María García-Parajó - Former memberJoost-Anne Veerman - Former member|
(Published in Physics Review Letters, 83, p2155 1999)
Single molecule detection is a powerful method to observe intriguing quantum phenomena that are
generally hidden in the ensemble: photon-bunching, discrete photobleaching, spectral and
rotational jumps and rare intermediate conformations. Moreover single molecules can be exploited
as reporters to probe the dynamics of the local nano-environment.
Recently we have observed molecular quantum jumps in real-time at room temperature using single
molecule detection at ms time resolution. Our experimental approach involves the excitation of
individual molecules by a local sub-wavelength light source, the aperture of a near-field optical
probe. Images are obtained by collecting fluorescence while scanning the probe. Alternatively we
position the sub-wavelength source directly above a molecule in order to record the photodynamics
of a single molecule in real-time. With a typical signal of 105-106 photon
counts/s about 50 µs time resolution is achievable. All molecules are illuminated and monitored
until their irreversible photobleaching after emitting typically 106 photons.
An individual carbocyanine molecule is shown in Figure 1, together with a 15 ms fluorescence
time trace. Immediately the on-off blinking of the emission is noted. The blinking can be
understood by considering the three-level scheme in Figure 2. Besides the repetitive transitions
between the singlet states, giving rise to fluorescence, the molecule has a small chance to make
a spin flip into the triplet state. The chance to leave the triplet state is equally small. The
fluorescence is interrupted as long as the triplet state remains occupied and only after decaying
to the singlet ground state fluorescence restarts. Thus the fluorescence photons are emitted in
bunches separated by dark periods: blinking.
During the total observation time, until the unavoidable photobleaching, thousands of triplet
excursions occur (Figure 3). A histogram of the duration of all dark periods yields an exponential
decay where the decay time represents the triplet lifetime of that particular molecule. For a
large set of molecules a distribution of lifetimes is obtained (Figure 4). The width of the
distribution reflects the spatial heterogeneity of the sample, resulting in a different local
environment for each investigated molecule.
Image of a single fluorescent carbocyanine molecule. The apparent FWHM of the molecule is
~ 100 nm, as determined by the aperture of the near field optical probe used for excitation.
While collecting the fluorescence line-by-line the molecule switches on and off, showing up as
discrete black dots in the molecular image. In the bottom inset the single molecule fluorescence
is plotted as a function of time with 50 µs time resolution. Clearly the fluorescence drops
repeatedly to a low level: the so-called "blinking".
Three-level energy scheme describing single molecule fluorescence. S0 and S1
are the singlet ground and excited states; T1 the first excited triplet state. The
repeated S1-S0 excitation-emission photo-cycle yields the fluorescent signal.
Occasionally the molecule can drop into the T1 state (intersystem crossing) where it
gets trapped because the T1 lifetime is much longer than the S1 lifetime.
Surprisingly, for a considerable fraction of the molecules we found a triplet lifetime that varied
in time (Figure 5). Fluctuations on a timescale of seconds were observed, reflecting the dynamic
heterogeneity of the environment. Finally we were pleased to find a nice correspondence between
the lifetime variation in space and time, a direct manifestation of the ergodic principle of
Time image with 3.5 s of fluorescence blinking. Bright streaks are due to S0-S1
cycling; dark streaks represent residence in the T1 state. At first impression one sees
noise, yet detailed analysis reveals stochastic quantum jumps governed by the triplet lifetime
and singlet-triplet crossing rate of the individual molecule.
Distribution of the occurrence of T1 lifetimes for a set of single carbocyanine
molecules in PMMA. Note that an order of magnitude difference in lifetime is observed, which
reflects the heterogeneity of the various molecular surroundings.
Trajectory of the T1 lifetime of a single carbocyanine molecule as a function of time.
The lifetime of the molecular state changes over a factor of 2 indicating fluctuations in the local
This work has been financed by the Netherlands Foundation for Fundamental Research of Matter (FOM).