Using Filters to Capture Your Signal

For epifluorescence microscopes, the excitation filter, dichroic beam-splitter, and emission filter will usually all be housed in the same cube and will be designed to match the spectra of a specific fluorophore or fluorescent protein. Occasionally the filters will not be in a cube, but will be on a wheel, referred to as a turret. Turrets allow you to mix and match your excitation and emission wavelengths. The ultimate goal is to collect the light you want from the fluorophore bound to your target and not any light from other parts of the system (ambient light, excitation source, etc.). We refer to unwanted light that makes it to the detector as background fluorescence.

The different kinds of filters used in epifluorescence imaging

In general, a filter set will be designed to capture the maximum excitation and emission wavelengths of a given fluorophore, but will not capture all of the fluorescence. Most modern filter sets are designed so that the excitation filter has a defined band of wavelengths that it allows through. This style of filter is referred to as a band-pass filter. Band-pass filters are normally identified by the middle-value wavelength and the width of a band. In a simple epifluorescence microscope setup, once the excitation light leaves the excitation filter it is reflected onto the target. The fluorophores in the target become excited and then emit light that is shifted towards the red end of the spectrum (compared to the excitation light). Not all of this emitted light will be captured by the detector—only what is allowed through the dichroic beam-splitter and also passes through the emission filter. Emission filters are usually band-pass or long-pass filters, depending on the specific fluorophore and imaging experiment. A long-pass filter may be desirable if you need all the light beyond a certain wavelength to pass through to the detector.

Figure 1. Excitation and emission spectra of a nuclear dye (DAPI) overlaid with the range of wavelengths passed through the filters for excitation (purple box) and emission (blue box). The black line depicts the transmission of the dichroic filter.