Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture

Abstract

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.

Fluorescence correlation spectroscopy with sub-diffraction-limited resolution using near-field optical probes

“…Recently, efforts have been made to observe smaller spatial scales with FCS in order to observe samples with higher concentrations. These methods include stimulated emission depletion, which has achieved a five times reduction in the observation volume, 1 the application of a supercritical angle objective, which reduces the observation volume in the axial direction at the cost of an increase in the radial direction, 2 the application of arrays of sub-wavelength-sized apertures ͓of-ten referred to as zero-mode wave-guides ͑ZMW͔͒ in thin films which produce attoliter 3,4 and even zeptoliter observation volumes ͑or observation areas in lipid membranes of 0.002-0.03 m 2 ͒, 5,6 and total internal reflection FCS to reduce the excitation volume along the optical axis. 7 Here, we report the combination of FCS with probes used for nearfield scanning optical microscopy ͑NSOM͒ to achieve subdiffraction-limited observation areas.…”

“…Although the radial excitation intensity profile created by the near-field tip is not well known, it is not expected to be Gaussian. 3,11 Therefore a different model is needed to describe the correlation data for diffusing particles acquired by FCS-NSOM.…”

Near-field optical fluorescence correlation spectroscopy

“…Therefore, an adhesive layer has to be coated on glass before the actual gold coating. In this work, an adhesive layer made out of chromium was used although titanium is another common adhesive layer of choice [80]. Due to the large light absorption of chromium the adhesive layer must not be coated too thick otherwise the intensity at the end of the probe becomes too small.…”

Near-field optical probes provide subdiffraction-limited excitation areas for fluorescence correlation spectroscopy on membranes

“…Although the excitation intensity profile created by the near-field tip is not well known, it is not expected to be Gaussian [6,27], and a different model from that used for 2D diffusion using confocal optics is required to describe the correlation data acquired by FCS-NSOM. We assume that the excitation intensity is constant over the 5-nm bilayer thickness and that the probability of detecting a fluorescence photon from a fluorophore is constant directly under the aperture but falls off sharply beyond the metal-glass boundary due to the well-defined aperture, as observed in our previous work [16].…”

“…Stimulated emission depletion (STED), a method based on the use of a second shaped laser pulse to stimulate emission of the outer edges of a diffraction-limited excitation spot created by an initial laser pulse, has achieved a five-fold decrease in the observation volume [5]. More substantial reductions have been obtained using subwavelength-sized apertures (holes) in thin films to produce attoliter (10 -18 l) [6,7], as well as zeptoliter (10 -21 l) observation volumes (the latter corresponding to an observation area in lipid membranes of 0.002-0.03 µm 2 ) [7][8][9]. Such approaches have significant promise for measurements in solution, as, for example, for single-molecule enzymology [9,10], and have also recently been applied to cellular imaging [7,11].…”