Friday, September 2, 2011

Fluorescence Microscopy

By using fluorescence or phosphorescence, a fluorescence microscope allows the study of organic or inorganic substances. Whereas traditional light microscopes use only reflection or absorption to view specimens, fluorescence microscopes illuminate a sample with light of a wavelength that causes fluorescence in the sample. This light is a longer wavelength than the illumination, and is then detected through the microscope objective.

Two filters are typically used in fluorescence microscopy:
  1. An illumination (excitation) filter – ensures the illumination is near monochromatic and at the correct wavelength.
  2. Second emission (detection) filter – ensures none of the excitation light source reaches the detector.
Fluorescence microscopy takes a different approach to creating a light microscope image to transmitted or reflected white light techniques such as phase contrast or differential interference contrast. These two contrasting optical microscopy methods provide very different, but complementary data.

So how does a fluorescence microscope work?

The specimen is illuminated with light of a specific wavelength that is absorbed by the fluorophores, causing them to emit light of longer wavelengths (in other words a different color than the absorbed light). The illumination light is separated from the weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope include:
  1. Light source (xenon arc lamp or mercury-vapor lamp)
  2. Excitation filter
  3. Dichroic mirror (or dichromatic beamsplitter)
  4. Emission filter
The filters and dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore (color) used to label the specimen. The distribution of a single fluorophore is imaged at one time.

Most fluorescence microscopes are “epi-fluorescence microscopes”.  Epi refers to the fact that the excitation and observation of the fluorescence are from above the specimen.

 Fluorescence microscope image of mouse embryo.