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

doi:10.1117/12.638559

21 February 2006 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

Author Affiliations +

Proceedings Volume 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV; 60880U (2006) https://doi.org/10.1117/12.638559
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

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.

Citation Download Citation

I. T. Young, Y. Garini, B. Vermolen, G. Liqui Lung, G. Brouwer, S. Hendrichs, M. el Morabit, J. Spoelstra, E. Wilhelm, and M. Zaal "Absolute fluorescence calibration", Proc. SPIE 6088, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV, 60880U (21 February 2006); https://doi.org/10.1117/12.638559

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