Fluorescence Near-Field Microscopy of DNA at Sub-10 nm Resolution

We demonstrate apertureless near-field microscopy of single molecules at sub-10 nm resolution. With a novel phase filter, near-field images of single organic fluorophores were obtained with $\sim$sixfold improvement in the signal-to-noise ratio. The improvement allowed pairs of molecules separated by $\sim 15\mathrm{nm}$ to be reliably and repeatedly resolved, thus demonstrating the first true Rayleigh resolution test for near-field images of single molecules. The potential of this technique for biological applications was demonstrated with an experiment that measured the helical rise of $A$-form DNA.

Figure 1

(a) Illustration of the microscope. The linearly polarized beam, passing through a mask with a wedged window (not shown), is at total internal reflection at the substrate-air interface (focus area $\sim 350\times 1000\mathrm{nm}$) to achieve a large field component along the tip axis. (b) Tip-oscillation phase histogram of the photons. The inset is a typical fluorescence time trace of a Cy3 molecule, where the vertical axis is the photon count per 0.01 s. (c) The background noise (standard deviation) obtained from the phase filter (solid curve) and from the unfiltered shot noise, $\sqrt{n}$ (dash curve). The horizontal axis is the same as (d). (d) The SNR calculated as the image pixel signal divided by the background noise from the phase filter (solid curve) and from the unfiltered shot noise (dash curve). The image pixel signal is $0.60fN/3$ ($f$: fluorescence enhancement; $N$: photon number per pixel emitted by a typical molecule) for the solid curve according to Eq. (1), and $0.75fN/3$, which is the direct sum of the near-field photons [Fig. 1b], for the dash curve. For both curves, we used $f=5$, $N=10$.

Figure 2

(a), (b) Near-field images of isolated Cy3 molecules. Each figure was extracted from a $1\times 1\mu \mathrm{m}$, $512\times 512$ pixel image. The SNR for (a) and (b) is 16.2 and 25.5, respectively. Scale bars: 25 nm. (c), (d) Histograms of FWHM measured along the minor and major directions, respectively.

Figure 3

(a) Simulated images with the tip radius at 10 nm and fluorescence enhancement at 5. Scale bar: 20 nm. (b)–(d) Experimental images ($150\times 150\mathrm{nm}$) showing the same patterns as the simulated ones. In these images, $S_{\mathrm{rn}}$ was used as the pixel signal.

Figure 4

(a)–(c) Near-field images of Cy3 pairs. The SNR is 12.4 and 15.9 for (a), 16.1 for (b), and 20.4 for (c). The insets show the profiles with line cut through the image centers (indicated by arrows), where the horizontal axis is in pixels ($1\mathrm{\text{pixel}}=1.95\mathrm{nm}$) and the vertical axis is the pixel signal. (d)–(f) AFM images corresponding to images (a)–(c), respectively. Scale bars: 50 nm. (g) Histogram of distances between the resolved Cy3 molecules. (h) Histogram of distances between the two artifactual lobes of single Cy3 molecules.