Structured Illumination Microscopy Imaging Research Articles

Solvatochromic Buffering Fluorescent Probe Resolves the Lipid Transport and Morphological Changes during Lipid Droplet Fusion by Super-Resolution Imaging.

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.

Digital micromirror device-based fast super-resolution lattice structured light illumination microscopy

Super-resolution structured illumination microscopy (SR-SIM) offers numerous advantages such as high temporal resolution, low photobleaching and phototoxicity, and no special requirements for fluorescent probes. It is particularly suitable for long-term SR imaging of living cells. By using two-dimensional lattice structured light serving as illumination, SR-SIM can achieve faster imaging speed and reduce phototoxicity, however, it is accompanied with system complexity increasing. To address this problem, in this work, we propose a fast SR lattice structured illumination microscopy imaging method based on a digital micromirror device (DMD), called DMD-Lattice-SIM. This method utilizes a DMD and synchronous time-sharing triggering with sCMOS to generate two-dimensional orthogonal lattice structured light. The proposed method only requires the collection of five phase-shifted raw images for SR image reconstruction, reducing the acquisition time by approximately 44.4% compared with the traditional SR-SIM method that requires nine phase-shifted raw images. In this work, we also introduce a rapid SR image reconstruction method called Lattice-JSFR-SIM, which combines the advantages of joint space and frequency reconstruction (JSFR)-SIM and Lattice-SIM. The raw images are pre-filtered in the frequency domain and then undergo SR reconstruction in the spatial domain. This approach reduces reconstruction time by approximately 55.6% compared with traditional frequency domain image reconstruction processing, within an imaging field of view of 512 pixels×512 pixels. The feasibility of the proposed method is demonstrated through experiments on cell microtubules and the observation of mitochondrial division and fusion in living cells. The findings presented in this paper hold great significance and application value for enabling real-time SR imaging of living cells.

Easily scalable multi-color DMD-based structured illumination microscopy.

Structured illumination microscopy (SIM) achieves super-resolution imaging using a series of phase-shifted sinusoidal illumination patterns to down-modulate high spatial-frequency information of samples. Digital micromirror devices (DMDs) have been increasingly used to generate SIM illumination patterns due to their high speed and moderate cost. However, a DMD micromirror array's blazed grating structure causes strong angular dispersion for different wavelengths of light, thus severely hampering its application in multicolor imaging. We developed a multi-color DMD-SIM setup that employs a diffraction grating to compensate the DMD's dispersion and demonstrate super-resolution SIM imaging of both fluorescent beads and live cells samples with four color channels. This simple but effective approach can be readily scaled to more color channels, thereby greatly expanding the application of SIM in the study of complex multi-component structures and dynamics in soft matter systems.

Super-resolution reconstruction of structured illumination microscopy using deep-learning and sparse deconvolution

Structured illumination microscopy (SIM) is one of the essential super-resolution microscopic imaging techniques, featuring fast imaging speed, low exposure dose, and having no special requirements for fluorescent probes. In this paper, we propose a super-resolution SIM reconstruction approach, which integrates an end-to-end multi-scale neural network (entitled Scale Richardson-Lucy Network, SRLN). SRLN fits the SIM imaging process through Richardson-Lucy deconvolution incorporated in the neural network. After being trained with sufficient data pairs, among which the ground-truth images were refined with sparse deconvolution, the SRLN network can yield super-resolution images with a spatial resolution of ∼70 nm from raw SIM intensity images. The feasibility and generalization capability of SRLN were experimentally demonstrated by reconstructing SR images for different biological structures, of which the raw images were acquired with different devices. The proposed network will be valuable for super-resolution imaging in the biology and medicine fields.

Projective oblique plane structured illumination microscopy

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.

Tandem aberration correction optics (TACO) in wide-field structured illumination microscopy.

Structured illumination microscopy (SIM) is a powerful super-resolution imaging technique that uses patterned illumination to down-modulate high spatial-frequency information of samples. However, the presence of spatially-dependent aberrations can severely disrupt the illumination pattern, limiting the quality of SIM imaging. Conventional adaptive optics (AO) techniques that employ wavefront correctors at the pupil plane are not capable of effectively correcting these spatially-dependent aberrations. We introduce the Tandem Aberration Correction Optics (TACO) approach that combines both pupil AO and conjugate AO for aberration correction in SIM. TACO incorporates a deformable mirror (DM) for pupil AO in the detection path to correct for global aberrations, while a spatial light modulator (SLM) is placed at the plane conjugate to the aberration source near the sample plane, termed conjugate AO, to compensate spatially-varying aberrations in the illumination path. Our numerical simulations and experimental results show that the TACO approach can recover the illumination pattern close to an ideal condition, even when severely misshaped by aberrations, resulting in high-quality super-resolution SIM reconstruction. The TACO approach resolves a critical traditional shortcoming of aberration correction for structured illumination. This advance significantly expands the application of SIM imaging in the study of complex, particularly biological, samples and should be effective in other wide-field microscopies.

Enhancing structural illumination microscopy with hybrid CNN-transformer and dynamic frequency loss

Deep learning techniques have augmented structured illumination microscopy (SIM) imaging by employing convolutional neural networks (CNNs) for the super-resolution reconstruction of SIM images. However, due to the intrinsic shortcomings of CNNs, i.e. limited receptive field and inadaptability to input content, their reconstruction faithfulness and imaging resolution are limited. Recently, another class of neural backbone, Transformer, has shown complementary advantages to CNNs. To this end, this work aims to borrow the recent Transformers to enhance CNN-based SIM reconstruction, thereby fully unleashing the power of neural networks for SIM imaging. Concretely, we first experimentally explore the pros and cons of CNNs and Transformers for SIM reconstruction. Then, we propose a Hybrid CNN-Transformer (HCT) model, to inherit merits of the both. To further encourage the recovery of high frequencies down-modulated by patterned illumination, we also devise a dynamic frequency loss based on the phase spectrum information, helping models focus on hard frequencies adaptively. Experiments on diverse subcellular structures demonstrate that the proposed method can evidently boost SIM reconstruction performance, even under low-light conditions or with fewer raw frames.

Multi-color two-photon scanning structured illumination microscopy imaging of live cells.

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.

High-speed spatially re-modulated structured illumination microscopy.

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.

260 Breast Cancer Biopsy Triage with Ex-Vivo Microscopy for Downstream Analysis

OBJECTIVES/GOALS: In this study, the ability of a pathologist to detect malignancy on digital pseudo-H&E slides obtained via structured illumination microscopy (SIM) imaging of fresh diagnostic breast biopsies was assessed. The speed of imaging and processing was also assessed for potential clinical implementation. METHODS/STUDY POPULATION: This study was conducted in accordance with an Ochsner Medical Center of New Orleans IRB. 200 patients undergoing either stereotactic or ultrasound-guided diagnostic breast biopsies were consented and an additional core from the suspicious lesion was collected for research use. Research biopsies were transported to the lab and stained with DRAQ5 and Eosin-Y and imaged with SIM before being submitted for histology processing. Imaging and digital processing times were recorded. The resulting SIM images and histology slides were given to a pathologist for blind review to assess accuracy. RESULTS/ANTICIPATED RESULTS: The ex-vivo structured illumination microscopy images and subsequent histology slides from 79 research cores have been assessed to date. Some samples were excluded from the total data set and not included in the final assessment due to technical failures of the imaging protocol. Of the current set, the pathologist has a specificity of 88% and a sensitivity of 65%, as well as an NPV of 88% and a PPV of 65%. Staining time for each biopsy was completed within 3 and a half minutes and imaging at 20x magnification took between 4 and 12 minutes, depending on size and implementation of autofocus to the imaging system. Image processing took approximately 5 minutes per biopsy and is a direct function of biopsy size. DISCUSSION/SIGNIFICANCE: Decreased time between cancer suspicion and treatment will improve the prognosis of breast cancer patients. SIM imaging of fresh breast biopsies could ultimately allow primary and secondary histology to be performed simultaneously and minimize histopathology time, thus allowing clinicians and patients to implement treatment course more quickly.

SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells

It is urgent to understand the infection mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for the prevention and treatment of COVID-19. The infection of SARS-CoV-2 starts when the receptor-binding domain (RBD) of viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) of the host cell, but the endocytosis details after this binding are not clear. Here, RBD and ACE2 were genetically coded and labeled with organic dyes to track RBD endocytosis in living cells. The photostable dyes enable long-term structured illumination microscopy (SIM) imaging and to quantify RBD-ACE2 binding (RAB) by the intensity ratio of RBD/ACE2 fluorescence. We resolved RAB endocytosis in living cells, including RBD-ACE2 recognition, cofactor-regulated membrane internalization, RAB-bearing vesicle formation and transport, RAB degradation, and downregulation of ACE2. The RAB was found to activate the RBD internalization. After vesicles were transported and matured within cells, RAB was finally degraded after being taken up by lysosomes. This strategy is a promising tool to understand the infection mechanism of SARS-CoV-2.

Nonblinking Core–Multishell InP/ZnSe/ZnS Quantum Dot Bioconjugates for Super-resolution Imaging

Colloidal quantum dots (QDs) are vital fluorescent probes with several optical features superior to those found in organic dyes for microscopy imaging. However, the emission blinking behavior of a single QD is an intrinsic drawback for stimulated emission depletion microscopy (STED) or structured illumination microscopy techniques. Herein, we present a rational strategy for the synthesis of a type of deterministic, environmentally friendly, small nonblinking core–multishell InP/ZnSe/ZnS QD using a hydrofluoric acid (HF) etching passivation. The strategy includes four steps: synthesis of the InP core, synthesis of oil-phase core–multishell InP/ZnSe/ZnS QDs using hydrogen fluoride and trioctylphosphine etching passivation, promotion of the water solubility of QDs modified by 3-mercaptopropionic acid (MPA), and enhanced biotargeting of core–multishell InP/ZnSe/ZnS QDs by surface coupling technology. The suppressed blinking mechanism was primarily attributed to surface wrapping shells and HF etching passivation of surface dangling bonds, which reduced surface nonradiative recombination. Nonblinking QDs exhibit high fluorescence efficiency (∼90%), good photostability (>20 min), a large Stokes shift (>135 nm), biocompatibility of the cell viability (>90%), and high targeting. Furthermore, super-resolution multifocal structured illumination microscopy imaging of subcellular structures labeled using these QDs confirmed a resolution improvement of greater than 2.6-fold compared to wide-field microscopy. The results indicate that the nonblinking QDs can function as excellent probes for use in the long-term super-resolution visualization of subcellular dynamics.

Structure Rigidification Promoted Ultrabright Solvatochromic Fluorescent Probes for Super-Resolution Imaging of Cytosolic and Nuclear Lipid Droplets.

Lipid droplets (LDs) containing cytosolic and nuclear LDs have recently received increasing attention because of their diverse biological roles in living systems. However, developing fluorescent probes for super-resolution visualization of these subcellular LDs still remains challenging due to insufficient fluorescence brightness and poor nuclear membrane permeability. Herein, we rationally synthesized a series of ultrabright solvatochromic fluorescent probes based on benzoboranils (BBAs) for LD-specific super-resolution imaging using structured illumination microscopy (SIM). The rigidly structured probes exhibit ultrahigh fluorescence quantum yields of up to 99.9% in low-polar solvents. They also show more significant fluorescence enhancements in lipid environments than commercial LD probes. Owing to these excellent merits, our lipophilic fluorescent probes can specifically light up subcellular LDs at ultralow concentrations down to 10 nM. Further use of BBA-CF3 for super-resolution SIM imaging of cytosolic and nuclear LDs and their fusion process was successfully achieved. The unprecedented spatial resolution for nuclear LDs with an FWHM value of 142 nm was also acquired. Collectively, our ultrabright fluorescent probes hold tremendous potential to unveil the mysterious roles of cytosolic and nuclear LDs in biological research using SIM.

Constructing D-π-A-π dye to obtain red-emission fluorescent probe for structured illumination microscopy imaging of lipid droplet dynamics

Lipid droplets (LDs) are dynamic organelles interacting with a variety of intracellular organelles. Tracking intracellular LD dynamics employing synthetic small molecules is crucial for biological studies. Fluorescence imaging in the red and near infrared (NIR) region is more suitable for biological imaging due to its low phototoxicity and high signal-to-noise ratio. However, available LD-dyes in the red region with remarkable environmental sensitivity, selectivity for LDs staining are limited. Here, we constructed a red-emission D-π-A-π type LD-dye LD 688P with higher environmental sensitivity and suitable “calculated log P” (Clog P) for LDs dynamic imaging. LD 688P was proved to be highly selective and photostable for tracing LD fusion including multiple consecutive fusions and fusions in a centrosymmetric manner by super-resolution microscopy. We believe that the D-π-A-π skeleton would be an efficient strategy to construct red and even NIR-emission dyes.

Glucose increases the length and spacing of the lattice structure of the axon initial segment.

The axon initial segment (AIS) plays an important role in maintaining neuronal polarity and initiating action potentials (APs). The AIS adapts to its environment by changing its length and distance from the cell body, resulting in modulation of neuronal excitability, which is referred to as AIS plasticity. Previous studies found an ~200 nm single periodic distribution of the key AIS components ankyrinG (AnkG), Nav 1.2, and βIV-spectrin, while it remains unclear how the lattice structure is altered by AIS plasticity. In this study, we found that the length of the AIS significantly increased, resulting in increased neuronal excitability, with high-concentration glucose treatment. Structured illumination microscopy (SIM) images of the lattice structure showed a dual-spacing periodic distribution (~200 nm and ~260 nm) of AnkG, Nav 1.2, and βIV-spectrin. Moreover, 480-kDa AnkG was crucial for AIS plasticity and increased lattice structure spacing. The discovery of new regulators for modulating AIS plasticity will help us to understand and manipulate the structure and function of the AIS. Glucose triggers axon initial segment (AIS) plasticity of cultured neurons. AIS lattice structure under glucose treatment shows an increased spacing by structured illumination microscopy imaging. 480-kDa AnkG contributes to AIS plasticity.

SCMOS Noise-Corrected Superresolution Reconstruction Algorithm for Structured Illumination Microscopy

Structured illumination microscopy (SIM) is widely applied due to its high temporal and spatial resolution imaging ability. sCMOS cameras are often used in SIM due to their superior sensitivity, resolution, field of view, and frame rates. However, the unique single-pixel-dependent readout noise of sCMOS cameras may lead to SIM reconstruction artefacts and affect the accuracy of subsequent statistical analysis. We first established a nonuniform sCMOS noise model to address this issue, which incorporates the single-pixel-dependent offset, gain, and variance based on the SIM imaging process. The simulation indicates that the sCMOS pixel-dependent readout noise causes artefacts in the reconstructed SIM superresolution (SR) image. Thus, we propose a novel sCMOS noise-corrected SIM reconstruction algorithm derived from the imaging model, which can effectively suppress the sCMOS noise-related reconstruction artefacts and improve the signal-to-noise ratio (SNR).

Fast and Lightweight Network for Single Frame Structured Illumination Microscopy Super-Resolution

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.

MAL2 mediates the formation of stable HER2 signaling complexes within lipid raft-rich membrane protrusions in breast cancer cells.

SUMMARYThe lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target.

Comparison of Two- and Three-Beam Interference Pattern Generation in Structured Illumination Microscopy

Structured illumination microscopy (SIM) provides wide-field optical sectioning in the focal plane by modulating the imaging information using fringe pattern illumination. For generating the fringe pattern illumination, a digital micro-mirror device (DMD) is commonly used due to its flexibility and fast refresh rate. However, the benefit of different pattern generation, for example, the two-beam interference mode and the three-beam interference mode, has not been clearly investigated. In this study, we systematically analyze the optical sectioning provided by the two-beam inference mode and the three-beam interference mode of DMD. The theoretical analysis and imaging results show that the two-beam interference mode is suitable for fast imaging of the superficial dynamic target due to reduced number of phase shifts needed to form the image, and the three-beam interference mode is ideal for imaging three-dimensional volume due to its superior optical sectioning by the improved modulation of the illumination patterns. These results, we believe, will provide better guidance for the use of DMD for SIM imaging and also for the choice of beam patterns in SIM application in the future.

Dual color DMD-SIM by temperature-controlled laser wavelength matching.

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.