Fluorescence Filter Types
Exciters, emitters, & dichroics - OH MY!
The primary filtering element in an epifluorescence microscope is the set of three filters housed in a fluorescence filter cube (or filter block): the excitation filter, the emission filter, and the dichroic beamsplitter.
The excitation filter (also called the exciter) transmits only those wavelengths of the illumination light that efficiently excite a specific dye. Although shortpass filter designs were used in the past, bandpass filter designs are now used almost exclusively. The emission filter (aka barrier filter or emitter) attenuates all of the light transmitted by the excitation filter and very efficiently transmits any fluorescence emitted by the specimen. This light is always of longer wavelength (more to the red) than the excitation color. These can be either bandpass filters or longpass filters. The dichroic beamsplitter (aka dichroic mirror or dichromatic beamsplitter) is a thin piece of coated glass set at a 45-degree angle to the optical path of the microscope. This coating has the unique ability to reflect one color, the excitation light, but transmit another color, the emitted fluorescence. Current dichroic beamsplitters achieve this with great efficiency, i.e., with greater than 90% reflectivity of the excitation along with approximately 90% transmission of the emission. This is a great improvement over the traditional gray half-silvered mirror, which reflects only 50% and transmits only 50%, giving only about 25% efficiency.
The spectra characteristics of a particluar fluorochrome can be overlayed with the spectral profile of a fluorecence filter set to aid in selecting the most appropriate filter set for any given application. Figure 7 illustrates what the spectral profile of an "ideal" filter set might look like for the given flourochrome excitation and emission specta. As you can see, the excitation filter transmits light spanning the absorbtion spectrum, and the emission filter transmits a band of wavelengths spanning the peak emission of the fluorochrome. The dichroic attenuates the exitation light and transmits only that from the emission.
Once an individual exciter, emitter, and dichroic is chosen for the circumstances, they are mounted in a filter cube and are ready for use in a microscope. The schematic of a typical filter cube for an inverted microscope is illustrated in figure 8, above. Most microscopes have a slider or turret that can hold from two to four individual filter cubes. It must be noted that the filters in each cube are a matched set, and one should avoid mixing filters and beamsplitters unless the complete spectral characteristics of each filter component are known.
Common filter blocks are named after the type of excitation filter: UV or U (Ultraviolet excitation for dyes such as DAPI and Hoechst 33342), B (Blue excitation for FITC and related dyes), and G (Green excitation for TRITC, Texas Red®, etc.). Common barrier filter colors are blue or pale yellow in the U-block, green or deep yellow in the B-block, and orange or red in the G-block.