Dynamic structured illumination microscopy: Focused imaging and optical sectioning for moving objects

.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.

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.

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.

The performance of a tunable three-dimensional (3D) structured illumination microscope (SIM) system and its ability to provide simultaneously super-resolution (SR) and optical-sectioning (OS) capabilities are investigated. Numerical results show that the performance of our 3D-SIM system is comparable with the one provided by a three-wave interference SIM, while requiring 40% fewer images for the reconstruction and providing frequency tunability in a cost-effective implementation. The performance of the system has been validated experimentally with images from test samples, which were also imaged with a commercial SIM based on incoherent-grid projection for comparison. Restored images from data acquired from an axially-thin fluorescent layer show a 1.6× improvement in OS capability compared to the commercial instrument while results from a fluorescent tilted USAF target show the OS and SR capabilities achieved by our system.

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.

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.

Imaging with single-pixel detectors offers a valuable alternative to the conventional focal plane array strategy, especially for wavelengths where silicon-based sensor arrays exhibit lower efficiency. However, the absence of optical sectioning remains a challenge in single-pixel microscopy. In this paper, we introduce a single-pixel microscope with optical sectioning capabilities by integrating single-pixel imaging (SPI) techniques with structured illumination microscopy (SIM) methods. A spatial light modulator positioned at the microscope's input port encodes a series of structured light patterns, which the microscope focuses onto a specific plane of the 3D sample. Simultaneously, a highly sensitive bucket detector captures the light reflected by the object. Optical sectioning is achieved through a high-frequency grating positioned at the microscope's output port, which is conjugated with the spatial light modulator. Utilizing SPI reconstruction techniques and SIM algorithms, our computational microscope produces high-quality 2D images without blurred out-of-focus regions. We validate the performance of the single-pixel microscope (SPM) by measuring the axial response function and acquiring images of various 3D samples in reflected bright-field configuration. Furthermore, we demonstrate the suitability of the optical setup for single-pixel fluorescence microscopy with optical sectioning.

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.

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.

Reflection interference contrast microscopy (RICM, also known as interference reflection microscopy) and related techniques have become of wide interest to the biophysical, soft matter, and biochemistry communities owing to their exquisite sensitivity for characterizing thin films or individual nanoscopic objects adsorbed onto surfaces, or for monitoring cell-substrate interactions. Over the recent years, striking progress has been made to improve the sensitivity and the quantitative analysis of RICM. Its use in more complex environments, with spurious reflections stemming from a variety of structures in the sample, remains however challenging. In this paper, we demonstrate two optical sectioning methods that effectively reduce such background and can be readily implemented in a conventional RICM setup: line confocal detection and structured illumination microscopy. We characterize experimentally the benefits to image quality and demonstrate the use of the methods for quantitative imaging of complex biological and biomimetic samples: cellular membranes, thin organic films, biofunctional surfaces. We then discuss the benefits of each method and provide guidelines to arbitrate between sectioning and signal-to-noise ratio. Finally, we provide a detailed description of our experimental setup and a home-written image acquisition and processing software that should allow the interested reader to duplicate such a setup on a home-built or commercial microscope.

This study presents a partially coherent illumination based (PCI-based) SIM apparatus for dual-modality (phase and fluorescent) microscopic imaging. The partially coherent illumination (PCI) is generated by placing a rotating diffuser on a monochromatic laser beam, which suppresses speckle noise in the dual-modality images and endows the apparatus with sound sectioning capability. With this system, label-free quantitative phase and super-resolved/sectioned fluorescent images can be obtained for the same sample. We have demonstrated the superiority of the system in phase imaging of transparent cells with high endogenous contrast and in a quantitative manner. In the meantime, we have also demonstrated fluorescent imaging of fluorescent beads, rat tail crosscut, wheat anther, and hibiscus pollen with super-resolution and optical sectioning. We envisage that the proposed method can be applied to many fields, including but not limited to biomedical, industrial, chemistry fields.

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) works well to produce depth information by removing undesired light from out of focus planes within a specimen. However, it generally requires multiple images in order to produce sectioning. We show that, under the right conditions, we can leverage Spatial Frequency Domain Imaging (SFDI) to produce both sectioning and relative depth information about a specimen using only a single image. After processing, we find that the axial resolution is comparable to that of traditional 3 phase SIM. With this technique, we are able to produce real-time videos in-vivo at 21 frames per second. Additionally, no moving parts are needed to produce sectioning at depth.

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 demonstrate the intensity distributions in the axial direction and the prospective application to biological systems.

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