Optical Sciences

Biomolecules and nanostructures

The Optical Sciences group studies the interaction of light and matter. Our current focus is on detection and sensing/imaging with an emphasis on the development of integrated photonics. We are part of Twente University's Department of Science and Technology and member of the MESA+ institute.


Photodynamics of individual green fluorescent proteins (GFP)

  • María García-Parajó - Former member

  • (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 s, 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.
    We have analyzed fluorescence trajectories of 64 individual S65T with an excitation intensity of ~14 kW/cm2. For each trace a set of values &Delta;ton for the bright intervals and the &Delta;toff dark intervals was collected. In our analysis &Delta;ton is the time width of a photon burst, and &Delta;toff 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 &Delta;ton (a) and &Delta;toff (b) distributions. Each histogram is fitted with a single exponential decay with a characteristic decay time &tau;. The analysis renders &tau;on = 80 18 ms and &tau;off = 1.6 0.2 s, corresponding to a relative "on" fraction &tau;on / (&tau;on + &tau;off) 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).
    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 &phi;q and the absorption cross-section &sigma;. We obtain [&phi;q x &sigma;] ~ 4.2 x 10-17 cm2. Taking &phi;q = 0.64 we calculate an absorption cross-section &sigma; = 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.
    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 = <&Delta;ton> / (<&Delta;ton> + <&Delta;toff>) 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).


    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
    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

    Garcia-Parajo MF, Segers-Nolten GMJ, Veerman JA, Greve J, van Hulst NF
    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
    vol 78 issue 1: p2264Pos part 2 JAN 2000

    Visualising individual green fluorescent proteins with a near field optical microscope

    Garcia-Parajo MF, Veerman JA, Segers-Nolten GMJ, de Grooth BG, Greve J, van Hulst NF
    vol 36 issue 3: p239-p246 JUL 1 1999
    Printable version