Photodynamics of individual green fluorescent proteins (GFP)
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MarÃa GarcÃa-Parajó - .$globalfunctie.
(Published in the Proceeding of the National Academy of Sciences USA, 97, p7237, 2000)
One area of research in our group concerns the photodynamical study of individual auto-fluorescent
proteins, such as the green fluorescent protein, GFP from the jellyfish Aequorea victoria.
At the individual molecular level the properties of the GFP differ dramatically from those of
ensemble measurements. Indeed, investigations with single molecules of GFP mutants T303Y and
T203F in polyacrylamide gels done by Moerner's group reported pH independent, spontaneous
"blinking" and "flickering" on a time scale of seconds.
We have applied real-time single molecule fluorescence detection to study the light driven
dynamics of the fluorescence emission in the S65T (Ser65 -> Thr65) mutant. We have combined
confocal microscopy and near-field scanning optical microscopy (NSOM) to obtain information on
the photodynamics of GFP. Our experiments show conclusively that excitation intensity has a
dramatic effect on GFP blinking, with a reduction of the fraction of molecules being in the
"on" state upon increasing excitation intensity. Correspondingly, we find that the
on-times become shorter at high intensity. We accurately fit our results assuming a
three level system, where the molecule transforms between an emissive and a non-emissive state.
From the statistical analysis we determine an optimum excitation condition at which the GFP
will be preferentially in an "on" state while simultaneously delivering enough
fluorescent signal for individual detection. This result has implications for the use of GFP
as marker in the study of dynamic biological processes at the single molecular level. Provided
that an efficient detection scheme is used, excitation of the GFP at low intensities
(<1.5 kW/cm2) results in a molecule with high probability to be in an emissive
state.
Figure 1 shows a near-field fluorescence image of individual S65T-GFP molecules embedded in a
polyacrylamide (PAA) gel. Spatially distributed molecules with fluorescent spot size of 70 nm FWHM
are observed in the image. Interesting features in the image are the sudden cease of emission of
some of the molecules during imaging, and the defined in-plane orientation of all the molecules,
evidenced by the constant color per individual spot. Figure 2 shows a typical real-time
fluorescence trajectory of an individual S65T, during ~ 60 s with continuous excitation at
~14 kW/cm2. With dwell times of 100 µs, our dynamic range is of more than five orders
of magnitude. This example illustrates that photon emission does not occur in a constant fashion
but as bursts of fluorescence rising in between long dark intervals (photon bunching). Also, in
many cases, the count rate detected within a photon bunch did not stay constant but showed
fluctuations up to 50%.
Figure 1,
Near-field fluorescence images of individual S65T-GFP molecules embedded in a PAA gel. Color in
the images indicates the orientation of the chromophore emission dipole moment. The scan area is
1.8 x 1.8 mm2. The image is 300 x 300 pixels. |
Figure 2,
Real-time fluorescence trajectory of a S65T protein. The fluorescence time trace shows mainly
background signal with occasional GFP fluorescence bursts.
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We have analyzed fluorescence trajectories of 64 individual S65T with an excitation intensity of
~14 kW/cm2. For each trace a set of values Δton for the bright
intervals and the Δtoff dark intervals was collected. In our analysis
Δton is the time width of a photon burst, and Δtoff
is defined as the time between two consecutive photon bursts, including those dark times present
between the start of the experiment and the first fluorescent burst. Figure 3 shows the
Δton (a) and Δtoff (b) distributions.
Each histogram is fitted with a single exponential decay with a characteristic decay time τ.
The analysis renders τon = 80 ± 18 ms and τoff = 1.6 ± 0.2 s,
corresponding to a relative "on" fraction τon / (τon +
τoff) as small as 5% for this excitation intensity. Additionally, the average
number of counts obtained during each bright interval has been extracted from the data. With a
collection efficiency of 7 % in our set-up we extract Non ~ 6 x 104 photons per photon
burst.
Figure 3,
(a) Histogram of the length of the bright, on-periods obtained for 64 proteins observed
continuously during ~ 60 s excited at ~ 14 kW/cm2.
(b) Histogram of the length of the dark, off-periods obtained for the same set of molecules as in (a).
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Statistical analysis over a large number of individual molecules at different excitation
intensities was performed to gain quantitative understanding of the light driven behavior of the
S65T. Figure 4a shows the fluorescence emission of the bright interval as a function of the
excitation intensity obtained for 140 molecules. A clear linear dependence of the count rate
as a function of excitation intensity is obtained. This linear relation is understood in terms
of a two level-system, specifically a singlet ground, and a singlet excited state. Within each
emissive interval the molecule is cycling between the first excited singlet-state and the singlet
ground state, emitting photons at a rate proportional to the illumination intensity. The linear
relation indicates that the molecule is below saturation of the singlet state transition. The
slope of the curve is a direct measure for the quantum yield φq and the absorption
cross-section σ. We obtain [φq x σ] ~ 4.2 x 10-17
cm2. Taking φq = 0.64 we calculate an absorption cross-section σ
= 6.5 x 10-17 cm2 (± 10 %).
Figure 4,
(a) Fluorescence emission during the bright interval versus excitation intensity as obtained for
140 molecules. Excitation and emission follow a linear dependence, indicating that when the
molecule is in an emissive state, its emission rate is proportional to the excitation intensity.
(b) Relative residence of the molecule in the emissive on-state as a function of the excitation
intensity. The fraction of time the molecule is in the emissive state decreases dramatically with
increasing excitation. Fitting of the data has been done using Fon = Is / (Is + I) with
Is = 1.5 kW/cm2.
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We also analyzed the relative residence of the molecule in the emissive and non-emissive states
at different excitation intensities. Figure 4b shows the fractional on-time, defined
as Fon = <Δton> / (<Δton> +
<Δtoff>) against excitation intensity, showing that Fon decreases
dramatically with increasing excitation intensity. We find excellent agreement between
experimental data and our model that assumes a 3-level system with the existence of a long-lived
dark state. At present we are conducting more experiments to elucidate the origin of the dark
state.
This work has been financed by the Royal Netherlands Academy of Arts and Sciences (KNAW) and the
Netherlands Foundation for Fundamental Research of Matter (FOM). |
Articles
The following articles have been published regarding this project:
Single molecule studies of living colours.
(full pdf)Garcia-Parajo MF, Koopman M, de Bakker BI, van Hulst NF
BIOPHYSICAL JOURNAL
vol 80 issue 1: p655.12 part 2 JAN 2001
Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules
(abstract) Garcia-Parajo MF, Segers-Nolten GMJ, Veerman JA, Greve J, van Hulst NF
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (PNAS)
vol 97 issue 13: p7237-p7242 JUN 20 2000
Real time light-driven dynamics of the fluorescence emission in individual green fluorescent proteins
Garcia-Parajo MF, Segers-Nolten GMJ, Veerman JA, van Hulst NF
BIOPHYSICAL JOURNAL
vol 78 issue 1: p2264Pos part 2 JAN 2000
Visualising individual green fluorescent proteins with a near field optical microscope
(abstract) Garcia-Parajo MF, Veerman JA, Segers-Nolten GMJ, de Grooth BG, Greve J, van Hulst NF
CYTOMETRY
vol 36 issue 3: p239-p246 JUL 1 1999
MicroCOR
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