Super-resolution imaging by random adsorbed molecule probes

Abstract

Single molecule localization (SML) is a powerful tool to measure the position and trajectory of molecules in numerous systems, with nanometer accuracy. This technique has been recently utilized to overcome the diffraction limit in optical imaging. So far, super-resolution imaging by SML was demonstrated using photoactivable or photoswitchable fluorophores, as well as diffusive fluorophore probes in solution. All these methods, however, rely on special fluorophore or object properties. In this Letter, we propose and demonstrate a new super-resolution technique attainable for a bio/dielectric structure on a metal substrate. A sub-diffraction-limited image is obtained by randomly adsorbed fluorescent probe molecules on a liquid-solid interface, while the metal substrate, quenching the unwanted fluorescent signal, provides a significantly enhanced imaging contrast. As this approach does not use specific stain techniques, it can be readily applied to general dielectric objects, such as nanopatterned photoresist, inorganic nanowires, subcellular structures, etc.

Figure 1

Principle of super-resolution imaging by randomly adsorbed molecular probes. (a) Schematic drawing shows nano-objects (gratings in this case) on a Au metal surface. Only the molecules (red) adsorbed on the dielectric surface emit a fluorescent signal to the far-field. The molecules on the metal surface (black) are quenched and cannot be detected. (b) Physical adsorption of molecules at a solid–liquid interface. A denotes the molecules diffusing freely in liquid, B denotes the surface vacancy, and C denotes the adsorbed molecules.

Figure 2

(a) Single molecule fluorescent image captured by the CCD. (b) 2D Gaussian function fitting of the experimental data in (a). The center position and intensity of each single molecule was derived from the fitting. (c) Position distribution of subsequence measurements of a fixed reference particle on the substrate. The standard deviation indicates the resolution of our method, which is about 18 nm. (d) Histogram of single molecule signal from the sample in Figure 3. A threshold was set to cut those measurements with too low S/N ratios.

Figure 3

Experimental demonstration of super-resolution imaging. (a) AFM image of the PMMA nanograting structure as the object. The thickness of the PMMA is 60 nm. (b) Computer-rendered super-resolution image from sample in panel a. Each single molecule was rendered using a 2D Gaussian shape PSF with 18 nm fwhm. (c) Fluorescent image from unprocessed data in (b). (d) Super-resolution image of 100 nm period PMMA nanograting. Left image is the experimental result, the top-right image is the AFM measurement, and the bottom-right image is the 2D Fourier transform of the left image (two distinct peaks corresponding to the special frequency of the grating are encircled by red lines).