An enhanced method of obtaining uniform excitation radiation for fluorescence microscopy

Chris F. Dimas; John J. Kuta; Manfred Hubert

doi:10.1117/12.543942

15 December 2003 An enhanced method of obtaining uniform excitation radiation for fluorescence microscopy

Chris F. Dimas, John J. Kuta, Manfred Hubert

Proceedings Volume 5260, Applications of Photonic Technology 6; (2003) https://doi.org/10.1117/12.543942
Event: Applications of Photonic Technology, 2003, Quebec City, Québec, Canada

Abstract

A novel lightsource to provide the excitation radiation for fluorescence microscopy is presented and its performance is compared to the current de factor standard in the field: mercury short arc lamps. This novel light source is remote to the microscope, and the radiation is coupled to the microscope via a liquid lightguide or fiber optic cable using special coupling optics. We present measurements made on some common fluorescent microscopes that show the new light source provides for higher overall optical power delivered to the sample and provides more uniform illumination of the microscopes' field of view in comparison to the standard short arc lamps. Using the definition of the Koehler illumination rules it is shown that the inherent design of the remote source makes it resistant to many non-uniformities and misalignments commonly enountered with the short arc lamp sources; thereby providing for a consistent, uniform irradiance and intensity distribution of the entrance pupil to the microscope. The experimental method used to quantitatively measure the uniformity of the excitation radiation at the microscope's objective plane is also discussed and shown to be far more reliable than other techniques which rely upon fluorescent radiation from synthetic samples placed at the objective plane.

Citation Download Citation

Chris F. Dimas, John J. Kuta, and Manfred Hubert "An enhanced method of obtaining uniform excitation radiation for fluorescence microscopy", Proc. SPIE 5260, Applications of Photonic Technology 6, (15 December 2003); https://doi.org/10.1117/12.543942

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