OPTICAL MODIFICATIONS ENABLING SIMULTANEOUS CONFOCAL IMAGING WITH DYES EXCITED BY ULTRAVIOLET-WAVELENGTH AND VISIBLE-WAVELENGTH LIGHT

Optical modifications to a confocal scanning laser microscope are described which allow simultaneous fluorescence imaging of living specimens excited by ultraviolet (UV)- and visible-wavelength light. Modifications to a Bio-Rad MRC 600 Lasersharp confocal microscope include the introduction of UV-path-specific lenses and a specially designed UV transmitting eyepiece and tube lens. Upon UV excitation these modifications provide similar resolution and field flatness when compared with visible confocal microscopy. The UV-path-specific optics could be adjusted to correct for varying amounts of longitudinal chromatic aberration in commercially available objectives. Eyepiece and tube lenses were chromatically corrected for UV through visible wavelengths to minimize lateral chromatic error. With these modifications, UV-wavelength light may be used to excite ratioing dyes to quantify intracellular ion concentrations, or as an energy source to release caged compounds in a spatially restricted volume, while simultaneously imaging with dyes excited by visible-wavelength light.