Novel approach for label free super-resolution imaging in far field

Abstract

Progress in the emerging areas of science and technology, such as bio- and nano-technologies, depends on development of corresponding techniques for imaging and probing the structures with high resolution. Recently, the far field diffraction resolution limit in the optical range has been circumvented and different methods of super-resolution optical microscopy have been developed. The importance of this breakthrough achievement has been recognized by Nobel Prize for Chemistry in 2014. However, the fluorescence based super-resolution techniques only function with fluorescent molecules (most of which are toxic and can destroy or lead to artificial results in living biological objects) and suffer from photobleaching. Here we show a new way to break the diffraction resolution limit, which is based on nano-sensitivity to internal structure. Instead of conventional image formation as 2D intensity distribution, in our approach images are formed as a result of comparison of the axial spatial frequency profiles, reconstructed for each image point. The proposed approach dramatically increases the lateral resolution even in presence of noise and allows objects to be imaged in their natural state, without any labels.

Figure 1. Results of numerical simulation.

(a) Simulated object, d1 = 50 nm, d2 = 50 nm, d3 = 250 nm. (b) Lateral intensity distribution of the reflected light from the object before convolution. (c–f), Intensity distributions in image plane using conventional microscopy. (g–j) Correlation coefficient distributions in image plane using srSESF approach. (c,d,g,h)– without noise; (e,f,i,j)—with noise. (d,f,h,j) are magnified portions of (c,e,g,i).

Figure 2. Results of numerical simulation.

(a–e) for d1 = 0.31 μm, d2 = 0.31 μm, d2 = 1.55 μm and (f–j) for d1 = 0.56 μm, d2 = 0.56 μm, d2 = 2.8 μm. (a,f)–Lateral intensity distributions of the reflected light from the object. (b,c,g,h) Intensity distributions in the image plane using conventional microscopy. (d,e,i,j) Correlation coefficient distributions in the image plane using srSESF approach. (b,d,g,i)– without noise; (c,e,h,j)—with noise. PSF—point spread function for objective lens with NA = 0.9, wavelength 600 nm.

Figure 3. Images of the nanosphere aggregates:

(a) scanning microscopy and (b) srSESF microscopy. Images (a,b) were formed using the wavelength range 1230 nm–1370 nm, NA = 0.5. Size of magnified portions in the images (a,b) is 1000 nm × 1000 nm. (c) Conventional bright field image using visible light, NA = 0.9. Scale bar is 2 microns.

Figure 4. Images of collagen fibres:

(a) and (d), scanning microscopy images with interference fringe noise; (b), (e) and (f), srSESF microscopy images formed using the wavelength range 1230 nm – 1370 nm, NA = 0.5, reveal the horizontal fibers; (c) and (g) high resolution conventional bright field images using visible light, NA = 0.8. The scale bar is 2 microns.

Figure 5. Schematic of the scanning microscope experimental setup with image acquisition.

(a) – microscope, where SLD—superluminescent diode 1230 nm–1370 nm, OC—optical coupler, DG—diffraction grating. (b) – axial spatial period profiles for different lateral locations, (c) – srSESF image.