Bio-Rad VersaFluor™ Fluorometer - Manual (Page 10)

Section 4
Introduction
4.1 Fluorometer System
The VersaFluor Fluorometer measures the concentration of various samples (analytes)
by fluorescence. By labeling a molecule with a fluorescent dye, the molecule can be accurately
quantitated by measuring the amount of fluorescence. A fluorescent molecule has the ability
to absorb energy at particular wavelengths and emit this energy at longer wavelengths.
Fluorescent dyes are classified by their excitation and emission wavelengths. Figure 4.1 shows
a typical excitation and emission spectrum. The excitation curve is at a shorter wavelength than
the emission curve. It is desirable to have no overlap between the two curves, insuring that all
the energy in the emission spectrum is a result of fluorescence.
The excitation and emission spectrums vary from dye to dye. The VersaFluor Fluorometer
has the capability to work with various dyes by using removable optical filters which select
for the specific excitation and emission wavelengths of a particular dye.
Fig. 4.1. Excitation and emission spectra of fluorescein.
For example, fluorescein has an excitation maxima at 490 nm and an emission maxima
at 520 nm (Figure 4.1). The EX490/10 excitation filter and the EM520/10 emission filter,
both of which have a 10 nm band pass, are used with fluorescein. The excitation filter allows
only light between 485 and 495 nm to pass through the sample. The emission filter removes
the excitation light and allows only the emitted light with a wavelength between 515 and 525 nm
to reach the detector.
The optical system of the VersaFluor Fluorometer is diagrammed in Figure 4.2. The
quartz halogen lamp emits light in the 350 nm to 900 nm range. Light emitted by the quartz
halogen lamp passes through a small aperture in the lamp housing before passing through the
excitation filter. The excitation filter allows only specific wavelengths of light to pass through.
450 500 550 600 400
Wavelength (nm)
Excitation
Emission
7
Wavelength (nm)
Excitation
400 450 500 550 600
Emission
Relative
Fluorescence