Use of UV excitation for confocal fluorescence microscopy in a conventional beam-scanning instrument

By making only minor modifications, we have adapted a conventional confocal scanning laser microscope for the recording of UV-excited fluorescence. An external argon ion laser provides the wavelengths 334, 351, and 364 nm for specimen illumination. In addition to substituting some optical components to obtain improved transmission and reflection properties in the UV, we have also adjusted the ray-path to compensate for the severe chromatic aberration of most conventional microscope objectives in the UV. We have also tested mirror objectives, which are inherently free from chromatic aberrations. However, since these objectives are not of the immersion-type, and furthermore have rather low numerical apertures, they are of limited value in biomedical applications. Using the modified instrument, we have recorded specimens labeled with AMCA and Fluoro-Gold. At present the instrument is capable of recording optical sections with a thickness of 1.5 micrometers when using an oil-immersion objective with a numerical aperture of 1.25.