating excitation light fluxes for quantitative light microscopy in cell biology. Nat Protoc. 2008;3(11):1809-14. doi: 10.1038/nprot.2008.180. Link to article on publisher's site


At the time of publication, David Grünwald was not yet affiliated with the University of Massachusetts Medical School.

Related Resources
Link to Article in PubMed
PubMed ID
Citation Information
David Grunwald, Shailesh M. Shenoy, Sean Burke and Robert H. Singer. "Calibrating excitation light fluxes for quantitative light microscopy in cell biology" Vol. 3 Iss. 11 (2008) ISSN: 1750-2799 (Linking)
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