Calibrating excitation light fluxes for quantitative light microscopy in cell biology.

David Grünwald, Shailesh M. Shenoy, Sean Burke, Robert H. Singer

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1809-1814
Number of pages6
JournalNature Protocols
Volume3
Issue number11
StatePublished - 2008

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Cytology
Optical microscopy
Cell Biology
Microscopy
Fluxes
Light
Microscopes
Fluorescence microscopy
Electric lamps
Light sources
Experiments
Fluorescence Microscopy
Calibration
Research Personnel

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Calibrating excitation light fluxes for quantitative light microscopy in cell biology. / Grünwald, David; Shenoy, Shailesh M.; Burke, Sean; Singer, Robert H.

In: Nature Protocols, Vol. 3, No. 11, 2008, p. 1809-1814.

Research output: Contribution to journalArticle

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