Principles of Fluorescence Spectroscopy

174 FREQUENCY-DOMAIN LIFETIME MEASUREMENTS
Figure 5.19. Frequency response for IR-144 in ethanol (open symbols)
and DOTCI in ethanol (filled symbols). Best one-component fits
to each data are indicated by the lines. IR-144 and DOTCI were excited
with laser diodes at 790 and 670 nm, respectively. Revised and
reprinted with permission from [79], Copyright © 1992, American
Chemical Society.
complex laser source can be replaced with laser diodes, 78–79
and light-emitting diodes. 80–86 Frequency-domain measurements
have also been accomplished with electroluminescent
devices, 87 and even a modulated deuterium lamp. 88
Given the rapid advances with pulsed LDs and LEDs these
devices are likely to be the dominant excitation source for
FD measurements in the near future.
5.5.1. Laser Diode Excitation
The output of laser diodes can be modulated up to several
GHz. Hence, laser diodes can be used for FD excitation
without the use of a modulator. Data are shown in Figure
5.19 for two laser dyes—IR-144 and DOTCI—which were
excited with 791- or 670-nm laser diodes, respectively (Figure
5.19). Frequency-doubled laser diodes have also been
used to obtain shorter excitation wavelengths near 400
nm. 89 However, the need for frequency-doubled LDs has
diminished given the availability of LDs and LEDs with
fundamental outputs ranging from 280 to 820 nm.
5.5.2. LED Excitation
It is now known that LEDs can be modulated to several
hundred MHz. 85–86 Hence, LEDs are becoming an alternative
to modulated arc lamps. The modulated output of a
390-nm LED output was used to measure the 3.2-ns decay
time of green fluorescent protein (not shown) and the 11.8ns
lifetime of 9-cyanoanthracene 85 (Figure 5.20).
The use of a simple light source such as an LED is likely
to find use in analytical chemistry and clinical chemistry.
84–85 This is illustrated in Figure 5.21 for the potassi-
Figure 5.20. Intensity decay of 9-cyanoanthracene in ethanol using
the 390-nm output of a UV LED as the excitation source. Revised
from [85].
um-sensitive probe CD 222. This probe has a lifetime of
0.15 ns in the absence of potassium and 0.67 ns in the presence
of bound potassium. The frequency response could be
measured up to 300 MHz using a modulated UV LED at
373 nm. The possibility of measuring nanosecond decay
times using modulated LEDs, and the availability of a wide
range of wavelengths, suggests these light sources will be
used for low-cost FD instruments in the near future.
Another application of LEDs will be for excitation of
the longer-lived metal–ligand complexes (Chapter 20). The
LEDs are ideal because the 450-nm output is centered on
the 450-nm charge-transfer absorption of the ruthenium
complexes. The shorter wavelengths are suitable for excitation
of the higher-quantum-yield rhenium complexes (Figure
5.22). In this case the entire frequency response of
[Re(dpphen)(CO) 3 ) (4-COOHPy)] + was measured in the
absence (10.24 :s) or presence of oxygen (598 ns). 84
Figure 5.21. Frequency response of the potassium probe CD 222
measured with a 373-nm LED ("). From [84].