Principles of Fluorescence Spectroscopy

170 FREQUENCY-DOMAIN LIFETIME MEASUREMENTS
and modulation of the background. A least-squares fit of the
phase and modulation data for the background is performed
and for the parameter values to calculate φ TB and m TB . This
latter procedure is useful if the background file is not measured
at the same modulation frequencies as the sample. In
the presence of background the observed values of N T obs
and D T obs are given by
N obs
ω (1 f B)m ω sin φ ω f Bm ωB sin φ ωB
D obs
ω (1 f B) m ω cos φ ω f B m ωB sin φ ωB
(5.20)
(5.21)
In these equations f B is the fraction of the total signal due to
the background, and φ T and m T are the correct phase and
modulation values in the absence of background. The corrected
values of N T and D T are given by
Nω Nobs ω fBNωB 1 fB Dω Dobs ω fBDωB 1 fB (5.22)
(5.23)
In using these expressions the values of φ TB and m TB are
known from the measured frequency response of the background.
The value of f B is found from the relative steadystate
intensity of the blank measured under the same instrumental
conditions and gain as the sample. Except for
adjusting the gain and/or intensity, the values of φ TB and
m TB should be measured under the same conditions as the
sample. The corrected values of N T and D T can be used in
eqs. 5.9 and 5.10 to calculate the corrected phase and modulation
values. An important part of this procedure is propagation
of errors into the corrected data file. Error propagation
is straightforward but complex to describe in detail. 69
5.4. REPRESENTATIVE FREQUENCY-DOMAIN
INTENSITY DECAYS
5.4.1. Exponential Decays
It is informative to examine some typical frequency-domain
measurements. 71 Frequency-domain intensity decays for
anthracene (AN) and 9-cyanoanthracene (9-CA) are shown
in Figure 5.12. The samples were in equilibrium with
atmospheric oxygen. The emission was observed through a
long pass filter to reject scattered light. Magic-angle polarizer
conditions were used, but this is unnecessary for such
samples where the emission is completely depolarized. The
excitation at 295 nm was obtained from the frequency-doubled
output of a rhodamine 6G dye laser, cavity dumped at
3.8 MHz. This pulse train provides intrinsically modulated
excitation over a wide range of frequencies (Section 5.6).
Experimental FD data are shown in Figure 5.12 (upper
panel). The increasing values are the frequency-dependent
phase angles (φ T ) and the decreasing values are the modulation
values (m T ). The solid lines are calculated curves for
the best single-decay-time fits. In the single-exponential
model the decay time is the only variable parameter. The
shape of a single-decay-time frequency response is always
the same, except that the response is shifted to higher frequencies
for shorter decay times.
The lower panels show the deviations between the data
(!, ") and the fitted curve (solid line). In this case the deviations
are presented in units of degrees and percentage
modulation. For calculation of χ R 2 the standard errors were
taken as δφ = 0.2 and δm = 0.005. The fact that the values
of χ R 2 are close to unity reflects an appropriate choice for
the values of δφ and δm. In analysis of the FD data the
absolute values of χ R 2 are variable due to the unknown
Figure 5.12. Single-exponential decays of anthracene (AN) and 9cyanoanthracene
(9-CA). Samples were equilibrated with atmospheric
oxygen. δφ = 0.2 and δm = 0.005. From [71].