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important. Today, software often needs to be sufficiently flexible to adapt to varied appli-
cations, yet simple enough for the increasing number of users with limited spectroscopic
backgrounds. More sophisticated software packages provide users with several options in
data display and analysis. This must include the possibility to record, display, calculate, and
then utilize spectral response curves on the excitation and emission channels. In addition,
the software must be suitably flexible to provide these correction curves under a variety
of different operating conditions such as changes in spectral bandpass, scan steps, signal
levels, and so on. The task is not straightforward and attention to detail is critical. Too often
the instrument software from manufacturers will assume “black box” operation and pre-
sent a “result” without the rigor of knowing whether it is the “true” result or whether it is
influenced by the optical spectral variations in instrument to instrument caused by spectral
properties of the light source, the illumination and collection optics, and the wavelength
performance of the analysing spectrometer and detector.
It is the successful combination of the light sources, light discriminators, coupling
optics, and sampling and detection methods that provides an optimal system. This hard-
ware package, coupled with a complete robust software analysis package and the correct
instrument calibration and correction functions, is the path to a successful instrument and
successful fluorescence measurements.
5.4.8 Instrument Performance Validation
In many laboratories, the performance validation of a fluorimeter instrument is still in its
infancy, and the influence of the many parameters involved in the instrument operation is
often not considered principally because it is thought that the instrument manufacturers
have taken care of this problem in their software. In reality instrument performance valida-
tion must also be the responsibility of the users to ensure the instrument meets their needs
and has not drifted with time. With this in mind, users might undertake a series of standard
measurements using one or more of the following:
Instrument calibration standards - physical sources such as line and tungsten lamp emis-
sions, known instrument independent luminescence standards, and so forth
Application standards - such as spectrally matched and known fluorophores whose prop-
erties are close to those of the samples being measured.
In many cases, quantitative fluorescence assessment often consists of undertaking some
intensity calibration of the instrument response with respect to the concentration of some
“standard” fluorophore in a certain solvent. These titration type experiments are a common
way to “calibrate” an instrument but they rely critically on very precise determinations of
the fluorophore purity, solvent purity, initial concentration determinations, dilutions meth-
ods, pH and temperature control, and so forth. The whole process is fraught with possible
sources of both random and systematic errors and of course is applicable only to that par-
ticular instrument at that time.
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