Fluorescence microscope

An upright fluorescence microscope (Olympus BX61) with the fluorescent filter cube turret above the objective lenses, coupled with a digital camera.

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A fluorescence microscope (colloquially synonymous with epifluorescence 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.^[1][2]

An inverted fluorescence microscope (Nikon TE2000) with the fluorescent filter cube turret below the stage. Note the orange plate that allows the user to look at the sample while protecting his eyes from the excitation UV light.

Technique

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).^[1] 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.^[1] 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.^[1]

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 microscope and the total internal reflection fluorescence microscope (TIRF). The Vertico SMI combining localisation microscopy with spatially modulated illumination uses standard fluorescence dyes and reaches an optical resolution below 10 nanometers (1 nanometer = 1 nm = 1 × 10⁻⁹ m).

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, by minimizing illumination, or by introducing a scavenger system to reduce the rate of photobleaching.

Epifluorescence microscopy

Schematic of a fluorescence microscope.

Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences. The excitatory light is passed from above (or, for inverted microscopes, from below), through the objective and then onto the specimen instead of passing it first through the specimen. (In the latter case the transmitted excitatory light reaches the objective together with light emitted from the specimen). The fluorescence in the specimen gives rise to emitted light which is focused to the detector by the same objective that is used for the excitation. A filter between the objective and the detector filters out the excitation light from fluorescent light. Since most of the excitatory light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and this method therefore gives an improved signal to noise ratio. A common use in biology is to apply fluorescent or fluorochrome stains to the specimen in order to image a protein or other molecule of interest.

Gallery

Epifluorescent imaging of the three components in 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, and the images are captured sequentially using a digital CCD camera, then overlaid to give a complete image.

Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial (BPAE) cells

human lymphocyte nucleus stained with DAPI with chromosome 13 (green) and 21 (red) centromere probes hybrydized (Fluorescent in situ hybridization (FISH))

Yeast cell membrane visualized by some membrane proteins fused with RFP and GFP fluorescent markers. Imposition of light from both of markers results in yellow colour.

See also

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

References

Further reading

• Bradbury, S. and Evennett, P., Fluorescence microscopy., Contrast Techniques in Light Microscopy., BIOS Scientific Publishers, Ltd., Oxford, United Kingdom (1996).
• Rost, F., Quantitative fluorescence microscopy. Cambridge University Press, Cambridge, United Kingdom (1991).
• Rost, F., Fluorescence microscopy. Vol. I. Cambridge University Press, Cambridge, United Kingdom (1992). Reprinted with update, 1996.
• Rost, F., Fluorescence microscopy. Vol. II. Cambridge University Press, Cambridge, United Kingdom (1995).
• Rost, F. and Oldfield, R., Fluorescence microscopy., Photography with a Microscope, Cambridge University Press, Cambridge, United Kingdom (2000).

External links

• WikiScope
• Fluorophores.org - Database of fluorescent dyes.
• The Advanced Cell Classifier project (ETH Zurich)

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