Fluorescence filters that control which wavelengths of light are reflected or transmitted through a microscope system. They allow researchers to isolate and observe specific fluorescent signals that would otherwise be invisible under normal illumination.
By isolating excitation and emission wavelengths, the filters make it possible to detect specific structures or molecules tagged with fluorescent dyes.
Types of Fluorescence Filters
The primary filtering elements in a fluorescence microscope consist of three optical filters, typically mounted within a filter cube or slide:
Excitation Filter
Dichroic Beamsplitter (Mirror)
Emission Filter
In contemporary fluorescence microscopes, these filters are typically interference filters, providing high precision and wavelength selectivity.
The Excitation Filter
When fluorescent materials are illuminated by ultraviolet or visible light, they emit fluorescence. The excitation filter—also known as the exciter—selects specific wavelengths of light from the illuminator that are required to excite the fluorophore within the specimen.
Historically, excitation filters were short-pass types, but modern systems primarily use band-pass filters. These transmit only the wavelengths that correspond to the absorption band of the target fluorophore.
The transmission efficiency of the excitation filter determines the brightness of the resulting image. To produce bright, well-illuminated images, excitation filters should generally achieve at least 40% transmission at the desired wavelength.
The Emission Filter
Also known as the barrier filter or emitter, the emission filter blocks unwanted light outside the emission band of the fluorophore. It ensures that only the light emitted by the fluorophore passes through to the detector or eyepiece, effectively eliminating background noise and enhancing contrast.
Emission filters may be long-pass or band-pass, depending on the desired emission spectrum. By blocking stray and scattered light, these filters create a dark background, allowing the fluorescent signal to appear bright and distinct.
Emission filters vary in shape, size, and material composition—commonly made of optical glass, or metal-coated substrates. They are typically fixed components, meaning they require minimal maintenance and provide long-term reliability within the microscope system.
The Dichroic Beamsplitter
The dichroic beamsplitter, or dichroic mirror, is an edge filter that selectively reflects and transmits light based on wavelength. Positioned at a 45° angle of incidence, it reflects shorter excitation wavelengths toward the specimen while allowing longer emission wavelengths from the fluorophore to pass through to the detector.
This design enables efficient separation of excitation and emission light, a crucial function for high-quality fluorescence imaging. Dichroic mirrors are also used to combine or separate multiple light colors for multi-fluorophore imaging systems.
Reduce light scattering and optical noise
Block unwanted wavelengths and ambient light
Direct light precisely toward the specimen
Enable detection of multiple fluorophores by switching filters on a motorized filter wheel
Increase contrast for high-clarity imaging
Remove harmful UV or IR radiation
Adjust light intensity and correct optical path issues
Isolate excitation wavelengths for selective fluorophore activation
By combining excitation and emission filters, fluorescence microscopes can detect multiple fluorescent dyes in rapid succession with high precision — a key advantage in multi-channel imaging.
Excitation Filters: Denoted as EX or X; may include Ultraviolet Glass (UG) or Blue Glass (BG) types.
Emission Filters: Denoted as M or BA; may include Red Glass (R or RG) or Yellow Glass (Y or GG) variants.
Dichroic Filters: Also referred to as DM (Dichroic Mirror), BS (Beamsplitter), CBS (Chromatic Beamsplitter), RKP (Reflectance Short Pass)
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