What is the purpose of the excitation filter?

Excitation Filter: Purpose and Function

An excitation filter is a critical component in fluorescence microscopy and related optical systems that functions to selectively transmit a specific wavelength range of light towards a specimen that is fluorescently labeled.

Function of Excitation Filter

• Fluorophore Excitation: The primary function of the excitation filter is to allow only the light of a specific wavelength that matches the excitation spectrum of the fluorophore to pass through. This is crucial for ensuring that the fluorophore is efficiently excited.
• Reduction of Background Noise: By excluding other wavelengths that do not contribute to the fluorophore excitation, the filter minimizes background fluorescence and noise, which enhances the contrast and clarity of the images captured.
• Protection of the Sample: Excitation filters also protect the sample from exposure to unnecessary and potentially damaging wavelengths of light, which could cause photobleaching or photo-damage, preserving the integrity of the specimen.
• Improved Spectral Separation: In multi-label fluorescence applications, different excitation filters can be used in conjunction with dichroic mirrors and emission filters to cleanly separate the excitation and emission spectra of different fluorophores. This ensures that the signals from multiple fluorophores can be discriminated without overlap.

Example of Excitation Filter Usage

In the context of fluorescence microscopy, an excitation filter is placed within the light path before the light reaches the sample. When a mercury or xenon lamp is used as the light source, the broad spectrum of light is initially filtered by the excitation filter to provide only the wavelengths necessary to excite the specific fluorophore. This light then illuminates the sample, and the fluorophore absorbs and subsequently emits light at a longer wavelength which is filtered once more by an emission filter before being detected, resulting in a high-contrast image of the sample's fluorescence.