18 Apr 2022
Fluorescence filters are at the heart of every Fluorescence optical device, and high quality filter sets are essential for high definition imaging and fluorescence microscopy. At Shanghai Optics we produce fluorescence filters for a wide range of applications in the form of filter cubes and filter wheels.
Fluorescence microscopes are designed to detect the fluorophores in a sample; either naturally occurring fluorophores or those used as dyes and labels. These fluorophores absorb energy in specific wavelengths (excitation range), and emit it in a different wavelength (emission range). This allows us to identify fluorophores by absorption and emission.
More precisely, as each fluorophore has a unique excitation spectrum and a unique emission spectrum, each peaking at a specific wavelength, they can be distinguished by either their excitation spectrum, by their emission signal, or by their Stokes shift, which is the distance between peak excitation and peak emission. This is the basic principle behind all fluorescence microscopy systems, whether it be a simple system such as a epifluorescent microscope or a more complex setup such as those used in multiphoton systems.
Fluorescence Filter Sets
Each fluorescence filter cube features three distinct optical filters: an excitation filter a dichroic mirror and an emission filter.
The excitation filter, otherwise called an exiter, is a bandpass filter designed to pass only the wavelengths of light which are absorbed by the fluorophore. By doing so, it minimizes excitation that might be caused by the light source and other sources of fluorescence and also blocks excitation wavelength light in the flurorecence emission band. The minimum transmission of this filter is what will determine the brightness or brilliance of the final image. In general, an excitation filter should have a minimum of 40% transmission; transmission of greater than 85 percent will give a bright, well-lit image. Optical density determines the background darkness, and should be at minimum 3.0 but preferably closer to 6.0. The excitation filter should be chosen so that the center wavelength (CWL) is close to the peak excitation of the fluorophores, and the bandwith is completely within the fluorophores excitation range.
The dichroic mirror is a specialized edge filter sometimes called a dichromatic beamsplitter. When used at a 45 angle of incidence, it is able to reflect light in the wavelengths of the excitation band and transmit light from the emission band to the emitter and on to the detector.
The third component of the fluorescence filter is the emitter. This is also an bandpass filter, and passes exclusively light emitted by the fluorophore. Excitation light and all other light outside, both UV an IR, are blocked out effectively by the emitter filter.
1. A fluorescence filter set includes an excitation filter, an emission filter, and a dichroic filter. The figure above shows the coating curves of an excitation filter designed for passing excitation range 480nm~ 500nm, emission filter designed for passing emission range 515nm ~ 545nm, and dichroic filter (i.e. long pass filter) designed for reflecting excitation wavelength and passing emission wavelength.
2. The figure above shows a transmission plot of an excitation filter which is a narrowband pass filter designed for passing excitation range 420nm~ 500nm and blocking the wavelength range 550nm ~1100nm.
3. The figure above shows a transmission plot of an emission filter which is a narrow bandpass filter with FWHM of 40nm and center wavelength at 475nm.
Our standard high performance fluorescence filter cubes are carefully designed to pass the appropriate wavelengths for high-brightness imaging while blocking irrelevant light that might decrease contrast. Since both system autoFluorescence and excitation light is blocked, our filters are able to provide a high signal to noise ratio and the dark background needed for precise imaging.
When a bright image is more crucial than high contrast, a longpass edge filter may be used as an emission filter in place of the bandpass filter. Longer wavelengths of light will then be transmitted, producing a well-lighted brighter imager.
When a multiband excitation filter is combined with a multiband emission filter and a polychroic rather than dichroic beamsplitter image with all fluorescent tags will be formed. This type of filter set is referred to as full-multiband and can be used with multiple fluorophores.
Choosing a Fluorescence Filter
At Shanghai Optics we produce a wide range of fluorescence sets that are designed with the excitation and emission filters suitable for each commonly used fluorophore. For specialized applications, such as those using multiple fluorophores, alternate dichroic filters or laser sources, we are able to produce custom filter sets upon request. Please contact us to discuss the specific requirements of your application and to get a free consultation and a price quote.
Fluorescence Filter Factory Standards
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