New epi-fluorescence optical system for independent analysis of two different fluorochromes in microscopy

A new epi-fluorescence optical system is described that uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. The optical system was constructed for analysis of two different fluorochromes, e.g., DAPI and TRITC. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm, 546 nm, 435-500 nm, and 590-750 nm), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet-excitation/blue-detection and green-excitation/red-detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) separate positioning of lenses for compensation of chromatic aberrations. This system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was obtained with good temporal resolution. It has been shown that it is feasable to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected.