Fluorescence Microscope - Labster Theory

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.

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.