Chemistry Reference
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fluorescence spectrum peaking around 540 nm (Figure 12.22d). Such emission and
fluorescence decay time values of about 4 ns are characteristic for the PMI chromo-
phore [91,129]. The bleaching of TDI followed by emission from PMI is seen as a
change in the value of
F
from 1 to nearly 0 (Figure 12.22c, upper panel) and therefore
as a change in FRET efficiency from 100% to zero because of the disappearance
(photobleaching) of the acceptor chromophore.
In 5% of the probed single molecules of compound
, the bleaching of the TDI
core is not followed by the emission from PMIs but from an intermediate species with
a fluorescence spectrumpeaking around 590 nm and as a result of this
F
values around
0.5 are obtained for this intermediate species. This intermediate species is probably an
oxidized TDI chromophore with a reduced extent of conjugation and therefore with
spectrally blue-shifted absorption and emission properties compared to an undam-
aged TDI chromophore. After bleaching of the intermediate, fluorescence is mainly
detected in the PMI channel.
In order to probe the FRET dynamics from the NMI chromophores, single
molecules of compound
13
were excited with 360 nm laser light and the fluorescence
emitted by the PMI and TDI chromophores was simultaneously monitored. Within a
population of 100 single molecules, compound
13
showed either exclusive TDI
emission (80%, Figure 12.23a) or TDI emission followed by activity in both channels
(20%, Figure 12.23c). None of the interrogated compound
13
molecules showed
emission solely in the PMI channel. Also it was observed that, for similar excitation
power, the survival time of single molecules of compound
13
13
excited at 360 nm is by
far shorter than that of the same molecules excited at 490 nm. Figure 12.23b shows the
probability densities of the survival times for single molecules of compound
13
undergoing excitation at 360 nm (upper panel) and 490 nm (lower panel). Exponential
fits to the histograms from Figure 12.23b give time constants of 23 s (360 nm
excitation) and 158 s (490 nm excitation). This difference in survival times can be
explained by the difference in fluorescence quantum yield of 0.98 and 0.1 for PMI and
NMI, respectively. For NMI, assuming that nonradiative deactivation is mainly
related to triplet formation [133], for a quantum yield of fluorescence of about 0.1
and a fluorescence lifetime of 0.33 ns, the rate constant and the quantum yield for ISC
are estimated to be around 3
10
10
s
1
and 0.9, respectively. When NMI is part of the
triad and 360 nm excitation is used, NMI fluorescence is quenched from 0.33 to
0.04 ns by energy transfer to the TDI core, either directly or in a cascade fashion.
Therefore, for a single triad molecule, optical excitation of an NMI chromophore will
open competitionmainly between triplet formation and direct energy transfer. Having
such a high quantumyield of triplet formation, the probability of photobleaching of an
NMI chromophore on 360 nm excitation becomes considerably higher than the
probability of photobleaching of a PMI chromophore on 490 nm excitation. Conse-
quently, the survival time of a single triad molecule becomes substantially shorter on
360 nm excitation than on 490 nm excitation (Figure 12.23b).
Shown in Figure 12.23c is an example of a single triad molecule where, on 360 nm
laser excitation, bleaching of the TDI is followed by activity in both TDI and PMI
channels. For this molecule, bleaching of TDI leads to a drop in the fractional intensity
from 1 to 0.5 (Figure 12.23c), pointing to the intermediate species that was also
