Principles of Fluorescence Spectroscopy

146 TIME-DOMAIN LIFETIME MEASUREMENTS
Figure 4.58. Intensity decay of green fluorescence protein. From
[207].
GFPs are widely used as a tag to follow gene expression.
The intensity decay of GFP was measured with 365-nm
excitation, a 1.25-MHz repetition rate, and magic-angle
polarizer conditions. 207 The emission was detected above
500 nm, using an MCP PMT.
The intensity decay of GFP could be well fit to a single
exponential (Figure 4.58). The value of χ R 2 is slightly elevated,
and not consistent with a single exponential model.
However, the value of χ R 2 was not decreased by including a
second decay time (χ R 2 = 1.18). Examination of the deviations
(lower panel) reveals the presence of systematic oscillations
for which a second decay time does not improve the
fit. The failure of χ R 2 to decrease is typically an indication
of systematic error as the origin of the elevated value of χ R 2.
4.13.3. Picosecond Decay Time
The measurement of picosecond decay times remains challenging
even with the most modern instruments for TCSPC.
Figure 4.59 shows a schematic for a state-of-the-art instrument.
208 The primary source of excitation is a Ti:sapphire
laser, which is pumped by a continuous argon ion laser. The
repetition rate is decreased as needed by a pulse picker
(PP). Additional excitation wavelengths are obtained using
a harmonic generator (HG) for frequency doubling or
tripling, or an optical parameter oscillator (OPO). The pulse
Figure 4.59. Intensity decay of DASPI in methanol. The upper panel
shows a schematic of the instrument with a Ti:sapphire-OPO pump
source at 543 nm and an R3809U MPC PMT. DASPI is 2-[2-[3dimethylamino)phenyl]-ethyl]-N-methyl
pyridinium iodide (DASPI).
Revised from [208].
widths were near 1 ps. This instrument has an R3809U
MCP PMT that has one of the smallest available transient
time spreads.
The lower panel in Figure 4.59 shows the intensity
decay of DASPI in methanol. DASPI has a very short decay
time in this solvent. The intensity decay is not much wider
than the IRF, which has an FWHM below 28 ps. The decay
time recovered for DASPI is 27.5 ps. Comparison of the
IRF and intensity decay of DASPI shows the need for
deconvolution. In spite of the complex profile of these
curves, they are consistent with a single-exponential decay
of DASPI with a 27.5 ps lifetime.
4.13.4. Chlorophyll Aggregates in Hexane
The intensity decay for the tryptophan residues in the tet
repressor was relatively close to a single exponential. Intensity
decays can be much more heterogeneous. One example