Principles of Fluorescence Spectroscopy

172 FREQUENCY-DOMAIN LIFETIME MEASUREMENTS
Figure 5.14. Frequency-domain intensity decay data for GFPuv in
0.05 M phosphate buffer, pH = 7, for one-photon excitation at 400 nm,
and for two-photon excitation at 800 nm. From [72].
Figure 5.15. The frequency responses shift to higher frequencies
with increasing amounts of chloride, indicating a
decrease in lifetime. One can use the data to calculate the
decay times at each chloride concentration. These lifetimes
are 25.5, 11.3, 5.3, and 2.7 ns, for 0, 10, 30, and 70 mM
chloride, respectively (Problem 5.1).
5.4.5. Intensity Decay of NADH
Reduced nicotinamide adenine dinucleotide (NADH) is
known to display a subnanosecond decay time near 0.4 ns.
Figure 5.15. Frequency-domain intensity decays of SPQ in the presence
of 0, 10, 40, and 70 mM chloride.
Figure 5.16. Frequency response of NADH dissolved in 0.02 M Tris
(pH 8) 25°C. The excitation wavelength was 325 nm from an HeCd
laser, which was modulated with an electrooptic modulator. The emission
filter was a Corning 0-52. For the two-component analysis f 1 =
0.57 and f 2 = 0.43. Revised and reprinted from [2], Copyright © 1985,
with permission from Elsevier Science.
Its intensity decay is complex, with decay times near 0.3
and 0.8 ns. 77 Frequency-domain data for NADH are shown
in Figure 5.16. The presence of more than one lifetime is
immediately evident from the failure of the single exponential
fit (– – –) and the systematic deviations ("). Use of the
two decay time model resulted in a 50-fold decrease of χ R 2.
The frequency-domain data for NADH illustrate a limitation
of the commercially available instruments. An upper
frequency of 200 MHz is too low to determine the entire
frequency response of NADH or other subnanosecond
intensity decays. For this reason FD instruments were
developed to allow measurements at higher modulation frequencies.
5.4.6. Effect of Scattered Light
A critical component of any frequency-domain or timedomain
experiment should be collection of emission spectra.
One possible artifact is illustrated in Figure 5.17, which
shows the emission spectrum of 9,10-diphenylanthracene
(DPA) in a solution that also scattered light. 69 9,10-
Diphenylanthracene was dissolved in ethanol that contained
a small amount of Ludox scatterer. When the emission is