Question

In a fixed source/detector spectro-fluorometer, what can be done to increase the sensitivity of the optical emission signal?

Answer 1

Spectro-fluorometer is a highly sensitive analytical technique,
so one of the primary specifications to consider when
selecting a spectrofluorometer is its sensitivity. The overall
sensitivity of a spectrofluorometer is determined by many
factors including, but certainly not limited to, the optical
design, coupling optics, intensity of delivered excitation
light, efficiency of fluorescence collection, spectrometer
design, detector technology and much more. For
example, a system that has a higher wattage lamp, does
not guarantee that it offers better overall fluorescence
sensitivity.
Given that there are so many variables that go into
making a sensitive fluorometer, what is needed is a
standard reference test that can be performed by any
user, to properly compare one fluorometer with another.
In the past, some commercial manufacturers of
fluorometers used detection limits for specific fluorescent
molecules, such as quinine sulfate or fluorescein to
demonstrate sensitivity. However, today, the highest
sensitivity fluorometers are able to detect at such
low concentrations of fluorophores, that the ability
to accurately perform a serial dilution down to these
detection limits becomes questionable. As such, the
water Raman test has become an industry standard as a
good measure of the relative sensitivity between different
instruments. The water Raman test is also preferable
because ultrapure water is readily available around the
world, the sample is stable, the signal is relatively weak,
and the Raman band of water can be measured over the
entire wavelength range of the instrument allowing for
more robust comparisons, than is possible with a single
fluorescent probe.
This sensitivity specification is derived from an emission
spectrum of the Raman vibrational band for pure water.
Typically it is acquired with the excitation wavelength
selected at 350 nm, with an emission scan from 365
to 450 nm. In general, the sensitivity of the fluorometer
is expressed in a signal-to-noise ratio (SNR) that is a
comparison of a signal value in the presence of a signal,
with a value for system noise, in the absence of signal.