Principles of Fluorescence Spectroscopy

164 FREQUENCY-DOMAIN LIFETIME MEASUREMENTS
Figure 5.7. Schematic representation of the variable-frequency phasemodulation
fluorometer. P, polarizer; SB, Soliel-Babinet compensator;
F, frequency; δF, cross-correlation frequency; PMT, photomultiplier
tube.
Consequently, the early phase-modulation fluorometers
operated at only one to three fixed modulation frequencies.
The limited data these instruments provided were adequate
for measuring mean decay times, or for detecting the presence
of a complex decay. However, the data at a limited
number of modulation frequencies were not generally useful
for resolution of the parameters describing multi- or
non-exponential decays.
As has occurred for TCSPC, pulsed-laser diodes
(LDs), and light-emitting diodes (LEDs) are becoming the
preferred excitation source. The output of the LDs and
LEDs can be modulated directly by the electrical input.
This eliminates the need for the electrooptic modulator,
which adds cost and complexity to the FD instruments.
5.2.2. An MHz Frequency-Domain Fluorometer
Frequency-domain fluorometers are now in widespread use.
Most designs are similar to that shown in Figure 5.7. The
main differences are the laser light source, the light modulator,
and the associated radio-frequency electronics. Without
the use of LDs or LEDs it is difficult to obtain light
modulation over a wide range of frequencies. Amplitude
modulation of laser sources over a continuous range of frequencies
to 200 MHz is possible with electrooptic modulators.
Light can also be modulated with acoustooptic modulators.
However, acoustooptic modulators provide modulation
at discrete resonances over a limited range of frequen-
cies. 54–56 Most electrooptic modulators have long narrow
optical apertures, and electrooptic modulators are not easily
used with arc lamp sources. Initially, only laser sources
seemed practical for use with electrooptic modulators. Surprisingly,
it is now possible to use electrooptic devices to
modulate arc lamps to 200 MHz, which is done in commercial
FD instruments. The operational principles of the modulators
and the electronic parts needed to construct such an
FD instrument are discussed below, along with the rationale
for selecting the various components.
The light source for an FD instrument can be almost
any continuous-wave (cw) light source or a high-repetitionrate
pulse laser. The choice of source is based on the needed
wavelengths and power levels. The He–Cd laser is a convenient
cw source, providing cw output at 325 and 442 nm.
Unfortunately, these wavelengths are not suitable for excitation
of protein fluorescence. A very versatile source is the
Ar ion laser, which can now provide deep UV lines (-275
nm) for intrinsic fluorescent probes. However, only a limited
number of UV wavelengths are available. Studies of protein
fluorescence usually require 290 to 300 nm to avoid
excitation of tyrosine and to obtain high fundamental
anisotropies. These wavelengths are not available from an
argon ion laser. The argon ion laser at 514 nm is an ideal
source for pumping dye lasers. The 514 nm line can be
mode-locked to synchronously pump a picosecond dye
laser system (Section 4.4). An Nd:YAG laser can also be the
primary source, particularly for pumping dye lasers.
Appropriate electronics are needed to measure the
phase angle and modulation at high frequencies. The measurements
appear difficult because the resolution of multiexponential
decays requires accuracy near 0.2E in phase
and 0.5% (0.005) in modulation, and that this accuracy
needs to be maintained from 1 to 200 MHz. In fact, the
measurements are surprisingly easy and free of interference
because of cross-correlation detection. In cross-correlation
detection the gain of the detector is modulated at a frequency
offset (F + δF) from that of the modulated excitation (F).
The difference frequency (δF) is typically in the range of 10
to 100 Hz. This results in a low-frequency signal at δF that
contains the phase and modulation information in the original
high-frequency signal (Section 5.11.2). At all modulation
frequencies, the phase and modulation are measured at
the same low cross-correlation frequency (δF). The use of
cross-correlation detection results in the rejection of harmonics
and other sources of noise. The newer FD instruments
use signal processing boards that extract the values
from the digitized low-frequency signal.