Three-dimensional deconvolution based on axial-scanning model for structured illumination microscopy

Structured illumination microscopy (SIM) is an essential super-resolution microscopy technique that enhances resolution. Several images are required to reconstruct a super-resolution image. However, linear SIM resolution enhancement can only increase the spatial resolution of microscopy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function. The frequency of the pattern generated by the nonlinear response in samples is not limited; therefore, nonlinear SIM (NL-SIM), in theory, has no inherent limit to the resolution. In the present study, we describe a two-photon nonlinear SIM (2P-SIM) technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern, which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process. The theoretical models of super-resolution imaging were established through our simulation, which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the resolution. The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.

Structured illumination microscopy (SIM) is a promising imaging technique for high-resolution imaging with a wide field of view. Although a periodic nanostructure is a versatile platform for engineering the spatial frequency of structured illumination patterns in SIM, challenges remain, including artifacts from Fourier space gaps. We designed an all-dielectric super-lattice metasurface (ADSLM) to generate structured illumination patterns with enhanced spatial frequency and broadened spatial frequency coverage with no intermediate frequency gaps. Our numerical simulations reveal that ADSLM-based image reconstruction is capable of producing high-contrast, artifact-free images, resulting in enhanced spatial resolution up to 5.7-fold for coherent SIM at 450 nm. Our results show that the ADSLM-SIM technique may facilitate high-resolution imaging using CMOS-compatible substrates, offering potential for compact miniaturized imaging applications.

Because structured illumination microscopy (SIM) has the advantages of wide-field, rapid imaging, and biocompatibility, it is widely used for super-resolution imaging of living cells. Here, we demonstrate a new super-resolution imaging method—bulk plasmon polariton based SIM (BPPSIM), which combines the SIM with the hyperbolic metamaterials (HMMs). By utilizing an HMM composed of a multilayer metal/dielectric film, a deep subwavelength bulk plasmon polariton is obtained. The imaging resolution of BPPSIM can be improved by the uniform and wide-field structured illumination pattern with high spatial frequency, benefiting from the filtering effect of HMM for the spatial frequency spectrum. The resolution of the recovered images by BPPSIM has been increased to 1/8 of the fluorescence wavelength, which is 2.7-fold enhancement in resolution compared with traditional fluorescence microscopy. This proposed approach demonstrates the operability and flexibility of the structured illumination pattern and can be used in a simple, wide-field and super-resolution fluorescence microscope.

Structured illumination microscopy (SIM) is an attractive choice for fast super-resolution imaging. The generation of structured illumination patterns made by interference of laser beams is broadly employed to obtain high modulation depth of patterns, while the polarizations of the laser beams must be elaborately controlled to guarantee the high contrast of interference intensity, which brings a more complex configuration for the polarization control. The emerging pattern projection strategy is much more compact, but the modulation depth of patterns is deteriorated by the optical transfer function (OTF) of the optical system, especially in high spatial frequency near diffraction limit. Therefore, the traditional super-resolution reconstruction algorithm for interference-based SIM will suffer from much artifact in the case of projection-based SIM that possesses a low modulation depth. Here, we propose an alternative reconstruction algorithm based on image recombination transform (IRT), which provides an alternative solution to address this problem even in a weak modulation depth. We demonstrated the effectiveness of this algorithm in the multicolor super-resolution imaging of BPAE cells in our developed projection-based SIM system, which applies a computer controlled digital micro-mirror device (DMD) for fast fringe generation and multicolor LEDs for illumination. The merit of the system incorporated with the proposed algorithm allows for a low excitation intensity fluorescence imaging even less than 1W/cm2, which is beneficial for the long-term, in vivo super-resolved imaging of live cells and tissues.

We present a volumetric imaging method for biological tissue that is free of mechanically scanning components. The optical sectioning in the system is obtained by structured illumination microscopy (SIM) with the depth of focus being varied by the use of an electronic tunable-focus lens (ETL). The performance of the axial scanning mechanism was evaluated and characterized in conjunction with SIM to ensure volumetric images could be recorded and reconstructed without significant losses in optical section thickness and lateral resolution over the full desired scan range. It was demonstrated that sub-cellular image resolutions were obtainable in both microsphere films and in ex vivo oral mucosa, spanning multiple cell layers, without significant losses in image quality. The mechanism proposed here has the ability to be integrated into any wide-field microscopy system to convert it into a three-dimensional imaging platform without the need for axial scanning of the sample or imaging optics. The ability to axially scan independent of mechanical movement also provides the opportunity for the development of endoscopic systems which can create volumetric images of tissue in vivo.

Structured illumination microscopy (SIM) is an established technique that allows subdiffraction resolution imaging by heterodyning high sample frequencies into the system's passband via structured illumination. However, until now, SIM has been typically used to achieve subdiffraction resolution for intensity-based imaging. Here, we present a novel optical setup that uses structured illumination with a broadband light source to obtain noise-reduced, subdiffraction resolution, quantitative phase imaging (QPM) of cells. We compare this with a previous work for subdiffraction QPM imaging via SIM that used a laser source, and was thus still corrupted by coherent noise.

Structured illumination microscopy (SIM) is an important super-resolution-based microscopy technique that breaks the diffraction limit and enhances optical microscopy systems. With the development of biology and medical engineering, there is a high demand for real-time and robust SIM imaging under extreme low-light and short-exposure environments. Existing SIM techniques typically require multiple structured illumination frames to produce a high-resolution image. In this article, we propose a single-frame SIM (SF-SIM) based on deep learning. Our SF-SIM only needs one shot of a structured illumination frame and generates similar results compared with the traditional SIM systems that typically require 15 shots. In our SF-SIM, we propose a noise estimator that can effectively suppress the noise in the image and enable our method to work in the low-light and short-exposure environment without the need for stacking multiple frames for nonlocal denoising. We also design a bandpass attention module that makes our deep network more sensitive to the change of frequency and enhances the imaging quality. Our proposed SF-SIM is almost 14 times faster than traditional SIM methods when achieving similar results. Therefore, our method is significantly valuable for the development of microbiology and medicine.

Structured illumination microscopy (SIM) is one of the most powerful and versatile super-resolution methods due to its live-cell imaging ability of subcellular structures with high speed. In this paper, we propose an alternative SIM assisted by Bloch surface wave (BSW). Through replacing the conventional laser interference fringes with sub-diffraction BSW counterparts with higher spatial frequency, the imaging resolution of SIM could be enhanced greatly and the super-resolution imaging capability down to 80 nm could be achieved. Compared with traditional wide-field fluorescence microscopy, this BSW SIM demonstrates 2.78 times enhancement in imaging resolution, which surpasses general SIM with only 2 times improvement. Moreover, the structured illumination intensity could be boosted drastically, which is beneficial to nonlinear super-resolution imaging techniques, such as saturated SIM. This BSW SIM would provide a super-resolution, wide field of view approach for surface super-resolution fluorescent imaging while maintaining high imaging speed and good bio-compatibility.

Biological samples are three dimensional and, therefore, optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain super-resolution information if structures of higher frequency are projected onto the sample. This promising approach to super-resolution microscopy is also briefly discussed from a user's perspective.

.Significance: Confocal laser scanning enables optical sectioning in clinical fiber bundle endomicroscopes, but lower-cost, simplified endomicroscopes use widefield incoherent illumination instead. Optical sectioning can be introduced in these simple systems using structured illumination microscopy (SIM), a multiframe digital subtraction process. However, SIM results in artifacts when the probe is in motion, making the technique difficult to use in vivo and preventing the use of mosaicking to synthesize a larger effective field of view (FOV).Aim: We report and validate an automatic motion compensation technique to overcome motion artifacts and allow generation of mosaics in SIM endomicroscopy.Approach: Motion compensation is achieved using image registration and real-time pattern orientation correction via a digital micromirror device. We quantify the similarity of moving probe reconstructions to those acquired with a stationary probe using the relative mean of the absolute differences (MAD). We further demonstrate mosaicking with a moving probe in mechanical and freehand operation.Results: Reconstructed SIM images show an improvement in the MAD from 0.85 to 0.13 for lens paper and from 0.27 to 0.12 for bovine tissue. Mosaics also show vastly reduced artifacts.Conclusion: The reduction in motion artifacts in individual SIM reconstructions leads to mosaics that more faithfully represent the morphology of tissue, giving clinicians a larger effective FOV than the probe itself can provide.

We present a method to improve the isotropy of spatial resolution in a structured illumination microscopy (SIM) implemented for imaging non-fluorescent samples. To alleviate the problem of anisotropic resolution involved with the previous scheme of coherent SIM that employs the two orthogonal standing-wave illumination, referred to as the orthogonal SIM, we introduce a hexagonal-lattice illumination that incorporates three standing-wave fields simultaneously superimposed at the orientations equally divided in the lateral plane. A theoretical formulation is worked out rigorously for the coherent image formation with such a simultaneous multiple-beam illumination and an explicit Fourier-domain framework is derived for reconstructing an image with enhanced resolution. Using a computer-synthesized resolution target as a 2D coherent sample, we perform numerical simulations to examine the imaging characteristics of our three-angle SIM compared with the orthogonal SIM. The investigation on the 2D resolving power with the various test patterns of different periods and orientations reveal that the orientation-dependent undulation of lateral resolution can be reduced from 27% to 8% by using the three-angle SIM while the best resolution (0.54 times the resolution limit of conventional coherent imaging) in the directions of structured illumination is slightly deteriorated by 4.6% from that of the orthogonal SIM.

Structured illumination microscopy (SIM) improves resolution and optical sectioning capability compared to conventional widefield techniques. The main idea of this method is the illumination of the sample with a structured pattern of fixed spatial modulation frequency. Previously, a Fresnel biprism has been implemented in a structured illumination (SI) device providing tunable-frequency sinusoidal patterns. However, the use of this SI system introduces a tradeoff between the visibility and field of view of the illumination fringes. In this contribution, we analyze theoretically this tradeoff and propose the optimal design for the Fresnel biprism-based SIM system.

Structured illumination microscopy (SIM) is a method of non-invasively extracting depth information about a specimen. By using modulated light focused at a discrete depth, it is able optically section a single plane of focus, even in the presence of scattered light from other, out-of-focus planes. However, in order for images to be accurately extracted, the modulation pattern must be positioned into three discrete phases, whose alignment is critical to artifact-free reconstruction. Due to this constraint, SIM does not work well at depth, within optically turbid media, such as human skin. Our research considers two new methods of applying and processing SIM. Random modulation patterns are considered which do not rely on discrete phase differences and are therefore much more robust at depth. In parallel, a complimentary super-resolution method is applied to extract both depth information, as well as enhanced resolution that exceeds the diffraction limit of the system. Secondly, a new single image processing scheme, based on the Hilbert transform is developed to process traditional discrete frequency, modulation patterns. This method mitigates the need for phase alignment greatly increasing the depth of the sectioning, as well as allowing for real-time processing. Further, we produce these images using an LED source in reflectance, obviating the need for fluorescent markers, which we are able to demonstrate by producing in-vivo images on a human subject. This research extends the depth of SIM to 100um within a tissue sample bringing it much closer to other clinical tools, such as Confocal, at a fraction of the cost.

Structured illumination microscopy (SIM) allows non-invasive visualization of nanoscale subcellular structures. However, image acquisition and reconstruction become the bottleneck to further improve the imaging speed. Here, we propose a method to accelerate SIM imaging by combining the spatial re-modulation principle with Fourier domain filtering and using measured illumination patterns. This approach enables high-speed, high-quality imaging of dense subcellular structures using a conventional nine-frame SIM modality without phase estimation of the patterns. In addition, seven-frame SIM reconstruction and additional hardware acceleration further improve the imaging speed using our method. Furthermore, our method is also applicable to other spatially uncorrelated illumination patterns, such as distorted sinusoidal, multifocal, and speckle patterns.

Structured illumination microscopy (SIM) achieves superresolution in fluorescence imaging through patterned illumination and computational image reconstruction, yet current methods require bulky, costly modulation optics and high-precision optical alignment, thus hindering the widespread implementation of SIM. To address this challenge, this work demonstrates how nano-optical metasurfaces, rationally designed to tailor the far-field optical wavefront at sub-wavelength dimensions, hold great potential as ultrathin, single-surface, all-optical wavefront modulators for SIM. We computationally demonstrate this principle with a multipolar-resonant metasurface composed of silicon nanostructures that generate versatile optical wavefronts in the far field upon variation of the polarization or angle of incident light. Algorithmic optimization is performed to identify the seven most suitable illumination patterns for SIM generated by the metasurface based on three key criteria. We quantitatively demonstrate that multipolar-resonant metasurface SIM (mrm-SIM) achieves resolution gain that is comparable to conventional methods by applying the seven optimal metasurface-generated wavefronts to simulated fluorescent objects and reconstructing the objects using proximal gradient descent. Notably, we show that mrm-SIM achieves these resolution gains with a far-field illumination pattern that circumvents complex equipment and alignment requirements of comparable methodologies. The work presented here paves the way for a metasurface-enabled experimental simplification of structured illumination microscopy.