In fluorescence microscopy, the excitation light is generally passed through a bandpass interference filter to select a specific band of wavelengths that are used to illuminate the fluorophore. Depending upon the filter characteristics and the light source, the amount of light available for excitation can vary by a wide margin. This interactive tutorial is designed to enable the visitor to choose between various ZEISS filter sets and common microscope illumination sources to determine the optimum combination for a specific application.
The tutorial initializes with a metal halide lamp spectrum being displayed in the window overlaid by the bandpass region of a Zeiss 00 filter set (Texas Red). The relative intensity of the light source is listed on the ordinate while the spectral width of the tutorial (300 to 700 nanometers) constitutes the abcissa. To operate the tutorial, use the Filter Set pull-down menu to choose a different filter set and the Light Source menu to choose between the available sources (metal halide, mercury, xenon, and LEDs).
The emission profiles of mercury and metal halide arc discharge lamps are distinct from those of the xenon and LED sources in that several prominent emission lines are present in the ultraviolet, blue, green and yellow spectral regions. These spectral lines are significantly brighter (up to 100 times) than the continuous background. When selecting filter sets, those excitation filters that overlap a spectral line will produce a greater level of excitation. In selecting filters to match LED spectral profiles, those diodes that best overlap the filter bandpass are highlighted on the left-hand side of the tutorial window.