Label‐free ultra‐sensitive visualization of structure below the diffraction resolution limit

Abstract

For both fundamental study of biological processes and early diagnosis of diseases, information about nanoscale changes in tissue and cell structure is crucial. Nowadays, almost all currently known nanoscopy methods rely upon the contrast created by fluorescent stains attached to the object or molecule of interest. This causes limitations due to the impact of the label on the object and its environment, as well as its applicability in vivo, particularly in humans. In this paper, a new label-free approach to visualize small structure with nano-sensitivity to structural alterations is introduced. Numerically synthesized profiles of the axial spatial frequencies are used to probe the structure within areas whose size can be beyond the diffraction resolution limit. Thereafter, nanoscale structural alterations within such areas can be visualized and objects, including biological ones, can be investigated with sub-wavelength resolution, in vivo, in their natural environment. Some preliminary results, including numerical simulations and experiments, which demonstrate the nano-sensitivity and super-resolution ability of our approach, are presented.

Accessing depth-resolved high spatial frequency content from the optical coherence tomography signal

“…( 1 , 2 ), we can see that within moderate NA of the objective lens and limited spectral bandwidth, there is a one-to-one correspondence between axial spatial frequency and wavelength. A spectral encoding of spatial frequency (SESF) approach uses this to encode axial spatial frequency through spectral diversity, translate spatial information from the Fourier domain into the image domain as wavelengths, independently of the resolution of the optical imaging system, and map to each pixel of the 2D image 17 , 21 – 25 . The uncertainties in spatial frequencies/periods depending on NA of the objective lens were analysed previously 17 , 21 .…”

“…( 2 ), is v z = 1538 l/mm for n = 1, and the corresponding spatial period of the object’s structure is 650 nm. At the same time, the axial spatial frequency profiles are ultra-sensitive to structural changes 17 , 21 – 25 .…”

“…Previously, it was shown that the axial Fourier spectrum of an object is very informative and highly sensitive to structural changes 17 , 21 – 25 . Following the SESF approach and having information about both K -space and reconstructed object space, we can perform structural characterization for a given volume of interest within the object with nanoscale sensitivity using a simple method utilizing the characteristics of the spatial frequency support in K -space.…”

Noninvasive detection of nanoscale structural changes in cornea associated with cross‐linking treatment

“…Scattering potential of a 3D object can be reconstructed from the distribution of the complex amplitude of the light scattered by the objective in far zone via 3D inverse Fourier transform . Based on the theory of spectral encoding of spatial frequency approach , which was demonstrated for quantitative characterization of the structure with nanoscale sensitivity, nsOCT has been proposed to be a label‐free depth‐resolved sensing technique to probe structural changes at the nanoscale . Later nsOCT has been used for the study of the nanometer‐scale structural changes of the human tympanic membrane in otitis media .…”

Label Free Ultra-Sensitive Imaging with Sub-Diffraction Spatial Resolution

“…1). The theory of the SESF approach was published in [25,21,22] and implemented for superresolution imaging in [23,24]. It was demonstrated that in reflection configuration, axial spatial frequency components of the 3D Fourier transform of the scattering potential of the object, which provide information about small, sub-micron structure, can be spectrally encoded as corresponding wavelengths.…”