1.6 Theory of Operation
1.6 Theory of Operation
This section includes the following topics:
•
1.6.1: Introduction to Fluorometry
•
•
1.6.1 Introduction to Fluorometry
Fluorescence is the light emitted by certain substances resulting from the absorption of
incident radiation. Fluorescent materials, or materials that absorb light energy of a
characteristic wavelength (excitation), undergo an electronic state change, and
instantaneously emit light of a longer wavelength (emission). Most common fluorescent
materials have well-characterized excitation and emission spectra.
The first figure below shows an example of excitation and emission spectra for a
fluorophore. In general, excitation and emission bands are broad, with half-bandwidths
of approximately 40 nm, and wavelength differences between the excitation and emission
maxima (the Stokes shift) is relatively small, about 30 nm. Exceptions to this rule include
the lanthanide dyes used in time-resolved fluorescence: they have a Stokes shift that is
typically about 300 nm. There is considerable overlap between the excitation and
emission spectra (gray area) when a small Stokes shift is present.
Excitation
Maximum
Emission
Maximum
Stokes
Shift
1.0
0.5
0
500
550
600
650
Wavelength (nm)
Relative Fluorescence
Excitation and Emission Spectra
Because the intensity of the excitation light is usually many tens of thousands of times
greater than that of the emitted light, some type of spectral separation is necessary to
reduce the interference of the excitation light with detection of the emitted light. The
FlexStation II instrument incorporates many features designed to restrict interference
from reflected excitation light. Among these features is a set of band-pass cutoff filters
that may be set automatically by the system or manually by the user. In addition, if the
Stokes shift is small, it may be advisable to choose an excitation wavelength that is as far
FlexStation II Operator’s Manual – Rev. D
19
Summary of Contents for FlexStation II
Page 6: ...FlexStation II Operator s Manual Rev D vi ...
Page 92: ...4 Applications 86 FlexStation II Operator s Manual Rev D ...
Page 102: ...5 Service and Maintenance Procedures 96 FlexStation II Operator s Manual Rev D ...
Page 124: ...6 Troubleshooting Procedures 118 FlexStation II Operator s Manual Rev D ...
Page 134: ...7 Appendices 128 FlexStation II Operator s Manual Rev D ...
