Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 177
Figure 5.25. Frequency response of 4-dimethylamino-4'-bromostilbene
(DBS) up to 10 GHz. The vertical dashed lines are at 200 MHz,
2 GHz, and 10 GHz. From [3].
Figure 5.24 shows that the photodiode provides a higher
bandwidth than does any of the MCP PMTs. In fact, photodiode
detectors were used in several phase fluorometers
for measurements at high frequencies. 109–112 Unfortunately,
the small active area of photodiodes results in low sensitivity,
so that photodiodes are rarely used for fluorescence
spectroscopy.
The schematic for the 10-GHz instrument shown in
Figure 5.23 incorporates a ps laser as an intrinsically modulated
light source, and an MCP PMT as the detector. A
photodiode (PD) is adequate as the reference detector
because the laser beam can be focused on its small active
area. The use of cross-correlation allows measurement over
the entire frequency range from 1 MHz to 10 GHz without
any noticeable increase in noise. Cross-correlation allows
measurements at any modulation frequency at the same low
cross-correlation frequency, and avoids the need to measure
phase angles and modulation at high frequencies. A valuable
feature of cross-correlation is that the entire signal
appears at the measured frequency. Contrary to intuition,
one is not selecting one harmonic component out of many,
which would result in low signal levels. The use of crosscorrelation
provides absolute phase and modulation values
as if the excitation and detector were both modulated as
sine waves. A final favorable feature of this instrument is
that the modulation can be higher than 1.0, which is the
limit for sine wave modulation. At low frequencies where
the detector is fully responsive, the modulation can be as
high as 2.0. To understand this unusual result one needs to
examine the Fourier components for a pulse train.
5.6.1. Gigahertz FD Measurements
Several examples of gigahertz FD measurements will illustrate
the value of a wide range of frequencies. Short decay
times are needed to utilize the high-frequency capabilities.
113 Otherwise, the emission is demodulated prior to
reaching the upper frequency limit. A short decay time was
obtained using 4-dimethylamino-4'-bromostilbene (DBS)
in cyclohexane at 75EC (Figure 5.25). Because of the short
61-ps lifetime the phase and modulation data could be
measured to 10 GHz. The intensity decay was found to be a
single exponential. 3 The vertical dashed lines illustrate how
only a fraction of the frequency response could be explored
if the upper limit was 200 MHz, or even 2 GHz. It would be
difficult to detect additional components in the intensity
decay if the data stopped at 200 MHz, which would display
a maximum phase angle of 4.4E. An important aspect of
these measurements is that no measurable color effect has
been observed in the 10 GHz measurements. 106
5.6.2. Biochemical Examples of
Gigahertz FD Data
GHz measurements may seem exotic, but such data are
often needed for studies of routine biochemical samples.
One example is NADH. At 200 MHz the data only sampled
part of the frequency response (Figure 5.16). When measured
to higher frequencies one can more dramatically see
the difference between the one and two decay time fits (Figure
5.26). The decrease in χ R 2 for the two decay time model
is 400-fold. While we have not performed a support plane
analysis on these data, the α i and τ i values will be more
closely determined using the data extending above 200
MHz.
Another biochemical example is provided by the peptide
hormone vasopressin, which acts as an antidiuretic and