Principles of Fluorescence Spectroscopy

56 INSTRUMENTATION FOR FLUORESCENCE SPECTROSCOPY
Figure 2.46. Effects of optical density on the fluorescence intensity of
quinine sulfate. The solid line (——) shows the measured intensities,
and the dashed line (– – –) indicates the corrected intensities, according
to equation (2.6) with OD em = 0. These data were obtained in a 1cm
2 cuvette that was centrally illuminated.
upon the optical densities of the sample at the excitation
and emission wavelengths.
The data for quinine sulfate in Figure 2.46 illustrates
the effect of optical density on fluorescence intensity. The
measured intensity is proportional to optical density only to
an optical density of 0.05. The linear range of the fluorescence
intensities could be expanded by using off-center
illumination, which reduces the effective light path. These
intensities can be approximately corrected for the inner filter
effects as follows. Suppose the sample has a significant
optical density at both the excitation and emission wavelengths,
OD ex and OD em , respectively. These optical densities
attenuate the excitation and emission by 10 –0.5ODex and
10 –0.5ODem , respectively. Attenuation due to absorption of the
incident light or absorption of the emitted light are sometimes
called the primary and secondary inner filter effects,
respectively. 56–57 The corrected fluorescence intensity is
given approximately by
F corr F obs antilog ( OD ex OD em
2
(2.6)
The corrected intensities for quinine sulfate are shown
in Figure 2.46, and these calculated values are seen to
match the initial linear portion of the curve. For precise corrections
it is preferable to prepare calibration curves using
the precise compounds and conditions that will be used for
the actual experimentation. Empirical corrections are typi-
)
Figure 2.47. Effect of concentrations on the intensity of anthracene.
Short pass refers to a 1 mm x 10 mm cuvette. Revised from [61].
cally used in most procedures to correct for sample absorbance.
56–60
Figure 2.47 shows the effect of anthracene concentration
on its emission intensity as observed for several
geometries. TIRF refers to total internal reflection, which is
described in Chapter 23. Short pass refers to a cuvette, 1 by
10 mm in dimension. The highest signal levels were
obtained with the standard right-angle geometry. The linear
range can be somewhat extended by using other geometries.
Intuitively we may expect that with the front-face geometry
the intensity will become independent of fluorophore concentration
at high concentrations. 60,62 Under these conditions
all the incident light is absorbed near the surface of the
cuvette. Front-face illumination is also useful for studies of
optically dense samples such as whole blood, or a highly
scattering solution. The intensity is expected to be proportional
to the ratio of the optical density of the fluorophore
to that of the sample. 63 When front-face illumination is used
the total optical density can be very large (20 or larger).
However, high fluorophore concentrations can result in
quenching due to a variety of interactions, such as radiative
and non-radiative transfer and excimer formation. Fluo-