For fluorescence microscopy it is essential to separate light with different spectral properties for the excitation of a fluorophore and the detection of its emission. Filtering is used for instance to attenuate the intensity of excitation light, to select the desired frequencies for fluorophore excitation and to separate light that is being emitted from fluorescent probes for multi-channel image acquisition. Knowledge about the spectral properties of the used fluorophores as well as the choice of suitable filter settings is absolutely essential for a valid image acquisition and to avoid spectral crosstalk between different channels.
There are the following major groups of filter or separation devices used in modern light microscopes, conventional glass filters, acousto-optic tunable filters and spectroscopic devices.
Glass filters are coated with layers of material that has specific, defined optical properties. It absorbs or reflects specific wavelengths, whilst light of different wavelengths is being transmitted. Classically, spectral separation is achieved by using a set of sequential filters with suitable optical properties. The most widely used filters are:
Neutral density filters attenuate the transmitted light across the whole visible spectrum and are usually used to adjust light from excitation sources to the right intensity. They are usually used to adjust the amount of excitation light in conventional widefield system, e.g. at the outlet of a arc vapour lamp.
Bandpass filters transmit a defined spectral band of light whilst blocking all light frequencies below and above the band. These serve to narrow down the wavelengths for fluorophore excitation or for the final filtering step of the emitted light. According to the general nomenclature, a bandpass filter BP520-545 would transmit a spectrum of 520 to 545 nm.
Longpass and shortpass filters have similar properties as bandpass filters, except they exclusively transmit light at long or short wavelengths, respectively. The wavelength at which they cut on (LP) or cut off (SP, or KP in German systems) is usually indicated as the cut on/off frequency. A longpass filter LP620 would therefore absorb all light below and transmit light above 620 nm.
Dichromatic or polychromatic filters are reflective for some wavelengths, whereas they transmit others. These filters are used to separate excitation from emission light and to filter emitted light into different spectral channels for detection.
For filter-based microscope systems it is important to know that the filters should be treated with great care. They must never be touched and only cleaned by trained staff with lens cleaning tissue and liquid. The complex filter coating underlies an ageing process, which means they should be checked for their spectral properties on a regular basis and changed when they start deteriorating. If not, they might become 'leaky' and potential bleed-through problems might occur and render imaging results useless.
Acousto-optical filters (AOTFs) rely on a special crystal that changes its optical properties in the presence of an acoustic wave. Light that passes through this crystal can be diffracted by applying an acoustic wave, which causes a frequency-dependent change in the refractive index of the crystal.
For setting up the correct filtering configuration of the microscope you use for your fluorescence image acquisition, the knowledge of the following parameters and physical proeprties is essential to guarantee optimal results:
- excitation and emission spectra of the fluorophores
- laser line or filter setting for fluorophore excitation
- filtering of the emitted fluorescence
- potential intrinsic fluorescence background in the sample
Based on these parameters the filter configuration should be set up, tested and optimised for each experiment. Suitable configaurations can be saved on all of the systems for subsequent re-use.