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Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 10630–10635, September 1997
Biophysics
Fluorescence and photobleaching dynamics of single
light-harvesting complexes
MARTIN A. BOPP*, YIWEI JIA*, LIANGQUAN LI*, RICHARD J. COGDELL
†
, AND ROBIN M. HOCHSTRASSER*
‡
*Chemistry Department, University of Pennsylvania, Philadelphia, PA, 19104; and
†
Division of Biochemistry and Molecular Biology, Institute of Biomedical and
Life Sciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom
Contributed by Robin M. Hochstrasser, August 1, 1997
ABSTRACT Single light-harvesting complexes LH-2 from
Rhodopseudomonas acidophila were immobilized on various
charged surfaces under physiological conditions. Polarized
light experiments showed that the complexes were situated on
the surface as nearly upright cylinders. Their fluorescence
lifetimes and photobleaching properties were obtained by
using a confocal fluorescence microscope with picosecond
time resolution. Initially all molecules fluoresced with a
lifetime of 1 6 0.2 ns, similar to the bulk value. The photo-
bleaching of one bacteriochlorophyll molecule from the 18-
member assembly caused the fluorescence to switch off com-
pletely, because of trapping of the mobile excitations by energy
transfer. This process was linear in light intensity. On con-
tinued irradiation the fluorescence often reappeared, but all
molecules did not show the same behavior. Some LH-2 com-
plexes displayed a variation of their quantum yields that was
attributed to photoinduced confinement of the excited states
and thereby a diminution of the superradiance. Others showed
much shorter lifetimes caused by excitation energy traps that
are only '3% efficient. On repeated excitation some molecules
entered a noisy state where the fluorescence switched on and
off with a correlation time of '0.1 s. About 490 molecules were
examined.
The structure of the light-harvesting complex LH-2 from
Rhodopseudomonas acidophila was recently determined by
x-ray diffraction (1, 2). There are 18 bacteriochlorophyll
(BChl) molecules arranged symmetrically in a circular ring
structure supported by nine
ab
-dipeptides. The principal
electronic absorption of these BChls is at 850 nm (B850). The
assembly contains another ring of nine BChl a molecules which
absorb at 800 nm (B800). The spectroscopy of LH-2 and the
energy transfer rates between B800–B800, B800–B850, and
B850–B850 have been studied extensively (3–13). Energy can
flow from B800 to B850 in about 650 fs at room temperature
(13). In the absence of traps or other antenna complexes this
excitation leads to emission at longer wavelength (.870 nm).
Here we examine the photochemical properties of individual
LH-2 units.
Recent exciting developments that couple various forms of
optical spectroscopy with microscopy (14–20) have made
possible heretofore unprecedented visualization of processes
involving single biological molecules or assemblies (21–25).
Time-resolved detection has also been incorporated into these
approaches (15–18), thereby enabling studies of the dynamics
of molecular processes at the single-assembly level. Fluores-
cence lifetimes and spectra obtained from the repeated exci-
tation of a single molecule have been reported and character-
ized for a number of fluorescent dyes and for a few molecules
of biological importance (15–18, 24–26). Such single-molecule
studies can yield knowledge that is not obtainable from
investigations of macroscopic systems (27, 28).
Light-harvesting complexes are protected from photooxida-
tion by carotenoids mostly through direct quenching of the
triplet BChl and, to a lesser extent, through quenching the
singlet oxygen (29). These processes affect the entire light-
harvesting apparatus. The exciton coupling between pigments
in B850 is large enough to cause significant delocalization
(30–34). The 18 B850 chromophores form an exciton band that
derives from coupling between nine dimeric units (1, 2). The
nine BChls in the B800 band are much further apart and
exhibit more localized excitations (2, 13, 31). Because there is
rapid excitation transfer or delocalization throughout the 18
cofactors, the photodamage of individual BChls can have a
significant effect on the luminescence properties of the whole
assembly. Our study shows that this is indeed the case.
METHODS
Sample Preparation, Immobilization, and Imaging. Isolated
LH-2 assemblies were first immobilized on mica plates with
thicknesses less than 100
m
m. Mica is optically transparent,
slightly negatively charged, and hydrophobic. For each exper-
iment freshly cleaved mica formed the base of a solution cell
with a volume of '200
m
l. The assemblies were also immobi-
lized on HF-treated microscope cover glasses.
The sample was prepared from a stock solution of 0.2
m
M
LH-2 (Rps. acidophila strain 10050) in buffer [50 mM TriszHCl,
pH 7.8y0.1% lauryldimethylamine oxide (LDAO)]. This so-
lution was diluted with buffer by a factor of 10
5
in four steps.
Seven minutes after addition of 50
m
lofthe2310
212
M
solution to the cell, the sample was washed five times by 150-
m
l
aliquots of buffer and transferred to the microscope. To keep
the sample immersed during the whole experiment, 150
m
lof
buffer was added. All manipulations were performed in the
dark. These same procedures were also carried out using a
buffer of 50 mM potassium phosphate, pH 7.7y0.8% n-octyl
b
-D-glucopyranoside (OGP).
The imaging was carried out by positioning the sample in a
confocal microscope (26) with the illumination source at
l
5
800 nm (850 nm in a few experiments) from a dye-laser
synchronously pumped at 6.33 MHz. The diffraction-limited
spot diameter was 700 nm. The scanning area is 7.2 3 7.2
m
m
2
.
Dichroic mirrors and color glass filters rejected the exciting
light.
To prove that the LH-2 assemblies were immobilized on the
mica, the same sample section was scanned several times. The
first, fifth, ninth, and twelfth images from a typical series of
scans are shown in Fig. 1. The integration time per pixel was
4 ms and the total counts per molecule in one image ranged
from 1,000 to 1,400. The excitation power was 290 nW at
l
5
800 nm. The assemblies remain immobilized until they are
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424y97y9410630-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
Abbreviation: BChl, bacteriochlorophyll.
‡
To whom reprint requests should be addressed. e-mail: hochstra@
mail.sas.upenn.edu.
10630
finally destroyed by photobleaching. The concentrations in this
demonstration were chosen 3 times higher than those used for
the single-molecule measurements. At 2 3 10
212
M LH-2 there
were usually about five molecules per image. Care was taken
to reduce the background signal by using thin clean mica and
pure buffer and by minimizing the scattered light. At 100–200
Wycm
2
the background was reduced to 20 counts per s, mainly
limited by the dark counts of the detector of '15 counts per
s. At this intensity the detected fluorescence count rate from
a single molecule was 1,000–2,000 counts per s.
Photobleaching and Fluorescence Lifetimes. To record a
typical photobleaching response a 25-
m
m
2
image was scanned
with low excitation power at a fast scan rate of 4 Hz per line
(1 ms per pixel). The light was then blocked and the scanner
was moved to a position where a molecule was now known to
be located. The emission of this molecule was then recorded
during constant illumination. The data for the time-resolved
fluorescence decay were collected through another channel at
a rate of 1 Hz with a PCA II multichannel analyzer (Nucleus,
Oak Ridge, TN). These data were binned according to the
different regions in the photobleaching curve, and the fluo-
rescence lifetime for each region was obtained by standard
techniques (35). After complete photobleaching of the first
molecule, the same procedure was then applied to the remain-
ing molecules in this image without ever scanning the whole
image again. A sample could not be used for more than 1.5 hr,
after which the fluorescence was irreversibly lost. A total of
'490 molecules were studied. Fluorescence emission polar-
ization experiments were carried with a variable polarizer in
front of the detector or by rotating the polarization of the
exciting laser beam.
RESULTS AND ANALYSIS
All except '10% of the 490 molecules studied gave similar
fluorescent signals. These few that showed anomalously large
signals were discarded and attributed to aggregates. The
following results used 800 nm, but 850-nm excitation showed
the same bleaching patterns.
Most molecules (64%) went from an initial bright (fluores-
cent) state to a dark (nonfluorescent) state, from which they
recovered after up to several tens of seconds. This second
bright state usually showed different properties from the first
one, and several other steps of dark and bright states often
followed. The remaining 36% of the molecules exhibited the
transition to the dark state and no further recovery. Examples
for the various types of photobleaching curves are shown in
Fig. 2. The different states in the bleaching curves are cate-
gorized and discussed in the following presentation of the
results.
First we discuss the one-step bleaching of the initial bright
state, labeled A, into a completely dark state, labeled B. A
sudden switching off of the relatively constant A state fluo-
rescence was observed for '90% of the molecules. This is
proposed to be a result of photobleaching of one BChl
molecule in the assembly. The total time in state A was
measured for each of these molecules and plotted into histo-
grams for different excitation intensities. The incident inten-
sity was determined by the average number of counts mea-
sured in state A. The longevity of this initial state becomes
shorter with increasing excitation intensity. A plot of the mean
of the inverse of the lifetime as a function of the excitation
power in a range from 0.5 to 5
m
W suggests a linear depen-
dency of this first bleaching time on the excitation power (Fig.
4a). For higher power levels (.5
m
W) this method is difficult
to apply because the survival time in the state A gets consid-
erably shorter than 1 s. Measurements were also carried out
with about 100 LH-2 assemblies in the focal spot (Fig. 4b). At
low power the time constant is consistent with the statistical
result from single LH-2 assemblies. However, for higher
intensities (.6
m
W) the bleaching rate saturates, becoming less
dependent on the light intensity. This ‘‘bulk’’ measurement
detects not only those molecules that go from an A state to the
B state but also those that resume emitting photons, as
described below. This result shows clearly that the interstate
dynamics can be observed only through the study of single
molecules.
The lifetimes of the A states were all in the range 1.0 6 0.2
ns (Fig. 3), in excellent agreement with bulk measurements of
the fluorescence lifetime. The narrow distribution indicates
that the sample is relatively homogeneous. After the disap-
pearance of state A by photobleaching, the LH-2 assembly
must continue to absorb photons, but its fluorescence is
quenched beyond our detection limit. Traps are created that
quench all fluorescence regardless of the spatial distribution of
the excitations.
We investigated the properties of the first dark state B by
measuring the excitation intensity dependence of the mean
time between the photobleaching of the A state and its first
recovery into a bright state (see Fig. 4a). The survival time of
the first B state is essentially independent of the light intensity,
with a mean value of 15 s and a standard deviation of 19 s. The
recovery of this state is not dominantly light induced. In
another experiment, the excitation light was blocked for 90 s
immediately after appearance of the B state. After re-
illumination, most of the molecules showed no fluorescence.
However, about one-third of them did begin emitting after
prolonged irradiation. This indicates that even without irradi-
ation, the B states can undergo chemistries which lead to the
permanent disappearance of the fluorescence signal. Clearly,
FIG. 1. Examples from a series of fluorescence images of single LH-2 assemblies. The image size is 102 3 128 pixels and the integration time
per pixel was 4 ms. The excitation power was 290 nW at
l
5 800 nm. The molecule labeled 1 is an A state in a.ItgoestoaB9state in b and it
recovers to an A9 state in c. During the scanning of this image,aBstatewasformedfor'0.5 s before recovering to the A9 state. In d aPstate
is reached. The circle labeled 2 is one of the rare cases where a molecule suddenly appears. Probably it was bleached toaBstateduring the sample
preparation.
Biophysics: Bopp et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10631
certain steps in the recovery to a fluorescent state are light
assisted.
In 64% of all cases, exemplified by the photobleaching
curves of Fig. 2 b–d, emitting states reappeared before a
‘‘permanent’’ dark state, designated as P, was achieved. These
regenerated fluorescent states are labeled A9,B9, and N
(bright, dark, and noisy, respectively) representing the three
major fluorescence patterns.
In these A9 states, 20–100% of the initial A state counts and
a fluorescence lifetime close to that of the A state were
observed. This A9 state was found for 76% of all molecules that
exhibited recovery. A set of data obtained from a molecule
that exhibited this behavior is shown in Fig. 2d. There are four
bright states with the following relative (to A) number of
countsyfluorescence lifetimes: A, 1.0y1.0 ns; A9
1
, 0.6y0.9 ns; A9
2
,
0.41y0.9 ns; and A9
3
, 0.21y0.8 ns. These extremely interesting
states whose fluorescence yield appears to vary without much
change in lifetime will be discussed more fully below. In most
cases the A9 states that were formed afteraBstateexhibited
‘‘blinking’’ (e.g., A9 3 B 3 A9 3 or A9 3 B9 3 A9 3) with
up to 10 cycles. The A9 states also evolved from B9 states.
Approximately 30% of the measurements yielded a different
type of recovered state having a count rate reduced from A
state emission by a factor of more than 6, and a fluorescence
lifetime somewhat shorter than our resolution of 200 ps. The
luminescence was not fully quenched as in B states, but the
lifetime was nevertheless significantly reduced. These are
evidently strongly quenched states, so they are referred to as
B9. The lifetimes of the B9 states were shorter than the
instrument function, but they could be estimated from the
signal strengths in the time-correlated single-photon counting
experiments to be in the range of 20–50 ps. Most of the B9
states were formed fromaBstate, but some evolved directly
from A states. About half of the B9 states lead to B or P states,
and the other half generated A9 states. When the illumination
ofaB9molecule was interrupted for 90 s it mostly reappeared
inaB9state, indicating that light is needed to converts B9 to
other states.
Some regenerated bright states exhibited a fast switching
between bright and dark states. This noisy state, N, was seen
in only a few cases (18%), one of which is shown in Fig. 2c. The
mean fluorescence decay time of the N state was found to be
similar to that of the A state. The autocorrelation function of
the signal in the N state fits a single-exponential decay with
correlation time of '0.1–0.6 s. This time constant should be
viewed as the correlation time of the fluctuations between the
dark and bright states.
To reveal possible protein motions that influence the fluo-
rescence, we excited the LH-2 assemblies at a very low laser
power (50 nW) to obtain trajectories up to 400 s, which were
analyzed by autocorrelation. There were no decay time con-
stants between 4 ms and 4 s. However, the signal shows a very
slow variation on the time scale of tens of seconds. A control
measurement ruled out that this was due to drift of our
instrument. We also searched for the effects of spectral jumps
in the emitted light (25, 36, 37). A narrow band-pass filter
(Chroma Technology, 10-nm bandwidth, centered at
l
5 885
nm) was placed in front of the detector. The trajectories
obtained in this way were again analyzed with autocorrelation,
but no significant decay was found.
Several polarization-sensitive measurements were per-
formed to obtain information about the orientation of LH-2
assemblies. In a first set the emitted fluorescence was analyzed
with a polarizer in front of the detector. At a fixed excitation
polarization several images were recorded by alternatively
flipping the emission polarizer 90° after each image. No large
systematic changes in the count rates from molecules in the two
orientations were observed, suggesting that the emission is
approximately isotropic. This finding was confirmed by switch-
ing the polarization of the exciting laser beam and detecting
the total fluorescence emission. From measurements of the
count rates for 40 molecules at the two orientations, we found
an average polarization ratio of 0.99. The data also showed that
there was an equal chance of either polarization being the
largest. The variance of this series of measurements was found
to be 0.27. When 5 molecules with anomalous ratios were not
included in the calculation, the variance was reduced to 0.16.
When a number of molecules were viewed in an image they all
exhibited similar signals, independent of the choice of polar-
ization.
To discover whether the buffer influences the fluorescence
properties of the LH-2 assemblies, we carried out some control
measurements. When the Tris buffer and detergent were
changed to potassium phosphate and n-octyl glucopyranoside,
FIG. 2. Examples of bleaching curves of single LH-2 assemblies with a binning time of 0.25 s. The excitation power at
l
5 800 nm was 0.5
m
W,
3.7
m
W, 0.3
m
W, and 0.7
m
W for a, b, c, and d, respectively. The state labels are discussed in the text. The corresponding lifetime measurements
of the different states in d are given in Fig. 3.
10632 Biophysics: Bopp et al. Proc. Natl. Acad. Sci. USA 94 (1997)
respectively, measurements on single LH-2 assemblies showed
the same bleaching patterns as we reported above for Tris and
lauryldimethylamine oxide buffer on mica. We also replaced
the mica, which is slightly negatively charged, with an HF-
treated thin glass plate. Although the LH-2 assemblies them-
selves did not stick as well on this surface, we obtained enough
data to establish that the bleaching behavior was the same as
with the mica surface.
In summary, the light-induced destruction of individual
LH-2 assemblies involves a complex sequence of events. All the
molecules showed the initial bright state A and the final dark
state P, but there were many different intermediate bright and
dark states that we referred to as A9,B,B9, and N. Some
molecules showed only one intermediate state, whereas others
had several or all of them. The periods of time spent in the A
and A9 states were sometimes comparable, but they could be
quite different. The recovery time of the B states exhibited a
large variance and no apparent regularity. The first transition
fromanAtoaBstateinitiated a progression that inevitably
generatedaPstateinlessthan 3 min. If the excitation light was
blocked while the system was in an A state, this state could still
be found up to 30 min later. However, if the sample was left
on the mica plate in the dark for times in excess of 1–1.5 hr,
hardly any molecule could be detected in the focal spot. Thus
there are also nonphotochemical destruction processes occur-
ring.
DISCUSSION
When the LH-2 assembly absorbs a photon at 800 nm the B800
rings are excited, but they transfer their excitation in 0.65 ps to
the B850 ring, where they have a lifetime of 1 ns. Thus the
probability of photobleaching one of the nine B800 cofactor
molecules is about 10
3
times less than that of a B850 cofactor.
Therefore most of the observed dynamics occurs in the B850
ring. An excitation has equal probabilities of being located
anywhere in the B850 ring after about 250 fs (13). Therefore,
regardless of what mechanism transports the excitation around
the ring, we will expect that repeated excitations of a single
assembly eventually will sample all the B850 molecules with equal
probability during our measurements.
Bleaching was not influenced by either the buffer or the type
of surface on which the assemblies were immobilized, so we
seek explanations for it from properties of the immobilized
FIG. 3. Lifetime measurements on single LH-2 assemblies in the
states corresponding to those in Fig. 2d.
FIG. 4. Laser power dependence of bleaching. (a) The mean of the
inverse of the survival time in state A (^1y
t
A
&;
F
) and the mean of the
inverse of the time between state A and the first reemitting state
(^1y
t
B
&;
h
) are plotted against the excitation power at
l
5 800 nm. The
increase in ^1y
t
A
& is almost linear with the excitation power, whereas
^1y
t
B
& is almost independent of it. These results are based on the
analysis of 201 and 136 single-molecule measurements for ^1y
t
A
& and
^1y
t
B
&, respectively. (b) ‘‘Bulk’’ measurements on '100 molecules
were fitted by a double exponential. Only the fast time constant (90%)
is plotted against the excitation power. The error bars correspond to
a confidence level of 95%.
Biophysics: Bopp et al. Proc. Natl. Acad. Sci. USA 94 (1997) 10633
LH-2 on the surface immersed in buffer. Although the BChl
molecules of B850 have identical chances of being excited on
the time scale of our measurements, different cofactors may
undergo different photochemistry. The assembly consists of
nine
ab
-dipeptides, but the
a
and
b
BChls are not equivalent.
The
a
and
b
chains both have hydrophobic groups near the
BChls, but the histidine that binds the BChl is near two
phenylalanines in the
b
chain, whereas there are no aromatic
residues nearby in the
a
chain. This makes the possibilities for
photoreactions such as electron transfer very different for the
BChls in the
a
and
b
chains. The intrinsic asymmetry intro-
duces the possibility for at least two distinct types of photo-
bleaching, and there could well be others if each of the BChl
molecules in the assembly has a number of pathways for
photobleaching.
Polarization of Excitation and Fluorescence. The LH-2
assemblies can be viewed as cylinders with the radiative
transition dipoles of each BChl a molecule arranged in a circle
with its plane perpendicular to the axis of symmetry. If the
cylinders were upright on the mica surface, with their circular
absorption and emission oscillator plane parallel to the sur-
face, there should be no polarization preference when the
excitation beam is directed perpendicular to the surface of the
mica and the absorption should be isotropic. Because of the
subpicosecond energy transfer around the ring of BChls (13)
the emission for this geometry should also be isotropic. The
observed fluorescence signals suggest that the sample consists
of approximately upright cylinders. We can estimate their
orientation from the data. The polarization ratio obtained by
varying the excitation polarization and detecting all the emis-
sion, is given by (1 2 sin
2
u
cos
2
f
)y(1 2 sin
2
u
sin
2
f
), where
u
and
f
give the orientation of the cylinder axis in the laboratory
frame with the polar z-axis normal to the plane of the mica
sheet. The mean of many measurements should yield unity,
and a value of 0.99 was observed. The variance in the polar-
ization ratio provides an estimate of
u
. A numerical simulation
showed that the observed variance corresponds to an angle
u
of 20–30°. This assumed that the cylinder axes all have similar
u
angles and that there is no rapid rotational averaging.
Trapping. The loss of fluorescence is presumed to be the
result of trapping of the excitation by photobleached regions
of the assembly. Trapping of excitations in molecular aggre-
gates (38, 39) occurs when an impurity has excited states at
lower energy than the aggregate state. It may involve any of the
common energy transfer mechanisms. For LH-2 the 0.9-nm
separation between adjacent BChls suggests a Fo¨rster mech-
anism will be important in nonresonant trapping. Charge
carrier generation, where an excitation forms an electron and
a hole, can also occur. For low-energy excitations, electron
transfer to an acceptor should also be considered. In a crystal
this would be the unavoidable impurity such as a quinone
generated by oxidation, but in the LH-2 it could be the peptide
constituents. The bleaching then produces a BChl radical
cation which has an optical absorption band near 925 nm (40,
41) and could be a trap for excitations. The calculated Fo¨rster
energy transfer rate is 10
12
s
21
. Once created, the positive hole
would migrate around the assembly. The tendency of the
positive charge to delocalize is counteracted by self-trapping,
which creates a distortion of the structure, again in analogy
with molecular crystals (39). There is also the possibility of
oxidizing an amino acid residue. Atom or ion elimination and
even the disruption of the LH-2 assembly are also possible
contributors to the photobleaching. The corresponding charge
or atom recombination reactions could occur to regenerate
fluorescent assemblies. During a typical trajectory of repeated
excitations, the BChls in the single-molecule experiments are
each electronically excited on average about 10
6
times before
photobleaching. Thus the processes responsible for photo-
bleaching are extremely slow and have extremely small effi-
ciencies in bulk materials.
The high probability of occurrence of one-step bleaching
A 3 B can be understood as generation of a trap for electronic
excitations by photobleaching. If a trap were formed, the very
efficient and fast delocalization of the excitons would ensure
that it could be accessed by the excitations formed in subse-
quent light absorption by the ring. The inverse lifetime of the
A state depends linearly on the light intensity when B is the
product, suggesting that the A 3 B bleaching involves just one
trap.
Traps are also created as a result of interactions occurring
among singlet and triplet excitations. The fluorescence yield is
10% (32), and the triplet yield is '2% to 15% (42). The
assembly is excited about once per 3
m
s with the source at 1
m
W. Thus there is a negligible probability for two singlet
excitations being present in the ring at the same time. The
triplet lifetime of BChl a is known to be 10–20
m
sinthe
absence of carotenoids (43). The triplet lifetime of BChl a is
reduced to 20–30 ns in the presence of the carotenoids (29), so
the probability of a double triplet excitation of BChl a being
created in the ring is also negligible. However, the carotenoid
triplet state has a lifetime of 3–15
m
s (44) and is known to be
a very efficient trap for singlet excitations in the bacterial LH-1
and LH-2 complexes (13, 45, 46). Therefore at our excitation
rate 0.7% to 12% of the singlet excitations created in the
experiment will be rapidly quenched by triplet carotenoid and
will not contribute to the fluorescence signal. These dynamics
were not seen because of the relatively low total count rates.
However, the total number of counts is consistent with this
analysis: at 1
m
W incident power, a collection efficiency of
'7%, and a fluorescence yield reduced to 6%, we expect a
count rate of 1,250 s
21
, whereas 2,000 s
21
was observed. A
large proportion of the heat energy from these internal
nonradiative processes is deposited into the carotenoid by
repeated triplet–triplet and singlet–triplet transfer, suggesting
a likely source of thermal damage of the carotenoid. The
removal of the carotenoid will lengthen the LH-2 triplet
lifetime and leave the assembly unprotected from oxygen (29).
The bleaching process A 3 B9 yields a partially quenched
state of LH-2. Incomplete quenching requires a photochemical
product having a low probability of trapping the excitations,
which must nevertheless be able to recover to yield bright A9
type states. The B9 states may be generated at many stages in
the trajectory. An analysis of the fluorescence decay signal
amplitudes for B9 (see Fig. 3) suggests that the lifetime of the
LH-2 excitation is reduced to about 20–50 ps. According to the
energy delocalization parameters of neat LH-2 there should be
about 80–200 visits to a trap during this period. Therefore
there must be some barrier associated with trapping in this
case.
Fluorescence Lifetimes and Quantum Yields in the A*
States. In a given trajectory the bright A9 states have similar
lifetimes, yet they can exhibit widely different signals. These
intensity variations are suggested to be the result of fluores-
cence quantum efficiency changes. Nonradiative pathways
dominate the fluorescence rate coefficient, so variations in the
radiative rates are needed to alter the yield without causing
large changes in the lifetime. For example, if the nonradiative
yield is 90%, a fluorescence yield reduction by a factor of 2
requires a fluorescence lifetime change of only 5%. In LH-2
the inverse of fluorescence lifetime is 2–3 times larger than in
BChl as a result of exciton delocalization (32). Therefore
partial localization of the excitations by the destruction of the
ring structure is expected to cause a diminution of the count
rate by up to a factor of 3. The observed changes of the count
rate in the A9 states are consistent with this interpretation,
which implies that we are observing superradiant states of
single molecules (the A states): the superradiance is dimin-
ished in the A9 states that have reduced yields. Superradiance
arises when the BChls in the LH-2 ring all emit with a definite
phase relation such as would occur if the emitting state were
10634 Biophysics: Bopp et al. Proc. Natl. Acad. Sci. USA 94 (1997)
a superposition of the individual BChl states. The constructive
interference enhances the transition moment over that ex-
pected for 18 uncoupled monomers. This type of behavior is
well known in other systems (47, 48). The destruction of the
superradiance leading to lower yield states with similar life-
times would be caused by structural deformations that localize
the excitations. In other instances the recovery process gen-
erated A9 states with the same count rate as the A state. These
may be cases of charge recombination or other type of repair
process. The polarization measurements make it unlikely that
tilting of the transition dipole plane by light-induced tumbling
of the whole assemblies or large structural displacements of the
immobilized protein is the cause of the intensity variations in
these A9 states.
SUMMARY
Single light-harvesting complexes LH-2 from Rps. acidophila
were immobilized on various surfaces under physiological
conditions, and about 490 of them were examined for their
fluorescence lifetime and photobleaching properties by using
a confocal fluorescence microscope with picosecond time
resolution. The planes of the circular absorbers and the mica
plate were within '20–30°. The photocharacteristics were
independent of the immobilization procedure and the types of
buffer and detergent. The photobleaching of one BChl mol-
ecule from the 18-member assembly causes the fluorescence to
switch off completely because of trapping of the delocalized
excitations. This process is linear in light intensity. On con-
tinued irradiation the fluorescence often reappears with a
range of characteristics. Some LH-2 complexes show states
having a range of radiative rates, attributed to localization of
the excitations and diminution of the superradiance. Others
show much shorter lifetimes caused by excitation energy traps
that are only '3% efficient. On repeated excitation some
molecules enter a noisy state where the fluorescence switches
on and off with a correlation time of '0.1 s.
Note added in Proof. After submission of this paper a fluorescence
study of single, conjugated polymers was published (49) which shows
some effects similar to those reported here.
Thanks are due to G. J. Small for his help in the early stages of this
work. This research was supported by the National Institutes of Health
and the National Science Foundation, with instrumentation developed
under National Institutes of Health Grant RR03148 (R.M.H.); by the
Biotechnology and Biological Sciences Research Council, U.K.
(R.J.C); and by the Swiss National Science Foundation and the
Freiwillige Akademische Gesellschaft, Basel (M.A.B).
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