An array of planar apertures for near-field fluorescence correlation spectroscopy

Abstract

We have developed a method of performing near-field fluorescence correlation spectroscopy via an array of planarized circular apertures of 50 nm diameter. This technique provides 1 μs and 60 nm resolution on proximal samples, including live cells, without incorporating a scanning probe or pulsed lasers or requiring penetration of the sample into the aperture. Millions of apertures are created in an array within a thin film of aluminum on a coverslip and planarized to achieve no height distinction between the apertures and the surrounding metal. Supported lipid bilayers and plasma membranes from live cells adhere to the top of this substrate. We performed fluorescence correlation spectroscopy to demonstrate the sub-diffraction-limited illumination with these devices.

Figure 1

Transillumination though a nanoscale aperture provides near-field excitation of membrane-bound fluorophores for FCS, based on finite element analysis computations. An array of 50-nm-diameter SiO₂-filled apertures in a 100-nm-thick Al film is coated with 10 nm SiO₂. Transmission through each aperture illuminates a nanoscale region of the membrane; ω₀ = 50 nm within 20 nm vertically from the aperture.

Figure 2

Scanning electron micrograph of PANOMs 50 ± 4 nm in diameter. The four fused silica apertures are indicated by white arrows and are surrounded by the Al alloy film, which has noticeable metallic grain structure.

Figure 3

Normalized autocorrelation data from FCS of G_M1-Bodipy diffusion on biological membranes. Comparisons of FCS data and fit for live rat basophilic leukemia 2H3 cells and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine SLBs are shown for illumination areas determined by confocal, far-field optics and PANOMs. Error bars are the error of the mean from sequential 30-s measurements.