What filter is used in fluorescence microscopy?
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Filters Used in Fluorescence Microscopy
Fluorescence microscopy is a powerful tool used to visualize structures and processes in biological specimens. Its sensitivity and specificity rely on the use of filters that control the wavelengths of light reaching the specimen and the detector. These filters are essential for distinguishing between the fluorescence signal and the background light.
Types of Filters in Fluorescence Microscopy
- Excitation Filter: This filter is placed in the light path before the light reaches the specimen. It selectively allows only the wavelengths that can excite the fluorophores in the sample, while blocking others.
- Emission Filter: Situated in the path between the sample and the detector (like a camera or an eyepiece), the emission filter allows only the light emitted by the fluorophores to pass, thus improving the contrast of the image.
- Dichroic Mirror (or Beam Splitter): The dichroic mirror is a special filter that reflects one range of wavelengths while allowing another range to pass through. It is placed at an angle in the optical path and is responsible for redirecting the excitation light towards the specimen and then allowing the fluorescent light emitted by the specimen to pass through towards the detector.
Function of Filters in Fluorescence Microscopy
The primary function of these filters is to ensure that the light used to illuminate the specimen is of a specific wavelength or range of wavelengths. These filters help to separate the excitation light from the emitted fluorescence, thereby enhancing the quality of the final image. The excitation filter lets through only the wavelengths that match the excitation spectrum of the fluorophore, while the emission filter blocks the excitation wavelength and transmits only the specific wavelengths corresponding to the fluorescence of the specimen.
Without these filters, the emitted light would be overwhelmed by the excitation light and unwanted background fluorescence, making it difficult or impossible to discern the specific signal from the noise.