Introduction 

Fluorescent proteins and dyes provide a powerful toolkit to biological researchers. Fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) and red fluorescent protein (RFP) can be tagged to proteins of interest with a cell or organism. This then allows the visualisation and tracking of these proteins in a fixed (dead) or live context. In addition to fluorescent proteins a large range of fluorescent dyes are available to specifically mark cell types, organelles etc. The range of available fluorescent proteins and dyes is growing every year.
Whether a fluorophore is a fluorescent protein or dye the way it produces its fluorescence is the same.

Basic Physics

The fluorescence produced by a fluorophore is a result of electrons within the fluorophore being raised to a higher energy state by the excitation wavelength. When the electrons fall back to a lower energy state fluorescence of the emission wavelength is emitted.

The light emitted (fluoresced) will always be of a longer wavelength than that used for excitation (but still specific).  This difference in wavelength is known as the Stokes’ Shift. Below is an example fluorescent spectrum for eGFP. Notice the two distinct peaks; the difference between them represents the Stokes’ Shift.

Exitation and Emission

All fluorophores have a maximum excitation and emission wavelength. The maximum excitation wavelength is the wavelength that will result in the maximum emission of fluorescence. Excitation at other wavelength may result in fluorescence but it will not be optimal. To achieve optimal excitation and emission, filters made from coated glass are used to provide the specific wavelengths required.

Light for excitation is provided from either a mercury or xenon arc lamp (in confocal microscopy a laser of the excitation maximum is used). This light is passed through the microscope to a cube containing three filters; an excitation filter, a dichroic mirror and an emission filter. The following figures show the light path through a filter cube.

Light from the light source is passed through the excitation filter (blue). The excitation filter only allows light of the specific wavelength of excitation through, in this example blue light. The excitation light is then reflected off of a dichroic mirror (yellow). Dichroic mirrors will reflect some wavelengths of light while allowing others to pass through. The reflected light is passed through the objective of the microscope to the sample.

The light fluoresced from the sample passes back through the objective and through the dichroic mirror.

Finally the light passes through the emission filter (green), where it is “cleaned up”. The final light is specific for the emission wavelength of the fluorophore and can then be passed through to the ocular of the microscope or another type of detector (e.g. a camera).

These filters can be overlayed onto the previous eGFP spectra, as shown below. Only part of the spectrum is highlighted, the rest is rejected by the filters. This allows for precise illumination and detection of fluorescent molecules. Filter sets exist that can detect two or more fluorophores at the same time. These filter sets still contain the same three components (emission, dichroic and excitation filters) but these filters are created to let multiple bands of light pass through.

Bleaching

All fluorophores will eventually bleach, this means it will no longer fluoresce. Some fluorophores (such as FITC) will bleach very rapidly while others (such as Alexa dyes) will be more resistant. To limit bleaching of the sample anti-fade reagents can be added to the mounting media (such as Prolong, CitiFluor etc).
To minimise bleaching the length of time the excitation light is shone on the sample should be kept as short as possible. Using high transmittance objectives and sensitive detection systems will help minimise bleaching.

Phototoxicity

When imaging live cells or tissue phototoxicity can become an issue. Free radicals are produced during the fluorescence process; these reactive oxygen species can then kill the cell. Phototoxicity can be reduced, or eliminated, by reducing the amount of fluorescent light applied to the cells or tissue. Additionally free radical scavengers, such as ascorbic acid or DTT, can be added to the culture media to reduce phototoxic effects.