Absolute fluorescence calibration

I. T. Young, Y. Garini, B. Vermolen, G. Liqui Lung, G. Brouwer, S. Hendrichs, M. El Morabit, J. Spoelstra, E. Wilhelm, M. Zaal

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review

7 Scopus citations


While fluorescence microscope systems remains an essential tool in modern biology and medical work, no compact instrumentation has been developed for the rapid calibration of such systems. Almost invariably results are presented in terms of the [AU], "arbitrary units". To remedy this situation we have developed a small, portable instrument - the size of a microscope slide - that uses low-power LEDs at different wavelengths to produce calibrated amounts of light. A computer controls the instrument-through a USB connector-so that the current to the selected LED can be swept through an increasing range of values. The amount of light measured by the microscope's total imaging system (lenses, filters, EO sensor, and digitizer) is then recorded to provide a "current in, digital value out" calibration. Further, the current can be translated easily to optical power and thus photons per second at the chosen LED wavelength. We have built and programmed such a system, tested it for accuracy and precision, and used it to calibrate several microscopes and microscope/lens combinations. The results will be presented.

Original languageEnglish
Title of host publicationProgress in Biomedical Optics and Imaging - Proceedings of SPIE
StatePublished - 2006
Externally publishedYes
EventImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV - San Jose, CA, United States
Duration: 23 Jan 200625 Jan 2006

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceImaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV
Country/TerritoryUnited States
CitySan Jose, CA


  • Fluorescence calibration
  • Fluorescence microscopy
  • LED sources
  • Quantitative microscopy


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