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 Single Molecules


  • Joost-Anne Veerman - Former member


  • With optical research at the nanometer scale (nano-optics) one enters the domain of sub-wavelength optics, evanescent and local fields. Using a nano-optical source individual nanoparticles or molecules can be excited. Alternatively sub-wavelength optical fields can be probed using a single emitter.
    Crucial in all these experiments is the efficient localization of the exciting optical field. Using focused ion beam (FIB) milling we are able to make high-definition flat-end aperture probes, combining: small aperture (typically 50-100 nm), high brightness (up to ~kW/cm2) and good polarization characteristics (ext. ratio > 100:1). Yet the efficiency is only 10-5-10-4 and localization below 10 nm is preferred. Currently we are exploiting the potential of FIB milling in order to:
    • increase the apex angle of the probe to shift the cut-off dimension closer to the final probe end,
    • fabricate a transmission line structure with high efficiency for a specific polarization component,
    • minimize the aperture size to such an extent that single molecule detection in absorption becomes feasible.
    By proper control of our technology, distinct advantages of near-field optics are exploited. (I) The nanometric excitation/emission volume (104-105 nm3), which provides high spatial resolution, localization of a single molecule within a few nm and reduced background. (II) The sensitivity for single molecule orientation in all three dimensions. (III) The high local brightness, allowing real-time single molecule detection down to ms resolution. (IV) The simultaneous co-localization with nanometric surface topography.
    Figure1
    (Figure 1)
    (a) FIB image of a FIB-etched near-field aperture probe with aperture diameter 70(5) nm. The probe has a flat end-face and an aperture with well-defined edges and circular symmetry. The principle of near-field single molecule detection is sketched. A single molecule is scanned in the x-y plane parallel to the probe end-face and excited if the molecular absorption dipole moment matches the local field vector of the aperture field. Generated fluorescence is collected in the far field.
    (b,c,d) Series of three NSOM fluorescence images of the same area of a sample of DiIC18 molecules embedded in a 10 nm thin film of PMMA, as measured with the 70 nm aperture probe in (a). The fluorescence signal is color-coded red/green according to the detected x/y polarization. The excitation polarization was changed between circular (b), linear along the vertical y-direction (c) and linear along the horizontal x-direction (d). The 3D optical near-field distribution and the molecular dipole orientation determine the appearance of the molecules. Molecules oriented in x, y and z direction are observed, where molecules oriented along z (out of plane) show up as circles and double lobes [Veerman et al., J.Microsc. 194, 477 (1999)].
    Figure2
    (Figure 2)
    Examples of near-field optical single molecule localization.
    (a-d) Series of four successive measurements of the same 440 x 440 nm sample area obtained with a 70 nm aperture probe. The fluorescence signal is color-coded red/green according to the detected x/y polarization. The linear excitation polarization direction was varied between (a) 90░ y-direction, (b) 0░ x-direction, (c) -45░ and (d) 45░. Two molecules are visible located within 10 nm from each other: one molecule in the sample plane along the x-direction (red in (b)), the other mainly perpendicular to the sample plane (yellow in (a)).
    (e-f) Series of images for two other molecules. The linear excitation polarization direction was varied between (e) 90░ y-direction and (f) 0░ x-direction. Combination of images (e) and (f) in (g) shows discrimination of the two molecules, one along the x-direction (red), the other along the y-direction (green). In graph (h) line traces of the molecular fluorescence signal in both polarization channels are plotted, showing the two molecules separated by 45(10) nm [van Hulst et al., J. Chem. Phys. 112, 7799 (2000)].

    Real-time quantum jumps between singlet and triplet state of an individual molecule are observed. Distributions for triplet state lifetime and crossing yield are determined. Both triplet state lifetime and crossing yield of a single molecule appear to vary in time, due to the local heterogeneity [Veerman et al., PRL 83, 2155 (1999)].

    Figure3
    (Figure 3)
    (a) Single molecule fluorescence time trace with 57 Ás integration time per point. The fluorescence drops repeatedly to a low level. This characteristic switching between "on" and "off" (blinking)corresponds to the molecule jumping between singlet and triplet state, respectively.
    (b) Real-time-image with 3.5 s of fluorescence blinking. Time runs continuously from line to line (left => right, top => bottom). The gray level represents the photon count rate during a 57 Ás bin: bright streaks are due to the S0-S1 singlet excitation-emission cycle; dark streaks represent residence in the T1 triplet state. Several thousands of triplet excursions occur during the 3.5 s observation time.

    Individual dendritic molecules containing a single fluorescent core are investigated. The dendritic assemblies are discriminated from free fluorescent cores on the basis of accurate simultaneous localization of both the fluorescent core and the topography of the surrounding dendritic shell. Intra-molecular rotational motion of the fluorescent core is observed [Veerman et al., J. Phys. Chem. A 103, 11264 (1999)].

    Figure4
    (Figure 4)
    (a) Near-field fluorescence image of individual dendritic molecules, with ~70 nm optical resolution. Scan range 1.65 x 1.65 Ám2 at 1 ms/pixel. The excitation light at l = 514.5 nm is circularly polarized. Fluorescence signal is typically 50 kcounts/pixel for an isolated spot. The fluorescence signal is color-coded red/green according to the detected x/y polarization, and thus indicating the in-plane orientation of the molecular emission dipole.
    (b) Simultaneously obtained shear-force image showing both isolated and clustered dendritic molecules on the glass surface. Contours of the fluorescence spots and clusters in (a) are overlayed to facilitate co-localization of fluorescence and topography. Both correlated and non-correlated spots are observed, indicating presence of dendritic molecules with fluorescent core, without fluorescent core, and isolated fluorophores. Scalebar: 450 nm.

    Individual green fluorescent proteins (GFPs) are visualized, both in fluorescence and topography. Photo-induced conformational changes to a non-emissive form of the protein are observed, leading to long dark intervals of several seconds [Garcia-Parajo et al., PNAS 97, 7237 (2000)].

    (Figure 5)
    Fluorescence signal of an individual S65T-GFP in time, during 26 s with 1.6 ms integration time, at 14 kW/cm2 excitation intensity.
    (a) Total fluorescence time trace, showing mainly background signal with occasional GFP fluorescence bursts
    (b,c,d) Magnification of the fluorescence bursts, containing a few thousand photo-counts, during typically 100 ms.
    Figure5


    Articles

    The following articles have been published regarding this project:

    Photoplastic near-field optical probe with sub-100 nm aperture made by replication from a nanomould

    (abstract) (full pdf)
    Kim GM, Kim BJ, Ten Have ES, Segerink F, Van Hulst NF, Brugger J
    JOURNAL OF MICROSCOPY-OXFORD
    vol 209: p267-p271 part 3 MAR 2003

    Near-field effects in single molecule emission

    (abstract)
    Gersen H, Garcia-Parajo MF, Novotny L, Veerman JA, Kuipers L, Van Hulst NF
    JOURNAL OF MICROSCOPY-OXFORD
    vol 202: p374-p378 part 2 MAY 2001

    Single molecules in the near field.


    van Hulst NF, Garcia-Parajo MF, Veerman JA, Gersen H
    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
    vol 221: p169-ANYL part 1 APR 1 2001

    Moulded photoplastic probes for near-field optical applications

    (abstract)
    Kim BJ, Flamma JW, Ten Have ES, Garcia-Parajo MF, Van Hulst NF, Brugger J
    JOURNAL OF MICROSCOPY-OXFORD
    vol 202: p16-p21 part 1 APR 2001

    Looking at the photodynamics of individual fluorescent molecules and proteins

    (abstract)
    Garcia-Parajo MF, Veerman JA, Kuipers L, van Hulst NF
    PURE AND APPLIED CHEMISTRY
    vol 73 issue 3: p431-p434 MAR 2001

    Influencing the angular emission of a single molecule

    (abstract) (full pdf)
    Gersen H, Garcia-Parajo MF, Novotny L, Veerman JA, Kuipers L, van Hulst NF
    PHYSICAL REVIEW LETTERS
    vol 85 issue 25: p5312-p5315 DEC 18 2000

    Analysis of individual (macro)molecules and proteins using near-field optics

    (abstract) (full pdf)
    van Hulst NF, Veerman JA, Garcia-Parajo MF, Kuipers L
    JOURNAL OF CHEMICAL PHYSICS
    vol 112 issue 18: p7799-p7810 MAY 8 2000

    Near-field scanning optical microscopy of single fluorescent dendritic molecules

    (abstract)
    Veerman JA, Levi SA, van Veggel FCJM, Reinhoudt DN, van Hulst NF
    JOURNAL OF PHYSICAL CHEMISTRY A
    vol 103 issue 51: p11264-p11270 DEC 23 1999

    Time-varying triplet state lifetimes of single molecules

    (abstract) (full pdf)
    Veerman JA, Garcia-Parajo MF, Kuipers L, van Hulst NF
    PHYSICAL REVIEW LETTERS
    vol 83 issue 11: p2155-p2158 SEP 13 1999

    Single molecule mapping of the optical field distribution of probes for near-field microscopy

    (abstract) (full pdf)
    Veerman JA, Garcia-Parajo MF, Kuipers L, Van Hulst NF
    JOURNAL OF MICROSCOPY-OXFORD
    vol 194: p477-p482 part 2-3 MAY-JUN 1999

    High definition aperture probes for near-field optical microscopy fabricated by focused ion beam milling

    (abstract) (full pdf)
    Veerman JA, Otter AM, Kuipers L, van Hulst NF
    APPLIED PHYSICS LETTERS
    vol 72 issue 24: p3115-p3117 JUN 15 1998