Tunable-frequency three-dimensional structured illumination microscopy with reduced data-acquisition.

.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.

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.

Structured illumination microscopy (SIM) is a valuable tool for three-dimensional microscopy and has numerous applications in bioscience. Its success has been limited to static objects, though, as three sequential image acquisitions are required per final processed, focused image. To overcome this problem we have developed a multicolored grid which when used in tandem with a color camera is capable of performing SIM with just a single exposure. Images and movies demonstrating optical sectioning of three-dimensional objects are presented, and results of applying color SIM for wide-field focused imaging are compared to those of SIM. From computer modeling and analytical calculations a theoretical estimate of the maximum observable object velocity in both the lateral and axial directions is available, implying that the new method will be capable of imaging a variety of live objects. Sample images of the technique applied to lens paper and a pigeon feather are included to show both advantages and disadvantages of CSIM.

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 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.

Optical sectioning structured illumination microscopy (OS‐SIM) is a fast, minimally invasive 3D imaging technique that has found widespread application in the biosciences. It is based on sample illumination with several illumination fringe patterns featuring distinct mutual phase shifts, from which an axially sectioned image is reconstructed. Its optical sectioning capability is commonly attributed to the attenuation of the fringe modulation of light collected from planes displaced from the focal plane. However, in addition to this effect, which is governed solely by the detection optics, optical sectioning can be further enhanced by confining the fringe modulation axially via partially coherent illumination (PCI). To establish guidelines for optimal illumination field shaping, both theoretically and experimentally are investigated, the optical sectioning strength of OS‐SIM upon variation of the two key parameters, modulation period and angular spectrum of the incident illumination. By using PCI with OS‐SIM, nearly fivefold and 1.4‐fold enhanced axial resolution are achieved for scattering (non‐fluorescent) and fluorescent samples, respectively. This work elucidates the optical sectioning mechanism of OS‐SIM and provides a perspective for further optimization.

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) 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.

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) 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.

SIMToolbox is an open-source, modular set of functions for MATLAB equipped with a user-friendly graphical interface and designed for processing two-dimensional and three-dimensional data acquired by structured illumination microscopy (SIM). Both optical sectioning and super-resolution applications are supported. The software is also capable of maximum a posteriori probability image estimation (MAP-SIM), an alternative method for reconstruction of structured illumination images. MAP-SIM can potentially reduce reconstruction artifacts, which commonly occur due to refractive index mismatch within the sample and to imperfections in the illumination. SIMToolbox, example data and the online documentation are freely accessible at http://mmtg.fel.cvut.cz/SIMToolbox. ghagen@uccs.edu Supplementary data are available at Bioinformatics online.

Optical manipulation and detection of biological particulates are crucial procedures in biophotonics. Optical sectioning (OS) opens the avenue to three-dimensional (3D) microscopy, but nonoptical approaches, including sample adhesion and mechanical scanning, have always been required in this technique, rendering it impossible to image suspended cells. Here, we develop optical tweeze-sectioning microscopy by coupling structured illumination microscopy (SIM) with holographic optical tweezers (HOTs). By sculpting light in HOTs, we demonstrate that the position fluctuations of suspended yeast cells can be optically squeezed to tens of nanometers, which is sufficient for the implementation of OS with SIM. Sample scanning is achieved through optical delivery of the cells, instead of translation stages as in the conventional way. Our work presents an all-optical solution for OS, broadening its application to nonadherent, suspended cells. It further furnishes the original technique that enables both SIM-based 3D imaging and optical manipulation.

Structured illumination microscopy (SIM) is one of the most significant widefield super-resolution optical imaging techniques. The conventional SIM utilizes a sinusoidal structured pattern to excite the fluorescent sample; which eventually down-modulates higher spatial frequency sample information within the diffraction-limited passband of the microscopy system and provides around two-fold resolution enhancement over diffraction limit after suitable computational post-processing. Here we provide an overview of the basic principle, image reconstruction, technical development of the SIM technique. Nonetheless, in order to push the SIM resolution further towards the extreme nanoscale dimensions, several different approaches are launched apart from the conventional SIM. Among the various SIM methods, some of the important techniques e.g. TIRF, non-linear, plasmonic, speckle SIM etc are discussed elaborately. Moreover, we highlight different implementations of SIM in various other imaging modalities to enhance their imaging performances with augmented capabilities. Finally, some future outlooks are mentioned which might develop fruitfully and pave the way for new discoveries in near future.

Imaging a volumetric sample in real time is required to directly observe biological anatomy and mechanisms. There are a variety of microscopic imaging systems, developed to achieve optical sectioning, such as confocal microscopy. However, the drawbacks of existing systems are long scanning time or strong laser power. To overcome such problems and to obtain high-speed optical sectioning images, we demonstrate HiLo structured illumination microscopy by the use of digital micro-mirror device (DMD) and the focal tunable lens (FTL). The proposed system is configured such that hybrid uniform and non-uniform pattern are projected onto DMD to reconstruct optical sectioning images. In addition, FTL is utilized to digitally change observation plane with constant magnification and resolution; thus, mechanical movement is eliminated. Furthermore, in vivo three-dimensional (3D) images of biological samples, including a live Canenorhabditis elegans, are experimentally demonstrated.