Ultra-high spatio-temporal resolution imaging with parallel acquisition-readout structured illumination microscopy (PAR-SIM)

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.

Structured illumination microscopy (SIM) is a fast and gentle super-resolution fluorescence imaging technique, featuring live-cell compatible excitation light levels and high imaging speeds. To achieve SIM, spatial modulation of the fluorescence excitation light is employed. This is typically achieved by interfering coherent laser beams in the sample plane, which are often created by spatial light modulators (SLMs). Digital micromirror devices (DMDs) are a form of SLMs with certain advantages, such as high speed, low cost and wide availability, which present certain hurdles in their implementation, mainly the blazed grating effect caused by the jagged surface structure of the tilted mirrors. Recent works have studied this effect through modelling, simulations and experiments, and laid out possible implementations of multi-color SIM imaging based on DMDs. Here, we present an implementation of a dual-color DMD based SIM microscope using temperature-controlled wavelength matching. By carefully controlling the output wavelength of a diode laser by temperature, we can tune two laser wavelengths in such a way that no opto-mechanical realignment of the SIM setup is necessary when switching between both wavelengths. This reduces system complexity and increases imaging speed. With measurements on nano-bead reference samples, as well as the actin skeleton and membrane of fixed U2OS cells, we demonstrate the capabilities of the setup.

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.

Structured illumination microscopy (SIM) is a widely used super resolution imaging technique that can down-modulate a sample's high-frequency information into objective recordable frequencies to enhance the resolution below the diffraction limit. However, classical SIM image reconstruction methods often generate poor results under low illumination conditions, which are required for reducing photobleaching and phototoxicity in cell imaging experiments. Although denoising methods or auxiliary items improved SIM image reconstruction in low signal level situations, they still suffer from decreased reconstruction quality and significant background artifacts, inevitably limiting their practical applications. In order to improve the reconstruction quality, second-order optimized regularized SIM (sorSIM) is designed specifically for image reconstruction in low signal level situations. In sorSIM, a second-order regularization term is introduced to suppress noise effect, and the penalty factor in this term is selected to optimize the resolution enhancement and noise resistance. Compared to classical SIM image reconstruction algorithms as well as to those previously used in low illumination cases, the proposed sorSIM provides images with enhanced resolution and fewer background artifacts. Therefore, sorSIM can be a potential tool for high-quality and rapid super resolution imaging, especially for low signal images.

The super-resolution imaging technique of structured illumination microscopy (SIM) enables the mixing of high-frequency information into the optical transmission domain via light-source modulation, thus breaking the optical diffraction limit. Correlative SIM, which combines other techniques with SIM, offers more versatility or higher imaging resolution than traditional SIM. In this review, we first briefly introduce the imaging mechanism and development trends of conventional SIM. Then, the principles and recent developments of correlative SIM techniques are reviewed. Finally, the future development directions of SIM and its correlative microscopies are presented.

Structured-illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in-principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency-space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two-dimensional (2D) illumination patterns or sequentially rotated one-dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal-to-noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background. LAY DESCRIPTION: Structured-illumination microscopy (SIM) is a high-resolution light microscopy technique that allows imaging of fluorescence at a resolution about twice the classical diffraction limit. There are various ways that the illumination can be structured, but it is not obvious how the choice of illumination pattern affects the final image quality, especially in view of the noise. We present a detailed performance analysis considering two illumination techniques: sequential illumination with line-gratings that are shifted and rotated during image acquisition and two-dimensional (2D) illumination structures requiring only shift operations. Our analysis is based on analytical theory, supported by simulations of images considering noise. We also extend our analysis to a nonlinear variant of SIM, with which enhanced resolution can be achieved, limited only by noise. This includes nonlinear SIM based on the light-induced switching of the fluorescent molecules between a bright and a dark state. We find sequential illumination with line-gratings to be advantageous in ordinary (linear) SIM, whereas 2D patterns provides a slight signal-to-noise advantage under idealised conditions in nonlinear SIM if there is no nonswitching background.

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.

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.

Superresolution microscopy techniques, light‐based imaging techniques that can surpass the resolution limit imposed by diffraction, are beginning to play an increasing role in studies of synaptic physiology and plasticity. Neuromodulatory proteins implicated in memory‐related synaptic plasticity reside in densecore granules (DCGs) that are transported to both pre‐ and postsynaptic sites. We are using structured illumination microscopy (SIM), a superresolution imaging technique that can be used for multi‐color fluorescence imaging, to evaluate the position of DCGs relative to the active zone, the site where synaptic vesicles cluster near their release machinery. Additionally, we are evaluating if DCGs are targeted to a recently identified exocytic microdomain that is positioned lateral to the postsynaptic density. The enhanced resolution of SIM allows individual DCGs to be distinguished from DCG clusters, and the multi‐color capabilities of SIM provide more detailed synaptic localization data than were previously available using standard wide‐field and laser scanning fluorescence microscopy. This work is supported by National Institutes of Health grant 2 R15 NS40425–03.

Abstract: As an early component of modern super-resolution (SR) imaging technology, structured illumination microscopy (SIM) has been developed for nearly twenty years. With up to ~60 nm and 564 Hz frame rate, it has achieved an optimal combination of spatiotemporal resolution in live cells recently. Despite these advantages, SIM also suffers disadvantages, some of which originated from the intrinsic reconstruction process. Here we review recent technical advances in SIM, including SR reconstruction, performance evaluation, and its integration with other technologies to provide a practical guide for biologists.

Structured illumination microscopy (SIM) can double the spatial resolution of a fluorescence microscope and video rate live cell imaging in a two-dimensional format has been demonstrated. However, rapid implementations of 2D SIM typically only cover a narrow slice of the sample immediately at the coverslip, with most of the cellular volume out of reach. Here, we implement oblique plane structured illumination microscopy (OPSIM) in a projection format to rapidly image an entire cell in a 2D SIM framework. As no mechanical scanning of the sample or objective is involved, this technique has the potential for rapid projection imaging with doubled resolution. We characterize the spatial resolution with fluorescent nanospheres, compare projection and 3D imaging using OPSIM and image mitochondria and ER dynamics across an entire cell at up to 2.7 Hz. To our knowledge, this represents the fastest whole cell SIM imaging to date.

Quantifying submillimeter flows using optical diagnostic techniques is often limited by a lack of spatial resolution and optical access. This review discusses two super-resolution imaging techniques, structured illumination microscopy and total internal reflection fluorescence or microscopy, which can visualize bulk and interfacial flows, respectively, at spatial resolutions below the classic diffraction limits. First, we discuss the theory and applications of structured illumination for optical sectioning, i.e., imaging a thin slice of a flow illuminated over its entire volume. Structured illumination can be used to visualize the interior of multiphase flows such as sprays by greatly reducing secondary scattering. Second, the theory underlying evanescent waves is introduced, followed by a review of how total internal reflection microscopy has been used to visualize interfacial flows over the last 15 years. Both techniques, which are starting to be used in fluid mechanics, could significantly improve quantitative imaging of microscale and macroscale flows.

The varied functions of lipid droplets, which encompass the regulation of lipid and energy homeostasis, as well as their association with the occurrence of various metabolic diseases, are intricately linked to their dynamic properties. Super-resolution imaging techniques have emerged to decipher physiological processes and molecular mechanisms on the nanoscale. However, achieving long-term dynamic super-resolution imaging faces challenges due to the need for fluorescent probes with high photostability. This paper introduces LD-CF, a "buffering probe" for imaging lipid droplet dynamics using structured illumination microscopy (SIM). The polarity-sensitive LD-CF eliminates background fluorescence with a "cyan filter" strategy, enabling wash-free imaging of lipid droplets. In the fluorescent "off" state outside droplets, the probes act as a "buffering pool", replacing photobleached probes inside droplets and enabling photostable long-term SIM imaging. With this probe, three modes of lipid droplet fusion were observed, including the discovery of fusion from large to small lipid droplets. Fluorescence intensity tracking also revealed the direction of lipid transport during the lipid droplet fusion.

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.

We propose a super resolution imaging technique called plasmonic structured illumination microscopy (PSIM), which combines the structured illumination microscopy technique with the tunable surface plasmon interference. Because of the high-resolution enabled by using surface plasmon interference as an illumination source, PSIM possesses higher image resolving power compared with conventional structured illumination microscopy. To demonstrate the technique, we present two specific types of plasmonic structure designs for PSIM. The final images from the simulations show 3-fold and 4-fold resolution improvement compared with conventional epi-fluorescence microscopy.