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Fluorescence microscope

A fluorescence microscope is similar to a light microscope. Instead of shining the whole spectrum of visible light through the specimen, the fluorescent microscope only shines a specific wavelength on the sample. This can be achieved via an excitation filter that only allows the light of a specific wavelength to reach the sample, or by using lasers which only emit a specific wavelength. The wavelength matches with the absorption spectrum of the fluorophore, which then emits a longer wavelength that is used to produce an image. The observation is best performed in the dark for clearer detection of fluorescent signals and to avoid bleaching the slide, since the fluorescent dyes are light-sensitive.

The elements of internal structure of the fluorescence microscope. Number 1 presents a lightbulb and is called a light source.Next to it, a rainbow coloured, transparent tube is shown, depicted by number 2. At the other end of the tube, a blue circular disc is shown, called an excitation filter. Purple arrow goes through that filter to the left and up to grey thin horizontally aligned rectangular with number 3, called sample objective. From that rectangular, blue arrow goes down on a slightly skewed yellow rectangular surface with number 4, called dichroic mirror. The arrow passes through the mirror, first on the yellow disc below it with number 5 that is called emission filter, then to another blue disc, then it goes further down to last, slightly skewed blue disc with number 6, called mirror. Here it gets reflected, and goes left towards the number 7 with the name 'eyepiece’. The path is shown in simplicity on the microscope image on the left. The arrow starts at number 1, goes left to number 2, goes up to number 3, turns left and down to number 4, then goes down through number 5 and 6, then turns left and up to number 7.

Figure 1: A schematic overview of the key components of a light microscope: light source, excitation filter, dichroic mirror, and emission filter.

Fluorescent microscopes are equipped with a carousel of filter cubes. The cubes consist of an excitation filter, a dichroic mirror, and an emission filter. The combination of these filters matches the excitation and emission wavelengths of certain fluorophores. Therefore, the filter cubes are specific for a certain stain.

From the light source, the light reaches an excitation filter that only allows light of the specific wavelength that excites the fluorophore to pass through. These light waves hit a dichroic mirror that reflects the excitation wavelength light towards the sample, where it excites the fluorophore. The light emitted shifts to a longer wavelength and is collected by the objective lens. When the light reaches the dichroic mirror, this longer wavelength light passes through the towards the eyepieces and/or camera. Scattered light of other wavelengths, however, is reflected by the dichroic mirror and cannot be detected via the eyepieces and/or camera. The emission filter between the dichroic mirror and the camera filters out all other (background) wavelengths.

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Article
Fluorescence Microscopy