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5 March 2008 Photonic calibration for fluorescence microscopy
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Based upon a collection of compact LEDs (light-emitting diodes) and a compact photodiode, we have developed a calibration tool for fluorescence microscopes that are used as digital imaging devices. The entire device (excluding a USB connector) measures 25 mm × 80 mm × 12 mm. Virtually all commonly-used fluorophores can be simulated with one of the six LEDs. An LED is chosen from the host computer and its current range is selected (digitally) so as to provide a test of the complete dynamic range of the imaging system. Thus by varying the current through an LED in a controlled way, a controlled amount of "emission" light can be produced, transmitted through the chosen optical path of the microscope, and measured by the image sensor. The digitized intensity can then be determined as a function of the LED current. Any other (fluorescence) intensity measured through the same electro-optical path can then be characterized (and thus calibrated) by an equivalent electrical current. The excitation light is calibrated by a photodiode which has a dynamic range of 10^5:1 and thus is suitable for a variety of light sources: mercury lamps, lasers, LEDs, etc. The integration time of the photodiode as well as its gain can be digitally selected from the host computer. Further, using a Spectralon® reflector, the inherent non-linearity of the LED emission versus current can be measured by the photodiode and used to provide a look-up table compensation independent of the image sensor used in the fluorescence microscope system.
© (2008) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Ian T. Young, Mohammed el Morabit, Guus Liqui Lung, and Bart J. Vermolen "Photonic calibration for fluorescence microscopy", Proc. SPIE 6859, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI, 685915 (5 March 2008);

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