How are fluorescence filters used?
Optical fluorescence occurs when a molecule known as a fluorophore (usually a fluorescent dye) absorbs light with wavelengths within its absorption band, and then nearly instantaneously emits light at longer wavelengths within its emission band. Fluorophores are specifically attached to biological molecules, to cell regions, and to other targets of interest to enable quantification, identification, and even real-time tracking of activity on a microscopic scale. Fluorescence is widely used in biotechnology and analytical applications due to its extraordinary sensitivity, high specificity, simplicity, and low cost compared to other analytical techniques.
Most fluorescence instruments, including fluorescence microscopes, use optical filters to control the spectra of the excitation light and emission light. Filters make it possible for the sample to "see" only light within the absorption band, and the detector to "see" only light within the emission band. Without filters, the detector would not be able to distinguish the desired fluorescence from scattered excitation light (especially within the emission band) and autofluorescence from the sample, substrate, and other optics in the system.
A system with a broadband light source, such as a fluorescence microscope, has three basic filters: an excitation filter, a
dichroic beamsplitter, and an emission filter. The exciter is typically a bandpass filter that passes only the wavelengths absorbed by the fluorophore, thus minimizing excitation of other sources of fluorescence and blocking light in the fluorescence emission band. The dichroic is an edge filter used at an oblique angle of incidence to efficiently reflect light in the excitation band and to transmit light in the emission band. The emitter is also typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band - especially the excitation light. Systems with laser illumination might or might not use an exciter or a dichroic, but most include some variation of these filters. The fluorescence filters function as a set to provide the optimum signal with minimal noise.
In most fluorescence instruments, the best performance is obtained with thin-film filters, as opposed to other types of fixed or tunable filters, such as those based on diffraction gratings. Thin-film filters comprise multiple thin layers of transparent materials with high and low indexes of refraction on a glass substrate. The complex layer structure determines the spectrum of light transmission by a filter - the more layers and the more precisely they are deposited, the more complex and accurately reproduced a desired spectrum can be made. Thin-film filters are simpler, are less expensive, and provide excellent optical performance: high transmission over an arbitrarily determined bandwidth, steep edges, and high blocking of undesired light over the widest possible wavelength range. Recent advances in thin-film filter technology permit even higher performance while resolving the longevity and handling issues that can affect filters made with older technology
What should I look for when selecting fluorescence filters?
