Applied Optics · 1994 · 184 citations · 6 references
Natural SciencesPhysicsBiomedical OpticsBiophotonicsOpticsChromatic AberrationConfocal MicroscopyFluorescence ImagingFluorescence MicroscopyLight MicroscopyOptical ImagingAxial Chromatic AberrationChromatic Focal PointChromatic-aberration InteractionFocal Plane OpticsFocusMicroscope ImagingMicroscopy
In the image of a confocal microscope, only those parts of an object appear bright that are located in the focal plane of the objective. Because of an axial chromatic aberration deliberately introduced into the microscope objective, the location of the focal plane depends on the wavelength used. By using a white-light source and examining an object with a depth variation less than the axial range of the chromatic focus, we find that all parts of the object appear sharp and bright in the image, but according to its height they appear in different colors. A camera with black-and-white film sequentially combines, with three selected chromatic filters, intensity and tone of color of each object point. For each tone of color one can assign a height by using a calibration curve. This assignment could be made unequivocal by the selection of filters with adequate chromatic transmission.
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