Fluorescence microscopy

In fluorescence microscopy, a specimen is typically labeled with a fluorescent molecule, or fluorophore. When illuminated with light of a specific wavelength, the fluorophore can be excited and will subsequently emit light of a longer wavelength. This emitted light is detected by a CCD, and the image is captured.

The light source for the fluorescence microscope often produces a wide spectrum of light, and an excitation filter can be used to limit the illumination light to a specific wavelength range appropriate for the sample. An emission filter is useful for blocking any light outside of the range of the emitted wavelength, thus improving the signal to noise ratio of the acquired image. The animation on the upper right illustrates the light path in an upright microscope. Using a filter wheel, as shown in the animation (center) allows for rapid imaging of two different fluorophores.

The CCD (charge-coupled device) is made up of a two-dimensional array of photodiodes which convert light into electrical charge. When the exposure is completed, the charges are transferred, one-by-one, into an amplifier and converted into a voltage. Each photodiode in the CCD gives rise to a pixel in the final image, where the recorded voltage values are converted to black/white pixel intensities. The resolution of the final image is directly proportional to the number of photodiodes in the CCD. This is illustrated in the animation below.

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Excitation & Emission
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Filter Wheel
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Many thanks to Jennifer Waters (HMS Nikon Imaging Center) for collaborating on this project.

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This work by Janet Iwasa is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.