Photonic calibration for fluorescence microscopy

Abstract

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.