Axial super-resolution evanescent wave tomography

Abstract

Optical tomographic reconstruction of a 3D nanoscale specimen is hindered by the axial diffraction limit, which is 2-3 times worse than the focal plane resolution. We propose and experimentally demonstrate an axial super-resolution evanescent wave tomography (AxSET) method that enables the use of regular evanescent wave microscopes like Total Internal Reflection Fluorescence Microscope (TIRF) beyond surface imaging, and achieve tomographic reconstruction with axial superresolution. Our proposed method based on Fourier reconstruction achieves axial super-resolution by extracting information from multiple sets of three-dimensional fluorescence images when the sample is illuminated by an evanescent wave. We propose a procedure to extract super-resolution features from the incremental penetration of an evanescent wave and support our theory by 1D (along the optical axis) and 3D simulations. We validate our claims by experimentally demonstrating tomographic reconstruction of microtubules in HeLa cells with an axial resolution of ∼130 nm. Our method does not require any additional optical components or sample preparation. The proposed method can be combined with focal plane super-resolution techniques like STORM and can also be adapted for THz and microwave near-field tomography.Optical tomography is a major tool in threedimensional visualization of sub-micrometer scale specimens in biology, material sciences and nano-fabrication technology [1][2][3]. Tomographic reconstruction is done by optical sectioning of the object in the focal plane followed by 3D stitching of the acquired z-stack of focal plane images. Resolution of the 3D tomographic reconstruction of an object is therefore governed by the focal plane resolution and the axial resolution of the underlying optical image acquisition. A wide range of well advanced fluorescence based super-resolution microscopy techniques have been reported and are currently in practice [4]. Super-resolution [12] have increased the resolution in the axial direction with an almost spherical PSF. More recently a triple-view capture and fusion approach [13] has been reported to improve volumetric resolution by a factor of two. However, these * sarang.pendharker@ualberta.ca techniques require imaging and illuminating the same focal plane from both sides of the sample and depend on extensive optical components and precise optical phase matching. 3D STED [1] and 3D STORM [2, 3] for threedimensional localization have also been reported. Axial super-resolution can also be achieved by placing a reflective mirror behind the sample to squeeze the PSF in the axial direction by interference from the reflected STED beam [14,15]. However, STORM and STED impose constraints on properties of the fluorescent probes, limiting their applicability to image photo-switchable fluorophores and samples with a sharp emission spectrum, respectively. Evanescent wave illumination techniques like Total internal Reflection Fluorescence (TIRF) microscopy [16,17], Plasmon enhanced TIRF [18], variableangle TIRF [...

Varifocal Metalens for Optical Sectioning Fluorescence Microscopy

“…As a clear demonstration of imaging performance for our Moirémetalens-based microscope, both standard resolution target and fluorescent microspheres are imaged, with a lateral resolution of ∼2 μm, as well as optical sectioning capability of ∼7 μm. The varifocal Moirémetalens with ultrathin size and compact design may replace its counterpart in any optical system that requires a focus-tunable lens, such as optical coherence tomography 50 and microendoscopy 19 for in vivo imaging.…”

Analysis of a hyperprism for exciting high- k modes and subdiffraction imaging

“…However, it allows only one-dimensional magnification and is further limited when the host index is not small due to the loss of negative spatial frequencies. In many ways this is not entirely surprising, as dielectric prisms are known as dispersive tools for exciting surface plasmons [20] or for frustrated total internal reflection tomography [37], more so than they are for imaging. Here we explore whether hyperprism applications mimicking a conventional prism, tunably coupling free-space plane waves to high-k modes, are more promising.…”

“…The sample is thus illuminated by only the evanescent field, which can allow for high longitudinal resolution. By scanning the angle of incidence and thus the penetration depth, a three-dimensional image with subwavelength longitudinal resolution can be reconstructed [37,44]. Total internal reflection microscopy is particularly useful to image within highly absorbing samples, for example, in water at THz frequencies [45].…”

One-step deconvolution for multi-angle TIRF microscopy with enhanced resolution

“…On the other hand, fluorescence microscopy techniques such as wide-field microscopy, confocal microscopy and most of super-resolution (SR) microscopy exhibited axial resolution inferior to the lateral axis. By rapidly switching among multiple incident angles of the TIRF illumination (MA-TIRF), it has been shown that an axial resolution of ~50 nm is possible in live cells [1,2]. However, given that raw images are always convolution of fluorescence emission image with the point spread function (PSF) of the microscopy, the lateral resolution in previous studies did not reach the diffraction limit yet, as neither method deblurred the raw image with a deconvolution step.…”