What is fluorescence?
Wavelength Spectra
Excitation Spectra
Stokes' Shift
Advantages of Fluorescence Microscopy
Light Sources
Filters
The Path of Light
Staining Specific Cell Structures
Twelve Cell DAPI Stained Embryo
Related Links
References

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What is fluorescence?

     Fluorescence is the property of some atoms or molecules to  absorb light of a particular wavelength and re-emit later at a longer wavelength.  The interval between absorption and re-emission is termed the fluorescence  lifetime.

Wave length Spectra

• Light can be described as either a particle or a wave.
• Light normally consisits of a mixture of electromagnetic waves of many different lengths.
• As a wave, the energy of light can be quantified by wavelength.
• E(energy in ergs)=h(Planck's constant)*v(frequency of light)
• Shorter wavelengths have higher amounts of energy than longer wavelengths.
• The absorption of light is an all or nothing phenomenom.
• Fluorescent molecules can only absorb light of specific wavelengths,  known as absorption bands.

Excitation Spectra

• Absorption of light occurs very quickly (10^-15sec).
• Absorption excites fluorescent molecules from the ground state to an excited state.
• Fluorescent molecules relax almost immediately (10^-11sec) to a position referred to as the lowest excited singlet state.
• Thermal energy is given off during this first drop in energy.
• Molecules can "live" in the singlet state for short periods of time (10^-9).
• Relaxation from the singlet state to ground state with the emission of a photon (of lower wavelength) is fluorescence.
• Fluorescent molecules can repeat the excitation-emission process many times before excitation processes bleach the molecule.

Stokes' Shift

• Emission is generally shifted to longer wavelengths than absorption because emission occurs from very low energy singlet states.
• Stokes' shift allows a low number of emission photons to be detected against a background of large numbers of excitation photons, it allows for filtration.

Advantages of Fluorescence Microscopy

  Fluorescent microscopy has many advantages over normal microscopy, including:

• Specificity
• Sensitivity
• Quantization
• Environmental Sensitivity
• High Temporal Resolution
• High Spatial Resolution

Light Sources

• Our lab uses a mercury lamp.
• Mercury lamps most closely match the reds, blues, and greens most commonly used by biologists.

Filters

• The separation of excitation and emission wavelengths is achieved by the selection of proper filters to block or pass specific wavelengths of light.
• The design of fluorescence illuminators is based on control of excitation light and emission light by readily changeable filter insertions in the light path on the way toward the specimen and then emanating from the specimen.

The Path of Light

   Light follows a specific path through the microscope.

• Light Source
• Heat Filter
• Excitation Filter
• Dichroic Mirror
• Specimen
• Dichroic Mirror
• Emission Filter
• Eye or Camera

Specific Cell Structures

   A great many specific cell structures can be stained and viewed with a fluorescence microscope. These cell structures include Microtubules, Actin, DNA, Mitochondria, Golgi and ER.

Twelve Cell DAPI Stained Embryo
   This image is a twelve cell C. elegans embryo stained with DNA-binding DAPI. The picture was taken under 63x magnification with a fluorescence microscope.




Related Links:

http://www.microscopy.fsu.edu/primer/java/lightpaths/bx51fluorescence/bx51.html

http://www.itg.uiuc.edu/publications/techreports/99-006/

http://www.espn.go.com

References

Herman, Brian. Fluorescence Microscopy. Springer Pub.; New York, 1998.

The above links were also quite handy.

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This page was written and compiled by Ben Byrd.