Fluorescence microscope

  A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption.  

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In most cases, a component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore (such as green fluorescent protein (GFP), fluorescein or DyLight 488). The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter (see figure below). The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. In this manner, a single fluorophore (color) is imaged at a time. Multi-color images of several fluorophores must be composed by combining several single-color images.

Most fluorescence microscopes in use are epifluorescence microscopes (i.e. excitation and observation of the fluorescence are from above (epi) the specimen). These microscopes have become an important part in the field of biology, opening the doors for more advanced microscope designs, such as the confocal laser scanning microscope and the total internal reflection fluorescence microscope (TIRF).

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care must be taken to prevent photobleaching through the use of more robust fluorophores or by minimizing illumination.

Epifluorescence microscopy

Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences. The excitatory light is passed from above through the objective onto the specimen instead through the specimen into the objective. Since only reflected excitatory light filters through, the transmitted light is filtered out, giving a much higher intensity. Fluorescent or fluorochrome stains are applied to the specimen to provide an estimated count.  

This image uses epifluorescence to image three components of a dividing human cancer cell. DNA is stained blue, a protein called INCENP is green and the microtubules are red. Each fluorophore is imaged separately using a different combination of excitation and emission filters. The images are captured sequentially using a digital CCD camera, then overlaid to give a complete image.

Epifluorescence microscopy can be used to find routine direct total counts of bacteria in water samples.

See also

• Microscope
• Mercury-vapor lamp
• Xenon arc lamp
• Stokes shift

References

• Bradbury, S. and Evennett, P., Fluorescence microscopy., Contrast Techniques in Light Microscopy., BIOS Scientific Publishers, Ltd., Oxford, United Kingdom (1996).
• Rost, F. and Oldfield, R., Fluorescence microscopy., Photography with a Microscope, Cambridge University Press, Cambridge, United Kingdom (2000).