Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 145
Figure 4.56. Global lifetime χ R 2 surface for the three-component mixture
of In, AA, and 2-AP. From [187].
length global analysis the uncertainties are significant. For
instance, the value of the 4.1-ns lifetime can range from
about 3.2 to 5.5 ns and still be consistent with the data.
4.13. APPLICATIONS OF TCSPC
The concepts described in the preceding sections can be
made more understandable by examination of some specific
examples.
4.13.1. Intensity Decay for a Single Tryptophan
Protein
The tet repressor controls the gene in Gram-negative bacteria
that provides resistance to the antibiotic tetracycline. 206
This protein usually contains two tryptophans, but a mutant
protein was engineered that contains a single tryptophan
residue at position 43. Intensity decays are shown in Figure
4.57. The light source was a frequency-doubled R6G dye
laser at 590 nm, frequency doubled to 295 nm. The dye
laser was cavity dumped at 80 kHz. The excitation was vertically
polarized and the emission detected through a polarizer
set 54.7E from the vertical. The use of magic-angle
polarization conditions is essential in this case because the
protein can be expected to rotate on a timescale comparable
to the intensity decay. A Schott WG 320 filter was used in
front of the monochromator to prevent scattered light from
entering the monochromator, which was set at 360 nm.
The emission was detected with an XP-2020 PMT.
This PMT shows a wavelength-dependent time response
and an afterpulse. To avoid color effects the authors used a
Figure 4.57. Intensity decay of trp-43 in the tet repressor protein F75
TetR at 360 nm. The calibration is 108 ps/channel. Revised and
reprinted with permission from [206]. Copyright © 1992, American
Chemical Society.
short-lifetime reference that shifted the wavelength to the
measurement wavelength with minimal time delay. 112 This
was accomplished with a solution of p-terphenyl highly
quenched by CCl 4 . The fact that the measurements were
performed with a dynode PMT is evident from the width of
the impulse response function, which appears to be near
500 ps. Some of this width may be contributed from the
short lifetime standard.
The intensity decay was fit to the one, two, and three
exponential models, resulting in χ R 2 values of 17, 1.6, and
1.5, respectively. Rejection of the single-exponential model
is clearly justified by the data. However, it is less clear that
three decay times are needed. The ratio of the χ R 2 values is
1.07, which is attributed to random error with a probability
of over 20% (Table 4.3). The fractional amplitude of the
third component was less than 1%, and the authors accepted
the double exponential fit as descriptive of their protein.
4.13.2. Green Fluorescent Protein:
Systematic Errors in the Data
Green fluorescent protein (GFP) spontaneously becomes
fluorescent following synthesis of its amino-acid chain.