EPFLSTILOBMarcel LeuteneggerAbout

FCS on sub-wavelength apertures

Near-field fluorescence spectroscopy

Excitation intensity above a 250nm aperture in a 150nm thick gold film

Topic

For the observation of single molecule dynamics with fluorescence fluctuation spectroscopy (FFS) very low fluorophore concentrations are necessary. For in vitro measurements, this requirement is easy to fulfill. In biology however, micromolar concentrations are often encountered and may pose a real challenge to conventional FFS methods based on confocal instrumentation. We show a higher confinement of the sampling volume in the near-field of sub-wavelength sized apertures in a thin gold film. The gold apertures have been measured and characterized with fluorescence correlation spectroscopy (FCS), indicating light confinement beyond the far-field diffraction limit. We measured a reduction of the effective sampling volume by an order of magnitude compared to confocal instrumentation.

Nano-aperture FCS setup
Figure 1: Photograph of our trans-illumination nano-aperture FCS setup.

Results

Fits and residuals
Figure 2: Auto-correlations and fits G versus lag time τ for aperture diameters of 125nm (blue circles), 490nm (red points) and for free liquid (black dotted) at a Cy5 concentration of 30nM. Inset: Fit residuals r = Gfit/G - 1.

Diffusion times
Figure 3: Diffusion time τd versus aperture diameter d. The data points show the average and the error bars the standard deviation of 10 measurements per aperture diameter. For clarity, the standard deviation is shown for one case only (12nM Cy5, 50µm pinhole). The black dotted line shows the trend. In free liquid, the diffusion time was 160µs to 170µs. Inset: concentration of Cy5 and pinhole diameter of all cases.

We demonstrated that FCS on single apertures is an interesting approach for molecular investigations at 10nM to 100nM concentration. Using apertures of various sizes, we calculated and engineered the excitation field. For small apertures, we verified that the diffusion time and the number of molecules are proportional to the aperture diameter. We measured a reduction of the effective sampling volume by an order of magnitude compared to the typical volume in confocal FCS.

Acknowledgements

We thank Benjamin Dwir at EPFL for electron-beam and nanostructuring assistance.

This work was funded by the Swiss National Science Foundation (SNSF #200021-103333).

Publications

  1. M. Leutenegger, M. Gösch, A. Perentes, P. Hoffmann, O. J.F. Martin, T. Lasser
    Confining the sampling volume for FCS using a sub-wavelength sized aperture
    Opt. Express 14, 956-969 (Jan 2006)
  2. A. Perentes, I. Utke, B. Dwir, M. Leutenegger, T. Lasser, P. Hoffmann, F. Baida, M. P. Bernal, M. Russey, J. Salvi, D. Van Labeke
    Fabrication of arrays of sub-wavelength nano-apertures in an optically thick gold layer on glass slides for optical studies
    Nanotech. 16, S273-S277 (2005)
  3. A. Perentes, A. H. Bachmann, M. Leutenegger, I. Utke, C. Sandu, P. Hoffmann
    Focused electron beam induced deposition of a periodic transparent nano-optic pattern
    Microel. Eng. 73-74, 412-416 sp. iss. SI, (Jun 2004)

© 2011 École Polytechnique Fédérale de Lausanne