High-resolution light-scattering imaging with two-dimensional hexagonal illumination patterns: system implementation and image reconstruction formulations (original) (raw)
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Optics Letters, 2011
A reflective light-scattering (RLS) microscope with structured illumination (SI) provides subdiffraction resolution and improves the image quality of gold nanoparticles in biological systems. The three-dimensional (3D)-structured pattern is rapidly and precisely controlled with a spatial light modulator and scrambled at the conjugate image plane to increase spatial incoherence. The reconstructed SI-RLS image of 100 nm gold nanoparticles reveals lateral and axial resolutions of approximately 117 and 428 nm. We present a high-resolution image of gold nanoparticles inside a HeLa cell, with improved contrast.
Isotropic image in structured illumination microscopy patterned with a spatial light modulator
Optics express, 2009
We developed a structured illumination microscopy (SIM) system that uses a spatial light modulator (SLM) to generate interference illumination patterns at four orientations - 0 degrees, 45 degrees, 90 degrees, and 135 degrees, to reconstruct a high-resolution image. The use of a SLM for pattern alterations is rapid and precise, without mechanical calibration; moreover, our design of SLM patterns allows generating the four illumination patterns of high contrast and nearly equivalent periods to achieve a near isotropic enhancement in lateral resolution. We compare the conventional image of 100-nm beads with those reconstructed from two (0 degrees +90 degrees or 45 degrees +135 degrees) and four (0 degrees +45 degrees +90 degrees +135 degrees) pattern orientations to show the differences in resolution and image, with the support of simulations. The reconstructed images of 200-nm beads at various depths and fine structures of actin filaments near the edge of a HeLa cell are presented to...
Structured illumination microscopy of a living cell
European Biophysics Journal, 2009
Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells.
Three-beam interference with circular polarization for structured illumination microscopy
Optics express, 2013
Three-dimensional structured illumination microscopy (3D-SIM) is a wide-field technique that can provide doubled resolution and improved image contrast. In this work, we demonstrate a simple approach to 3D-SIM - using three-beam interference with circular polarization to generate the pattern of structured illumination, so that the modulation contrast is routinely maintained at all orientations without a complicated polarization rotator or mechanical motion. We derive the resultant intensity distribution of the interference pattern to confirm the modulation contrast independent of orientation, and compare the result with those using interfering beams of linear polarization. To evaluate the influence of the modulation contrast on imaging, we compare the simulated SIM images of 100-nm beads. Experimental results are presented to confirm the simulations. Our approach requires merely a λ/4-wave plate to alter the interfering beams from linear to circular polarization. This simplicity tog...
Structured illumination microscopy for super-resolution and optical sectioning
Chinese Science Bulletin, 2014
Optical microscopy plays an essential role in biological studies due to its capability and compatibility of non-contact, minimally invasive observation and measurement of live specimens. However, the conventional optical microscopy only has a spatial resolution about 200 nm due to the Abbe diffraction limit, and also lacks the ability of three-dimensional imaging. Super-resolution farfield optical microscopy based on special illumination schemes has been dramatically developed over the last decade. Among them, only the structured illumination microscopy (SIM) is of wide-field geometry that enables it easily compatible with the conventional optical microscope. In this article, the principle of SIM was introduced in terms of point spread function (PSF) and optical transform function (OTF) of the optical system. The SIM for super-resolution (SIM-SR) proposed by Gustafsson et al. and the SIM for optical sectioning (SIM-OS) proposed by Neil et al. are the most popular ones in the research community of microscopy. They have the same optical configuration, but with different data postprocessing algorithms. We mathematically described the basic theories for both of the SIMs, respectively, and presented some numerical simulations to show the effects of super-resolution and optical sectioning. Various approaches to generation of structured illumination patterns were reviewed. As an example, a SIM system based on DMDmodulation and LED-illumination was demonstrated. A lateral resolution of 90 nm was achieved with gold nanoparticles. The optical sectioning capability of the microscope was demonstrated with Golgi-stained mouse brain neurons, and the sectioning strength of 930 nm was obtained.
Applied Optics, 2018
In two-dimensional structured illumination microscopy (2D-SIM), high-resolution images with optimal optical sectioning (OS) cannot be obtained simultaneously. This tradeoff can be overcome by using a tunable-frequency 2D-SIM system and a proper reconstruction method. The goal of this work is twofold. First, we present a computational approach to reconstruct optical-sectioned images with super-resolution enhancement (OS-SR) by using a tunable SIM system. Second, we propose an incoherent tunable-frequency 2D-SIM system based on a Fresnel biprism implementation. Integration of the proposed computational method with this tunable structured illumination (SI) system results in a new 2D-SIM system that is advantageous compared to other 2D-SIM systems with comparable complexity, because it provides high-resolution OS images independent of the objective lens used, without the presence of coherent noise and without reducing the contrast of the structured pattern, as in other incoherent implementations. Evaluation of our proposed system is demonstrated with comparative studies of simulated and experimental reconstructed images to validate our theoretical findings. Our experimental results show a simultaneous improvement of the lateral resolution by a factor of 1.8× with the desired OS capability achieved in the resulting OS-SR combination image. Our experimental results also verify that our system can provide better OS capability than the commercial Zeiss ApoTome-SIM system in the investigated study.
Application of Structured Illumination in Nano-Scale Vision
2003 Conference on Computer Vision and Pattern Recognition Workshop
We describe how structured illumination patterns can be used to increase the resolution of an imaging system for optical microscopy. A target is illuminated by a sequence of finely textured light patterns formed by the interference of multiple coherent beams. The sequence of brightness values reported from a single pixel of a CCD imager encodes the target contrast pattern with sub-pixel resolution. Fourier domain components at spatial frequencies contained in the probing illumination patterns can be recovered from the pixel brightness sequence by solving an over-determined set of linear equations.
Biophysical Journal, 2008
Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.
Structured illumination microscopy artefacts caused by illumination scattering
Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2021
Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artefacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artefacts caused by scattering in thick samples. This article is part of the Theo Murphy meeting issue ‘Super-...