Filters fall into three main classes: bandpass, long pass, or short pass. A bandpass filter transmits wavelengths within a region of wavelengths "cutting on" at a low wavelength and "cutting off" at a higher wavelength. Wavelengths are blocked below the cut-on or above the cut-off by absorption and/or reflection. The bandpass or bandwidth (BW) is the peak width in nanometers (nm) at 50% of the transmission maximum for a given peak. It may also be referred to as FWHM (full width half maximum) or HPBW (half power bandwidth). A long pass filter transmits wavelengths above a specified value and blocks wavelengths below this value.
A short pass filter does the opposite of the long pass and therefore transmits wavelengths below a specified value while blocking wavelengths above that specified value. Interference filters are made by vacuum deposition of thin films on a glass substrate. Thin films are comprised of alternating layers of metal or dielectric with high and low refractive indices. Interference filterscan be designed to be short pass, long pass, or bandpass filters. Colored glass filters are made from dye impregnated glass and generally are long pass or bandpass. Filters can contain multiple elements of the colored glass and/or interference type stacked and cemented to each other with optical epoxy (laminated). If a filter contains any interference elements it is referred to as an interference filter.
Interference filters are highly desirable with their narrow bandwidths, high transmission efficiency and extremely sharp cut-ons and cut-offs. These filters are more expensive, less durable and generally have higher signal/noise (S/N) than colored glass filters. Colored glass filters generally have a wide bandwidth (i.e., 40nm for FITC exciter), lower transmission efficiency and trailing cut-on and cut-offs. Colored glass filters are used because they are readily available, less costly and more durable.
Excitation filters are normally bandpass filters. Excitation filters can be of the colored glass or interference type. The greater the bandwidth the greater the excitation energy. As bandwidth increases there is the accompanying increase in emission intensity, but a greater chance of generating non-specific autofluorescence.
Dichroics are interference filters. Dichroics are manufactured using thin film technology, but since they need to be kept thin for high image quality they are not protected with a laminated cover glass. Unprotected dichroics are easily scratched and damaged. With Zeiss Axioline filters, the user must take care in mounting filter sets and only handle the dichroic by the edges with gloved hands.
Emission filters may be bandpass or long pass.
Emission filters can be the colored glass or interference type. Long pass filters pass the most light resulting in brighter, but noisier images with higher background and lower signal/noise (S/N). This increase in noise and background results from cell debris, the microscope slide, etc.