High-throughput Imaging Research Articles

Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens.

Stochastic transcriptional bursting is a universal property of active genes. While different genes exhibit distinct bursting patterns, the molecular mechanisms for gene-specific stochastic bursting are largely unknown. We have developed and applied a high-throughput-imaging based screening strategy to identify cellular factors and molecular mechanisms that determine the bursting behavior of human genes. Focusing on epigenetic regulators, we find that protein acetylation is a strong acute modulator of burst frequency, burst size and heterogeneity of bursting. Acetylation globally affects the Off-time of genes but has gene-specific effects on the On-time. Yet, these effects are not strongly linked to promoter acetylation, which do not correlate with bursting properties, and forced promoter acetylation has variable effects on bursting. Instead, we demonstrate acetylation of the Integrator complex as a key determinant of gene bursting. Specifically, we find that elevated Integrator acetylation decreases bursting frequency. Taken together our results suggest a prominent role of non-histone proteins in determining gene bursting properties, and they identify histone-independent acetylation of a transcription cofactor as an allosteric modulator of bursting via a far-downstream bursting checkpoint.

Optofluidic time-stretch imaging flow cytometry with a real-time storage rate beyond 5.9 GB/s.

Optofluidic time-stretch imaging flow cytometry (OTS-IFC) provides a suitable solution for high-precision cell analysis and high-sensitivity detection of rare cells due to its high-throughput and continuous image acquisition. However, transferring and storing continuous big data streams remains a challenge. In this study, we designed a high-speed streaming storage strategy to store OTS-IFC data in real-time, overcoming the imbalance between the fast generation speed in the data acquisition and processing subsystem and the comparatively slower storage speed in the transmission and storage subsystem. This strategy, utilizing an asynchronous buffer structure built on the producer-consumer model, optimizes memory usage for enhanced data throughput and stability. We evaluated the storage performance of the high-speed streaming storage strategy in ultra-large-scale blood cell imaging on a common commercial device. The experimental results show that it can provide a continuous data throughput of up to 5891 MB/s.

Practical considerations for birefringence microscopy of myelin structure: Microscope design and tissue processing for effective imaging

Abstract Despite the interest in studying and quantifying the structural integrity of myelin in postmortem brain tissue, current methods for high-resolution imaging of myelin with optical microscopy are not sufficient. While imaging methods must have adequate resolution and sensitivity to detect microstructural alterations to myelin that are relevant in aging and neurodegenerative disease, an equally critical aspect is to minimize myelin damage that is induced during tissue processing steps. Birefringence microscopy (BRM) is a powerful technique that leverages the structural anisotropy of myelin to provide detailed, label-free images of myelin at any diffraction-limited optical resolution, while maintaining a simple and low-cost setup. Building on our previous work, we have developed a new BRM system and image processing pipeline that enable efficient, high-throughput imaging of myelin structure at multiple scales. Here, we utilize this system to systematically assess the damage to myelin that is induced by several common tissue processing steps in brain sections from the rhesus monkey. Images taken of the same myelinated axons, before and after each tissue processing step, provide direct evidence that mishandling of tissue during sample preparation can cause significant structural alterations to myelin. First, we report on key advancements to our BRM system, imaging procedure, and image processing pipeline, which provide significant increases to the speed and efficiency of BRM. These include integrating fast piezoelectric rotational stages, minimizing the number of images required (to three images) for determining birefringence parameter maps, and implementing an analytical solution for directly determining birefringence parameter maps. Second, using this BRM system, we demonstrate that effective myelin imaging requires (1) the avoidance of prolonged drying or dehydration of tissue, (2) the selection of the optimal mounting medium (85% glycerol), (3) the avoidance of tissue permeabilization with detergents (i.e., Triton X-100 and Saponin), and (4) the selection of a suitable tissue-section thickness (15, 30 and 60 µm) based on the region of interest. In addition to serving as a guide for new users interested in imaging myelin, these basic experiments in sample preparation highlight that BRM is very sensitive to changes in the underlying lipid structure of myelin and suggest that optimized BRM can enable new studies of myelin breakdown in disease. In this work, we show that BRM is a leading method for detailed imaging and characterization of myelin, and we provide direct evidence that the structure of myelin is highly sensitive to damage during inadequate preparation of brain tissue for imaging, which has previously not been properly characterized for birefringence imaging of myelin. For the most effective, high-resolution imaging of myelin structure, tissue processing should be kept to a minimum, with sections prevented from dehydration and mounted in 85% glycerol. With proper preservation of myelin structure, BRM provides exquisitely detailed images that facilitate the assessment of myelin pathology associated with injury or disease.

Multi-scale characterisation of cold response reveals immediate and long-term impacts on cell physiology up to seed composition in sunflower.

Early sowing can help summer crops escape drought and can mitigate the impacts of climate change on them. However, it exposes them to cold stress during initial developmental stages, which has both immediate and long-term effects on development and physiology. To understand how early night-chilling stress impacts plant development and yield, we studied the reference sunflower line XRQ under controlled, semi-controlled and field conditions. We performed high-throughput imaging of the whole plant parts and obtained physiological and transcriptomic data from leaves, hypocotyls and roots. We observed morphological reductions in early stages under field and controlled conditions, with a decrease in root development, an increase in reactive oxygen species content in leaves and changes in lipid composition in hypocotyls. A long-term increase in leaf chlorophyll suggests a stress memory mechanism that was supported by transcriptomic induction of histone coding genes. We highlighted DEGs related to cold acclimation such as chaperone, heat shock and late embryogenesis abundant proteins. We identified genes in hypocotyls involved in lipid, cutin, suberin and phenylalanine ammonia lyase biosynthesis and ROS scavenging. This comprehensive study describes new phenotyping methods and candidate genes to understand phenotypic plasticity better in response to chilling and study stress memory in sunflower.

Depth-of-field extended Fourier ptychographic microscopy without defocus distance priori.

Fourier ptychographic microscopy (FPM) provides a solution of high-throughput phase imaging. Thanks to its coherent imaging model, FPM has the capacity of depth-of-field (DOF) extension by simultaneously recovering the sample's transmittance function and pupil aberration, which contains a defocus term. However, existing phase retrieval algorithms (PRs) often struggle in the presence of a significant defocus. In this Letter, different PRs with embedded pupil recovery are compared, and the one based on the alternating direction multiplier method (ADMM-FPM) demonstrates promising potential for reconstructing highly defocused FPM images. Besides, we present a plug-and-play framework that integrates ADMM-FPM and total variation or Hessian denoiser for pupil function enhancement. Both simulations and experiments demonstrate that this framework enables robust reconstruction of defocused FPM images without any prior knowledge of defocus distance or sample characteristics. In experiments involving USAF 1951 targets and pathologic slides, ADMM-FPM combined with the Hessian denoiser successfully corrected the defocus up to approximately 200 µm, i.e., extending the DOF to 400 µm.

Resolving Nonequilibrium Shape Variations among Millions of Gold Nanoparticles.

Nanoparticles, exhibiting functionally relevant structural heterogeneity, are at the forefront of cutting-edge research. Now, high-throughput single-particle imaging (SPI) with X-ray free-electron lasers (XFELs) creates opportunities for recovering the shape distributions of millions of particles that exhibit functionally relevant structural heterogeneity. To realize this potential, three challenges have to be overcome: (1) simultaneous parametrization of structural variability in real and reciprocal spaces; (2) efficiently inferring the latent parameters of each SPI measurement; (3) scaling up comparisons between 105 structural models and 106 XFEL-SPI measurements. Here, we describe how we overcame these three challenges to resolve the nonequilibrium shape distributions within millions of gold nanoparticles imaged at the European XFEL. These shape distributions allowed us to quantify the degree of asymmetry in these particles, discover a relatively stable "shape envelope" among nanoparticles, discern finite-size effects related to shape-controlling surfactants, and extrapolate nanoparticles' shapes to their idealized thermodynamic limit. Ultimately, these demonstrations show that XFEL SPI can help transform nanoparticle shape characterization from anecdotally interesting to statistically meaningful.

Application of the polyhedral template matching method for characterization of 2D atomic resolution electron microscopy images

High-throughput image segmentation of atomic resolution electron microscopy data poses an ongoing challenge for materials characterization. In this paper, we investigate the application of the polyhedral template matching (PTM) method, a technique widely employed for visualizing three-dimensional (3D) atomistic simulations, to the analysis of two-dimensional (2D) atomic resolution electron microscopy images. This technique is complementary with other atomic resolution data reduction techniques, such as the centrosymmetry parameter, that use the measured atomic peak positions as the starting input. Furthermore, since the template matching process also gives a measure of the local rotation, the method can be used to segment images based on local orientation. We begin by presenting a 2D implementation of the PTM method, suitable for atomic resolution images. We then demonstrate the technique's application to atomic resolution scanning transmission electron microscopy images from close-packed metals, providing examples of the analysis of twins and other grain boundaries in FCC gold and martensite phases in 304 L austenitic stainless steel. Finally, we discuss factors, such as positional errors in the image peak locations, that can affect the accuracy and sensitivity of the structural determinations.

Crosstalk of growth factor receptors at plasma membrane clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these nanoscale complexes in response to external cues remain unclear. Here, we use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures across the plasma membrane of human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with ligands. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces a capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR individually with drugs prevents the recruitment of both EGFR and FGFR. Our data reveals novel crosstalk between multiple unrelated receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.

Label-free, high-throughput holographic imaging to evaluate mammalian gametes and embryos†.

Assisted reproduction is one of the significant tools to treat human infertility. Morphological assessment is the primary method to determine sperm and embryo viability during in vitro fertilization cycles. It has the advantage of being a quick, convenient, and inexpensive means of assessment. However, visual observation is of limited predictive value for early embryo morphology. It has led many to search for other imaging tools to assess the reproductive potential of a given embryo. The limitations of visual assessment apply to both humans and animals. One recent innovation in assisted reproduction technology imaging is interferometric phase microscopy, also known as holographic microscopy. Interferometric phase microscopy/quantitative phase imaging is the next likely progression of analytical microscopes for the assisted reproduction laboratory. The interferometric phase microscopy system analyzes waves produced by the light as it passes through the specimen observed. The microscope collects the light waves produced and uses the algorithm to create a hologram of the specimen. Recently, interferometric phase microscopy has been combined with quantitative phase imaging, which joins phase contrast microscopy with holographic microscopy. These microscopes collect light waves produced and use the algorithm to create a hologram of the specimen. Unlike other systems, interferometric phase microscopy can provide a quantitative digital image, and it can make 2D and 3D images of the samples. This review summarizes some newer and more promising quantitative phase imaging microscopy systems for evaluating gametes and embryos. Studies clearly show that quantitative phase imaging is superior to bright field microscopy-based evaluation methods when evaluating sperm and oocytes prior to IVF and embryos prior to transfer. However, further assessment of these systems for efficacy, reproducibility, cost-effectiveness, and embryo/gamete safety must take place before they are widely adopted.

Allele-level visualization of transcription and chromatin by high-throughput imaging

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

Animal PET scanner with a large field of view is suitable for high-throughput scanning of rodents.

In preclinical studies, high-throughput positron emission tomography (PET) imaging, known as simultaneous multiple animal scanning, can reduce the time spent on animal experiments, the cost of PET tracers, and the risk of synthesis of PET tracers. It is well known that the image quality acquired by high-throughput imaging depends on the PET system. Herein, we investigated the influence of large field of view (FOV) PET scanner on high-throughput imaging. We investigated the influence of scanning four objects using a small animal PET scanner with a large FOV. We compared the image quality acquired by four objects scanned with the one acquired by one object scanned using phantoms and animals. We assessed the image quality with uniformity, recovery coefficient (RC), and spillover ratio (SOR), which are indicators of image noise, spatial resolution, and quantitative precision, respectively. For the phantom study, we used the NEMA NU 4-2008 image quality phantom and evaluated uniformity, RC, and SOR, and for the animal study, we used Wistar rats and evaluated the spillover in the heart and kidney. In the phantom study, four phantoms had little effect on imaging quality, especially SOR compared with that for one phantom. In the animal study as well, four rats had little effect on spillover from the heart muscle and kidney cortex compared with that for one rat. This study demonstrated that an animal PET scanner with a large FOV was suitable for high-throughput imaging. Thus, the large FOV PET scanner can support drug discovery and bridging research through rapid pharmacological and pathological evaluation.

Build and operation of a custom 3D, multicolor, single-molecule localization microscope.

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system's performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000-180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4-8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images.

FPM-WSI: Fourier ptychographic whole slide imaging via feature-domain backdiffraction

Fourier ptychographic microscopy (FPM) theoretically provides a solution to the trade-off between spatial resolution and field of view (FOV), and has promising prospects in digital pathology. However, block reconstruction and then stitching has become an unavoidable procedure for reconstruction of large FOV due to vignetting artifacts. This introduces digital stitching artifacts, as the existing image-domain optimization algorithms are highly sensitive to systematic errors. Such obstacles significantly impede the advancement and practical implementation of FPM, explaining why, despite a decade of development, FPM has not gained widespread recognition in the field of biomedicine. We report a feature-domain FPM (FD-FPM) based on the structure-aware forward model to realize stitching-free, full-FOV reconstruction. The loss function is uniquely formulated in the feature domain of images, which bypasses the troublesome vignetting effect and algorithmic vulnerability via feature-domain backdiffraction. Through massive simulations and experiments, we show that FD-FPM effectively eliminates vignetting artifacts for full-FOV reconstruction, and still achieves impressive reconstructions despite the presence of various systematic errors. We also found it has great potential in recovering the data with a lower spectrum overlapping rate, and in realizing digital refocusing without a prior defocus distance. With FD-FPM, we achieved full-color and high-throughput imaging (4.7 mm diameter FOV, 336 nm resolution in the blue channel) free of blocking-and-stitching procedures on a self-developed Fourier ptychographic microscopy whole slide imaging platform. The reported FD-FPM shows the value of FPM for various experimental circumstances, and offers physical insights useful for the developments of models for other computational imaging techniques. The reported platform demonstrates high-quality, high-speed imaging and low cost, and could find applications in many fields of biomedical research, as well as in clinical applications.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

Structural analysis of the female reptile reproductive system by micro-computed tomography and optical coherence tomography†.

Volumetric data provide unprecedented structural insight to the reproductive tract and add vital anatomical context to the relationships between organs. The morphology of the female reproductive tract in non-avian reptiles varies between species, corresponding to a broad range of reproductive modes and providing valuable insight to comparative investigations of reproductive anatomy. However, reproductive studies in reptilian models, such as the brown anole studied here, have historically relied on histological methods to understand the anatomy. While these methods are highly effective for characterizing the cell types present in each organ, histological methods lose the 3D relationships between images and leave the architecture of the organ system poorly understood. We present the first comprehensive volumetric analyses of the female brown anole reproductive tract using two non-invasive, non-destructive imaging modalities: micro-computed tomography (microCT) and optical coherence tomography (OCT). Both are specialized imaging technologies that facilitate high-throughput imaging and preserve three-dimensional information. This study represents the first time that microCT has been used to study all reproductive organs in this species and the very first time that OCT has been applied to this species. We show how the non-destructive volumetric imaging provided by each modality reveals anatomical context including orientation and relationships between reproductive organs of the anole lizard. In addition to broad patterns of morphology, both imaging modalities provide the high resolution necessary to capture details and key anatomical features of each organ. We demonstrate that classic histological features can be appreciated within whole-organ architecture in volumetric imaging using microCT and OCT, providing the complementary information necessary to understand the relationships between tissues and organs in the reproductive system. This side-by-side imaging analysis using microCT and OCT allows us to evaluate the specific advantages and limitations of these two methods for the female reptile reproductive system.

Tools and methods for high-throughput single-cell imaging with the mother machine

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, ‘what you put is what you get’ (WYPIWYG) – that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.

Tools and methods for high-throughput single-cell imaging with the mother machine.

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, 'what you put is what you get' (WYPIWYG) - that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.

Harnessing Microbial Effectors for Macrophage-Mediated Drug Delivery.

Macrophage-based drug delivery systems are promising, but their development is still in its infancy, with many limitations remaining to be addressed. Our aim was to design a system harnessing microbial effectors to facilitate controlled drug cargo expulsion from macrophages to enable the use of more toxic drugs without adding to the risk of off-target detrimental effects. The pore forming and actin polymerizing Listeria monocytogenes effectors listeriolysin-O (LLO) and actin assembly-inducing protein (ActA) were synthesized using a novel green fluorescent protein (GFP)-linked heterologous expression system. These effectors were coated onto polystyrene beads to generate "synthetic cargo" before loading into primary M1 macrophages. Bead uptake and release from macrophages were evaluated by using high-throughput quantitative imaging flow cytometry and confocal microscopy. In vitro results confirmed appropriate activity of synthesized effectors. Coating of these effector proteins onto polystyrene beads (simulated drug cargo) resulted in changes in cellular morphology, bead content, and intracellular bead localization, which may support an interpretation of the induced release of these beads from the cells. This forms the basis for further investigation to fully elucidate any potential release mechanisms. Bacterial effectors ActA and LLO successfully effectuated actin polarization and protrusions from cell membranes similar to those seen in cells infected with Listeria spp., illustrating the potential of using these effectors and production methods for the development of an endogenous drug delivery system capable of low-risk, targeted release of high potency drugs.

Large-field and fast super-resolution microscopic imaging method based on laser interferometry

In recent years, structured illumination microscopy (SIM) has been drawing great attention for both technique development and application. However, conventional SIM, which uses a spatial light modulator (SLM) for fringe projection, often has a limited field of view. To meet the demand for high-throughput microscopic imaging in biomedicine research, a large-field super-resolution (SR) fluorescence microscopic imaging method based on laser interferometry was proposed. The method that combines a two-dimensional (2D) grating for fringe pattern projection and an SLM for selecting fringe orientation can break the limitation of fringe number limited by the digital projection devices. A spatial-domain reconstruction algorithm was developed to improve the computational speed of super-resolution imaging. Finally, an experimental platform for SIM microscopy was established. A large-field view of 1380 μm × 1035 μm under a 20×/NA0.75 objective is experimentally demonstrated, and an enhancement of 1.8-fold resolution is realized. The spatial-domain reconstruction algorithm can significantly improve the computational speed by approximately 10 times faster compared to the traditional frequency-domain algorithm.

Analysis of Phase Mismatch for Mercurous Bromide-Based Non-Collinear AOTF Design in Spectral Imaging Applications.

The spectral and spatial characteristics of Acousto-Optic Tunable Filters (AOTFs), such as a tuning curve, spectral resolution, angular aperture, and diffraction efficiency, are determined by the device's acousto-optic crystal configuration and piezoelectric transducer. For high-throughput spectral imaging applications, it is essential to enlarge the spectral bandwidth and angular aperture during the design phase of AOTFs. Thus, phase mismatch due to incident angle or wavelength was studied analytically using phase diagrams in this paper. Additionally, a performance parameter analysis model was established based on the use of mercurous bromide crystals for large angular aperture AOTF device design, and the impact of crystal and transducer design parameters on the spectral bandwidth and angular aperture was evaluated. This also experimentally validates the diffraction capability of AOTFs made from mercurous bromide crystal, which possess a broad spectral transmission ability ranging from visible to long-wave infrared.