10 May 1996 New epifluorescence microscope providing pairs of specific fluorescence images of double-stained cells for simultaneous visual perception and for quantification
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Abstract
A new epi-fluorescence microscope for analysis of cells stained with two fluorochromes which can be spectrally isolated is described. The system makes it possible to perform independent and specific spectral selection of each dye (e.g. DAPI and CY3) while perceiving the two specific images simultaneously by eye. The optics uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm and 546 nm in the excitation light path, 435-500 nm and 590- 750 nm in the emission light beams), (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) the possible separate positioning of lenses for compensation of chromatic aberrations. The system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was attained with good temporal resolution. It has been shown that it is feasible to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected. Quantitative and independent measurements of the two fluorescence images by a CCD camera synchronized with the light chopper are feasible. In conclusion, this imaging system is outlined for highly specific visual analysis and exact quantitative measurement of double fluorescence labeled specimens in cytology and histology.
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Thomas Heiden, Thomas Heiden, Bernhard Tribukait, Bernhard Tribukait, } "New epifluorescence microscope providing pairs of specific fluorescence images of double-stained cells for simultaneous visual perception and for quantification", Proc. SPIE 2678, Optical Diagnostics of Living Cells and Biofluids, (10 May 1996); doi: 10.1117/12.239507; https://doi.org/10.1117/12.239507
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