Introduction to Fluorescence Filters

Fluorescence occurs when a molecule absorbs light at wavelengths within its absorption band, and then emits light at longer wavelengths within its emission band. For example, brightly fluorescent molecules (called fluorophores) can be attached to biologically significant molecules in e.g. cell membranes, in the brain, or even on subunits of DNA, whose structures become visible in a fluorescence microscope that allows us to track the way cells function in health and disease. Fluorescence is widely used in biology, biotechnology, and medicine, due to its extraordinary sensitivity, high specificity, and simplicity of usage.

Fluorescence microscopy, and most instruments that use fluorescence, including fluorescence microscopes, rely on optical filters. A typical microscope has three basic filters: an excitation filter (or exciter), a dichroic beamsplitter (or dichroic mirror), and an emission filter (or emitter). These three filters form what is referred to as a “filter set” and are often housed in a special assembly called a “filter cube” that can be quickly mounted in a microscope.

The excitation filter is usually a bandpass filter designed to pass only those light source wavelengths that are to be absorbed by the fluorophore, thereby minimizing excitation of other sources of fluorescence, and especially blocking excitation light in the fluorescence emission band.

The dichroic is an edge filter (usually passing longer wavelength light and reflecting shorter wavelength light) used at an oblique angle of incidence (typically 45°) to efficiently redirect light in the excitation band towards the sample and transmit light in the emission band towards the detector.

The emitter is usually a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light.

The main difference between an excitation filter vs emission filter? An excitation filter lets in light to excite a molecule, while an emission filter transmits the light the molecule emits.

An optimal optical filter set of the above three individual filters blocks unwanted excitation light (including in the UV and IR) as well as unwanted emitted light (including autofluorescence) to ensure high signal and low background.

The filter set described above can only be used for one fluorophore, since it has only one passband each for excitation and for emission. If for example two fluorophores are used, the set will need to have two passbands each for exciter, dichroic, and emitter. These are called multiband sets; for more information, review our White Paper.

What are important characteristics of filter sets?

The type of experiment being performed often determines which type of filter set should be used in the microscope:

• High Brightness: This type maximizes the detected signal, even at the expense of higher background.
• High Contrast: This type maximizes image contrast between bright areas and background, i.e., for high signal-to-background ratios.
• Narrow passbands: When using multiple fluorophores, reducing crosstalk, and/or decreasing background signal, narrow passbands ensure only the desired signal from the desired fluorophore is captured.
• Wide passbands: When using a weakly emitting fluorophore or imaging single molecules, wider passbands allow more excitation light to the sample and capture more emission.

To summarize, filter sets strike a balance between high brightness and high contrast, typically by using narrower or wider passbands in combination with center wavelength placement.