Wide-field Imaging Microscopy Research Articles

Isotropy frequency-domain extension imaging and its application in detection of nanostructures

A wide-field high resolution microscopy with sub-micron resolution and high contrast image was demonstrated by using the isotropy frequency-domain extension imaging (IFEI), which was successfully applied to submicron-scale structure detection of fused silica wafer for the first time. Employing the frequency-domain extension and the isotropic period matching, the IFEI extended the bandwidth of coherent transfer function of the conventional wide-field microscopy, thereby improving the resolution to break the diffraction limit. In this method, a quantitative measurement of 200 nm was achieved using fused silica wafer with prefabricated etched structure on the surface. By imaging 81 nm gold nanoparticles, the system resolution calibration was achieved. And corresponding system’s lateral resolution of approximately 179.5 nm was obtained, which was 1.72 times higher than the conventional wide-field microscopy image. Moreover, the reconstructed IFEI images of ultra-precision processed fused silica optics were demonstrated to present the finer structures of sub-micron digs and scratches formed in the optics manufacturing process.

Segmentation with Residual Attention U-Net and an Edge-Enhancement Approach Preserves Cell Shape Features.

The ability to extrapolate gene expression dynamics in living single cells requires robust cell segmentation, and one of the challenges is the amorphous or irregularly shaped cell boundaries. To address this issue, we modified the U-Net architecture to segment cells in fluorescence widefield microscopy images and quantitatively evaluated its performance. We also proposed a novel loss function approach that emphasizes the segmentation accuracy on cell boundaries and encourages shape feature preservation. With a 97% sensitivity, 93% specificity, 91% Jaccard similarity, and 95% Dice coefficient, our proposed method called Residual Attention U-Net with edge-enhancement surpassed the state-of-the-art U-Net in segmentation performance as evaluated by the traditional metrics. More remarkably, the same proposed candidate also performed the best in terms of the preservation of valuable shape features, namely area, eccentricity, major axis length, solidity and orientation. These improvements on shape feature preservation can serve as useful assets for downstream cell tracking and quantification of changes in cell statistics or features over time.

CometAnalyser: A user-friendly, open-source deep-learning microscopy tool for quantitative comet assay analysis

Comet assay provides an easy solution to estimate DNA damage in single cells through microscopy assessment. It is widely used in the analysis of genotoxic damages induced by radiotherapy or chemotherapeutic agents. DNA damage is quantified at the single-cell level by computing the displacement between the genetic material within the nucleus, typically called “comet head”, and the genetic material in the surrounding part of the cell, considered as the “comet tail”. Today, the number of works based on Comet Assay analyses is really impressive. In this work, besides revising the solutions available to obtain reproducible and reliable quantitative data, we developed an easy-to-use tool named CometAnalyser. It is designed for the analysis of both fluorescent and silver-stained wide-field microscopy images and allows to automatically segment and classify the comets, besides extracting Tail Moment and several other intensity/morphological features for performing statistical analysis. CometAnalyser is an open-source deep-learning tool. It works with Windows, Macintosh, and UNIX-based systems. Source code, standalone versions, user manual, sample images, video tutorial and further documentation are freely available at: https://sourceforge.net/p/cometanalyser.

Deep learning 2D and 3D optical sectioning microscopy using cross-modality Pix2Pix cGAN image translation.

Structured illumination microscopy (SIM) reconstructs optically-sectioned images of a sample from multiple spatially-patterned wide-field images, but the traditional single non-patterned wide-field images are more inexpensively obtained since they do not require generation of specialized illumination patterns. In this work, we translated wide-field fluorescence microscopy images to optically-sectioned SIM images by a Pix2Pix conditional generative adversarial network (cGAN). Our model shows the capability of both 2D cross-modality image translation from wide-field images to optical sections, and further demonstrates potential to recover 3D optically-sectioned volumes from wide-field image stacks. The utility of the model was tested on a variety of samples including fluorescent beads and fresh human tissue samples.

Perspective on fast-evolving photoacoustic tomography.

:Significance: Acoustically detecting the rich optical absorption contrast in biological tissues, photoacoustic tomography (PAT) seamlessly bridges the functional and molecular sensitivity of optical excitation with the deep penetration and high scalability of ultrasound detection. As a result of continuous technological innovations and commercial development, PAT has been playing an increasingly important role in life sciences and patient care, including functional brain imaging, smart drug delivery, early cancer diagnosis, and interventional therapy guidance.Aim: Built on our 2016 tutorial article that focused on the principles and implementations of PAT, this perspective aims to provide an update on the exciting technical advances in PAT.Approach: This perspective focuses on the recent PAT innovations in volumetric deep-tissue imaging, high-speed wide-field microscopic imaging, high-sensitivity optical ultrasound detection, and machine-learning enhanced image reconstruction and data processing. Representative applications are introduced to demonstrate these enabling technical breakthroughs in biomedical research.Conclusions: We conclude the perspective by discussing the future development of PAT technologies.

Metalens-integrated compact imaging devices for wide-field microscopy

Metasurfaces have demonstrated unprecedented capabilities in manipulating light with ultrathin and flat architectures. Although great progress has been made in the metasurface designs and function demonstrations, most metalenses still only work as a substitution of conventional lenses in optical settings, whose integration advantage is rarely manifested. We propose a highly integrated imaging device with silicon metalenses directly mounted on a complementary metal oxide semiconductor image sensor, whose working distance is in hundreds of micrometers. The imaging performances including resolution, signal-to-noise ratio, and field of view (FOV) are investigated. Moreover, we develop a metalens array with polarization-multiplexed dual-phase design for a wide-field microscopic imaging. This approach remarkably expands the FOV without reducing the resolution, which promises a non-limited space-bandwidth product imaging for wide-field microscopy. As a result, we demonstrate a centimeter-scale prototype for microscopic imaging, showing uniqueness of meta-design for compact integration.

Fluorescence polarization filtering for accurate single molecule localization

Single-Molecule Localization Microscopy (SMLM) has become one of the most important methods of super-resolution fluorescence microscopy. It is based on the precise localization of single molecules in wide-field microscopy images. It is well known that the localization accuracy can show a significant bias if the imaged molecules have a fixed orientation and are located either close to an interface or not exactly within the focal plane of the microscope. In this Letter, we propose a simple solution to this problem, which is based on polarization-resolved imaging. This method can be easily implemented into any existing SMLM setup, and we demonstrate its performance by imaging single dye molecules embedded into a polymer film, which fixes their orientation in space.

Interferometric spatial frequency modulation imaging.

Interferometric spatial frequency modulation for imaging (I-SPIFI) is demonstrated for the first time, to our knowledge. Significantly, this imaging modality can be seamlessly combined with nonlinear SPIFI imaging and operates through single-element detection, making it compatible for use in scattering specimens. Imaging dynamic processes with submicrometer axial resolution through long working distance optics is shown, and high contrast images compared to traditional wide-field microscopy images. Finally, enhanced lateral resolution is achieved in I-SPIFI. To our knowledge, this is the first single platform that enables multimodal linear and nonlinear imaging, with enhanced resolution, all of which can be performed simultaneously.

Visualization of Neuronal Structures in Wide-Field Microscopy Brain Images.

Wide-field microscopes are commonly used in neurobiology for experimental studies of brain samples. Available visualization tools are limited to electron, two-photon, and confocal microscopy datasets, and current volume rendering techniques do not yield effective results when used with wide-field data. We present a workflow for the visualization of neuronal structures in wide-field microscopy images of brain samples. We introduce a novel gradient-based distance transform that overcomes the out-of-focus blur caused by the inherent design of wide-field microscopes. This is followed by the extraction of the 3D structure of neurites using a multi-scale curvilinear filter and cell-bodies using a Hessian-based enhancement filter. The response from these filters is then applied as an opacity map to the raw data. Based on the visualization challenges faced by domain experts, our workflow provides multiple rendering modes to enable qualitative analysis of neuronal structures, which includes separation of cell-bodies from neurites and an intensity-based classification of the structures. Additionally, we evaluate our visualization results against both a standard image processing deconvolution technique and a confocal microscopy image of the same specimen. We show that our method is significantly faster and requires less computational resources, while producing high quality visualizations. We deploy our workflow in an immersive gigapixel facility as a paradigm for the processing and visualization of large, high-resolution, wide-field microscopy brain datasets.

Comparison of different microscopy approaches to quantification of inhibitory effect on thrombus formation under flow conditions by the example of adenosine receptor agonist HE-NECA

IntroductionThrombus formation in vitro in flow conditions and its visualization and quantification with the use of microscopy are widely utilized to evaluate activity of compounds with a potential antithrombotic activity. Visualization and quantification of thrombi can be performed with the use of wide-field or confocal microscopy. Acquiring reliable numerical data from wide-field microscopy images of objects which have a complex three-dimensional structure is strongly influenced by the methods used for image analysis. This can be a possible source of inaccuracy in assessment of antithrombotic activity of a tested substance. We aimed to verify how different approaches to the quantification of wide-field images can affect the evaluation of an antiplatelet effect of a tested substance. MethodsWe compared three algorithms of image analysis to evaluate an effect of 2-hexynyl-5′-ethylcarboxamidoadenosine (HE-NECA), a compound of a moderate antiplatelet activity on thrombus formation, and of abciximab - a potent antiplatelet compound. Also, we studied how the results obtained in a wide-field imaging correspond to those obtained by means of confocal imaging. ResultsThree algorithms for analysis of wide-field images showed antiplatelet effect of HE-NECA or abciximab. Absolute values of thrombus area and outcomes of the evaluation of inhibition efficacy of HE-NECA were significantly different between the algorithms. Analysis of volumes and heights of thrombi obtained by confocal imaging confirmed inhibitory effect of HE-NECA, but the evaluated levels of inhibition were significantly different from that obtained by wide-field imaging. DiscussionWe conclude that wide-field imaging provides reliable qualitative data on an inhibitory effect on thrombus formation, despite differences which can emerge from various approaches to image analysis. However, quantitative evaluation and comparison of the efficacy of inhibitors on the basis of total area occupied by thrombi obtained by wide-field microscopy should be made with caution. To obtain a reliable quantitative assessment of the effect of a tested compound on thrombus structure the use of confocal microscopy is inevitable.

Supported Tubulated Bilayers: A Novel System for Evaluating Protein-Mediated Membrane Remodeling

Fusion and fission of cellular membranes involve dramatic, precisely regulated changes in membrane curvature mediated by a number of proteins whose mechanisms are not well understood. Despite several recent advances, current methods for investigating curvature sensing and generation in real time using well-controlled systems remain limited. We have developed a novel system based on supported lipid bilayers (SLBs) in which high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the bilayer, which results in the spontaneous formation of lipid tubules after sequentially washing with water and physiological ionic strength buffer solutions. We find that the process of tubule formation is the result of an ion-dependent spreading of the SLB; addition of a physiological ionic strength buffer solution free of divalent ions leads to expansion of the bilayer and formation of tubules, likely due to increased membrane tension. Conversely, the addition of divalent ions results in contraction of the membrane and a proportional loss of tubules. These ionic conditions can be tuned for each experiment, allowing investigators to control the extent of tubulation. We show the utility of these supported tubulated bilayers, which we term “STuBs,” with an investigation of Sar1, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1 to tubulated bilayers results in both further tubulation and tubule fission, of which fission is shown to be more dominant based on quantification of wide-field fluorescence microscopy images. Individual tubule formation events are observed with polarized total internal reflection fluorescence microscopy (pTIRFM), an imaging method that allows for semi-quantitative measurements of membrane deformations. Overall, STuBs is a simple experimental system, useful for monitoring solute- and protein-mediated effects on membrane topology in aqueous media and in real-time, using widely available instrumentation.

Organic nanoparticles with ultrahigh quantum yield and aggregation-induced emission characteristics for cellular imaging and real-time two-photon lung vasculature imaging.

Fluorescent organic nanoparticles based on small molecules have emerged as an attractive class of fluorescent agents for bioimaging in recent years. Herein, we report orange light-emitting BTPEBD based organic nanoparticles (BTPEBD NPs) with a large Stokes shift (>135 nm), ultrahigh quantum yield (>90% in water) and aggregation-induced emission characteristics. Single nanoparticle analysis studied by wide field microscopy imaging further proves that the BTPEBD NPs exhibit high brightness and good photostability. Both in vitro and in vivo experiments reveal that the BTPEBD NPs are promising fluorescent agents for cellular imaging and real-time two-photon lung vasculature imaging.

Accurate Detection of Dysmorphic Nuclei Using Dynamic Programming and Supervised Classification.

A vast array of pathologies is typified by the presence of nuclei with an abnormal morphology. Dysmorphic nuclear phenotypes feature dramatic size changes or foldings, but also entail much subtler deviations such as nuclear protrusions called blebs. Due to their unpredictable size, shape and intensity, dysmorphic nuclei are often not accurately detected in standard image analysis routines. To enable accurate detection of dysmorphic nuclei in confocal and widefield fluorescence microscopy images, we have developed an automated segmentation algorithm, called Blebbed Nuclei Detector (BleND), which relies on two-pass thresholding for initial nuclear contour detection, and an optimal path finding algorithm, based on dynamic programming, for refining these contours. Using a robust error metric, we show that our method matches manual segmentation in terms of precision and outperforms state-of-the-art nuclear segmentation methods. Its high performance allowed for building and integrating a robust classifier that recognizes dysmorphic nuclei with an accuracy above 95%. The combined segmentation-classification routine is bound to facilitate nucleus-based diagnostics and enable real-time recognition of dysmorphic nuclei in intelligent microscopy workflows.

自适应宽场高分辨率显微成像技术的研究进展

自适应宽场高分辨率显微成像技术的研究进展

Abstract 1998: Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH)

Abstract Normal expression of the minor oncogene EIF5A2 in the hypusine pathway is restricted to brain and testis. However aberrant expression has been noted in multiple cancers, including ovarian gastric, colorectal and pancreatic, and artificial overexpression causes tumor growth in naked mice. Unlike cDNAs from the homologous gene EIF5A1, which are readily translated into protein upon transient transfection, EIF5A2-mRNAs only associate with monosomes without production of eIF5A-2 protein. Here we present data from multiple EIF5A2-positive cell lines on the steady state levels of the four different EIF5A2-mRNAs that arise through differential polyadenylation. While considering qPCR and RNA-Seq, we chose to apply Stellaris single molecule RNA fluorescence in situ hybridization with four unique probe sets, each reporting on the dependent A, B, C, or D segments. The probe sets were designed with multiple tiled singly labeled 18-22-mer oligonucleotides with balanced GC content, and hence similar hybridization characteristics, which in turn enabled the simultaneous quadruplex smRNA FISH. Stellaris RNA FISH leaves the cell intact, and also does not require RNA isolation or nucleic acid amplification that may introduce an otherwise hidden experimental bias. The single molecule spots revealed by widefield microscopy and subsequent image analysis co-localized with a high degree of confidence. Taking advantage of the added spatial dimension that RNA FISH affords, we also determined the localization of each mRNA variant and recorded the cell-to-cell variability and the actual range of mRNA copy numbers per cell. Our data is consistent with previous gene expression analysis data, but also provide actual counts of each mRNA variant. The results presented here have direct applications for the expression of the minor oncogene EIF5A2, and the regulated protein synthesis of its mRNAs, as well as of other mRNAs in polyamine and hypusine pathways. Further, the techniques and probes developed here can be directly transferred to the analysis of other mRNA variants that may arise through alternative transcription start site use, alternative splicing or alternative 3’ end processing in normal and aberrant cells. Citation Format: Hans E. Johansson, Raymund H. Yin, Swati Mandal, Myung-Hee Park. Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1998.

Portable lensless wide-field microscopy imaging platform based on digital inline holography and multi-frame pixel super-resolution.

In this paper, an irregular displacement-based lensless wide-field microscopy imaging platform is presented by combining digital in-line holography and computational pixel super-resolution using multi-frame processing. The samples are illuminated by a nearly coherent illumination system, where the hologram shadows are projected into a complementary metal-oxide semiconductor-based imaging sensor. To increase the resolution, a multi-frame pixel resolution approach is employed to produce a single holographic image from multiple frame observations of the scene, with small planar displacements. Displacements are resolved by a hybrid approach: (i) alignment of the LR images by a fast feature-based registration method, and (ii) fine adjustment of the sub-pixel information using a continuous optimization approach designed to find the global optimum solution. Numerical method for phase-retrieval is applied to decode the signal and reconstruct the morphological details of the analyzed sample. The presented approach was evaluated with various biological samples including sperm and platelets, whose dimensions are in the order of a few microns. The obtained results demonstrate a spatial resolution of 1.55 µm on a field-of-view of ≈30 mm2.

Semi-quantitative monitoring of confluence of adherent mesenchymal stromal cells on calcium-phosphate granules by using widefield microscopy images.

The analysis of cell confluence and proliferation is essential to design biomaterials and scaffolds to use as bone substitutes in clinical applications. Accordingly, several approaches have been proposed in the literature to estimate the area of the scaffold covered by cells. Nevertheless, most of the approaches rely on sophisticated equipment not employed for routine analyses, while the rest of them usually do not provide significant statistics about the cell distribution. This research aims at studying confluence and proliferation of mesenchymal stromal cells (MSC) adherent on OSPROLIFE(®), a commercial biomaterial in the form of granules. In particular, we propose a Computer Vision approach that can routinely be employed to monitor the surface of the single granules covered by cells because only a standard widefield fluorescent microscope is required. In order to acquire significant statistics data, we analyse wide-area images built by using MicroMos v2.0, an updated version of a previously published software specific for stitching brightfield and phase-contrast images manually acquired via a widefield microscope. In particular, MicroMos v2.0 permits to build accurate "mosaics" of fluorescent images, after correcting vignetting and photo-bleaching effects, providing a consistent representation of a sample region containing numerous granules. Then, our method allows to make automatically a statistically significant estimate of the percentage of the area of the single granules covered by cells. Finally, by analysing hundreds of granules at different time intervals we also obtained reliable data regarding cell proliferation, confirming that not only MSC adhere onto the OSPROLIFE(®) granules, but even proliferate over time.

Oxygen-Sensitive Phosphorescent Nanomaterials Produced from High-Density Polyethylene Films by Local Solvent-Crazing

Discrete solid-state phosphorescent oxygen sensors produced by local solvent-crazing of high density polyethylene films are described. The simple spotting of dye solution followed by tensile drawing of the polymer substrate provides uniform nanostructures with good spatial control, effective encapsulation of dye molecules, and quenchability by O2. The dye-polymer composite sensors prepared using toluene as a solvent and stabilized by annealing at high temperature, show moderate optical signals, near-optimal sensitivity to O2 (RSD at 21 KPa 1.9%), and reproducible phosphorescence lifetime readings. Calibration experiments performed over 0-25 kPa O2 and 10-30 °C temperatures ranges reveal linear Stern-Volmer plots and temperature dependences and minimal effect of humidity on sensor calibration. The high degree of lateral and in-depth homogeneity of these O2-sensitive materials was confirmed by high-resolution atomic force and wide-field optical microscopy, including 2D and 3D phosphorescence lifetime imaging.

Visualization of peroxynitrite-induced changes of labile Zn2+ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes.

Zn(2+) plays essential roles in biology, and the homeostasis of Zn(2+) is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn(2+), and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn(2+), ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N3O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable Kd values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn(2+) binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn(2+) thus providing evidence for a direct connection between ER stress and ER Zn(2+) homeostasis.

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone