Application Of Fluorescence Microscopy Research Articles

Far-field superresolution of thermal sources by double homodyne or double array homodyne detection

We propose two schemes for estimating the separation of two thermal sources via double homodyne and double array homodyne detection considering the joint measurement of conjugate quadratures of the image plane field.By using the Cramér–Rao bound, we demonstrate that the two schemes can estimate the separation well below the Rayleigh limit and have an advantage over direct imaging when the average photon number per source exceeds five.For arbitrary source strengths, double homodyne detection is superior to homodyne detection when the separation is above 25/4σ/N s , σ is the beam width, N s is the average photon number per source.A larger separation can be estimated better via double array homodyne detection with the superiority of flexible operation compared with other schemes. High-speed and high-efficiency detection enables the measurement schemes with potential practical applications in fluorescence microscopy, astronomy and quantum imaging.

Dual-wavelength terahertz two-dimensional phase gratings based on all dielectric metasurfaces

Efficient and accurate phase gratings hold immense significance in the realization of large format heterodyne array receivers at terahertz frequencies. Metallic phase gratings have made substantial advancements in terms of operating wavelength and the number of diffraction beams. Like most other diffractive optical devices, metallic phase gratings are primarily optimized to operate at one specific wavelength. Metasurfaces compositing arrays of subwavelength nanostructures have been demonstrated with various optical functions, by freely modifying the polarization, phase, and amplitude of light. In this study, we present an approach to create a multi-wavelength phase grating compositing segments that incorporate multiple nanostructures. The resulting transmission phase grating not only exhibits uniform diffraction beams (2 × 2) but also achieves the same diffraction angles at both 1.31 and 2.7 THz. The measured total power efficiency of the diffraction beam pattern is 53.2% for 1.31 THz and 42.4% for 2.7 THz. These devices can be applied in terahertz astronomical observations and fluorescence microscopy applications, where multi-wavelength operation is necessary.

Investigating the Degradation of Mycenaean Glass Artifacts Using Scientific Methods

Mycenaean vitreous artifacts, such as beads and relief plaques, are highly susceptible to degradation, which can significantly alter their visual characteristics and pose challenges to their taxonomy. The visual manifestation of corrosion on vitreous artifacts, especially glass and faience, has often led to their misclassification, which, in turn, has a significant impact on their interpretation by researchers, often resulting in misleading notions. The present paper constitutes part of an overall study, implemented within the framework of the project, Myc-MVP: Mycenaean Vitreous Production, A Novel Interdisciplinary Approach Towards Resolving Critical Taxonomy Issues, which has employed a combination of established, state-of-the-art scientific methods to analyze and identify the specific compositional changes occurring at different spatial dimensions within surface layers, with the overarching aim of contributing to our understanding of the degradation mechanisms of vitreous artifacts and the relevant implications for the archaeological record. Importantly, these findings will yield useful data in devising strategies for the proper classification, management, and preservation of vitreous artifacts in the future. The present study focuses on investigating the relationship between the compositional changes in a subset of 12 (of the overall 126 objects entailed in the project) corroded vitreous artifacts from Mycenaean contexts in the Aegean and the way these are manifested visually, with the application of X-ray fluorescence and LED microscopy. We aim to decipher the nature of corroded objects with the aid of focused analysis. This study delves into degradation processes in glass artifacts, highlighting preservation variations and environmental influences like burial. Coloration, attributed to copper and cobalt oxide, shows some correlation with preservation quality. These chromophore agents potentially induce thermal stresses and corrosion. The complex interplay between chemical composition, environmental conditions, and preservation status underscores the need for comprehensive research. Analyzing the full artifact set using complementary techniques promises deeper insights for secure material classification and cultural heritage preservation.

5‐Aryl Substituted 2‐(2‐Methoxyphenyl)benzoxazoles with Large Stokes Shifts: Synthesis, Crystal Structures and Optical Properties

Abstract2‐(2‐methoxyphenyl)benzoxazole (MBO) displays similar photophysical properties to 2‐(2‐hydroxyphenyl)benzoxazole (HBO), but it lacks the large Stokes shifts necessary for fluorescence microscopy applications. In this paper, we report the 5‐substitution of MBO with aryl groups to produce 2‐(4‐methoxy‐[1,1’‐biphenyl]‐3‐yl)benzoxazoles (MBBOs) with large Stokes shifts. The synthesis of MBBOs 6–15 was accomplished in moderate to good yields by microwave‐assisted Sonogashira cross couplings. Crystal structures for compounds 6–9 and 15 are provided. MBBOs 6–10, 12, and 14 displayed considerably blue‐shifted absorption maxima and slight bathochromic shifts of their fluorescence maxima relative to the unsubstituted MBO.

Application of fluorescent microscopy and Fourier-transform infrared spectroscopy for analysis of microplastics in Can Gio seawater

Microplastics (MPs) pollution has become a global challenge due to their persistent properties and harm to nature and human health. Observation of MPs pollution, especially in marine environments, is a critical concern in environmental analysis. In this work, we validate the method for detecting the presence of MPs in seawater by using fluorescence microscopy with Nile Red dye combined with Attenuated Total Reflectance Fourier-transform infrared spectroscopy (ATC-FTIR). The specificity and accuracy of this method were evaluated by analyzing the standard spiked samples. Five standard MPs and chitin samples used in this study were polyamide (PA), polyethylene (PE), polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), and polyvinyl chloride (PVC) with a diameter from 5 to 300µm. The obtained recovery was 88.3-96.7%, with the related standard deviation (RSD%) in the range of 4.6-11.0%. This method was applied to determine microplastics in 27 seawater samples at three locations in Can Gio Sea, Ho Chi Minh City. The results showed the pollution level of each type of MP was significantly varied, with PE and PET being the highest, while there was almost no PMMA in seawater samples. ® 2023 Journal of Science and Technology - NTTU

Practical applications of total internal reflection fluorescence microscopy for nanocatalysis

A ‘pocket guide’ to applications of total internal reflection fluorescence in the field of chemistry.

Super-resolution fluorescence microscopy for investigating bacterial cell biology.

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.

Application of fluorescent microscopy and Fourier-transform infrared spectroscopy for analysis of microplastics in Can Gio seawater

Microplastics (MPs) pollution has become a global challenge due to their persistentproperties and harm to nature and human health. Observation of MPs pollution,especially in marine environments, is a critical concern in environmental analysis. Inthis work, we validate the method for detecting the presence of MPs in seawater byusing fluorescence microscopy with Nile Red dye combined with Attenuated TotalReflectance Fourier-transform infrared spectroscopy (ATC-FTIR). The specificity andaccuracy of this method were evaluated by analyzing the standard spiked samples.Five standard MPs and chitin samples used in this study were polyamide (PA),polyethylene (PE), polyethylene terephthalate (PET), poly(methyl methacrylate)(PMMA), and polyvinyl chloride (PVC) with a diameter from 5 to 300μm. Theobtained recovery was 88.3-96.7%, with the related standard deviation (RSD%) in therange of 4.6-11.0%. This method was applied to determine microplastics in 27seawater samples at three locations in Can Gio Sea, Ho Chi Minh City. The resultsshowed the pollution level of each type of MP was significantly varied, with PE andPET being the highest, while there was almost no PMMA in seawater samples.® 2023 Journal of Science and Technology - NTTU

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.

Expert- and Nonexpert-friendly Framework for Deep Learning Image Segmentation Demonstrates Successes Across Applications in vEM, CryoEM, MicroCT and Fluorescence Microscopy.

Expert- and Nonexpert-friendly Framework for Deep Learning Image Segmentation Demonstrates Successes Across Applications in vEM, CryoEM, MicroCT and Fluorescence Microscopy.

Application of fluorescence microscopy for histological discrimination of golden camellias leaves

Histological analysis, which aims to investigate the microscopic anatomy of biological tissues, has been a simple and powerful technique for plant taxonomy. Sectioning followed by staining methods is widely used in observing histological structures. However, the staining techniques often destroy tissue and provide low-quality images due to nonspecific reactions with the dyes making further analysis difficult. In this report, we propose an applicable non-staining histology protocol based on auto-fluorescence characteristics of plant tissues and its application in the anatomical discrimination of six similar-appearance species of golden camellias as a case study. We compared the images from the same tissue under a bright field with the staining step and under fluorescence directly without the staining step in the sample preparation. The images were taken from Eclipse Ni-U microscopy (Nikon, Japan) with a color DS-Ri2 camera (Nikon, Japan) and NIS-ELEMENTS Basic Research Imaging software. The non-staining method demonstrated significant advantages compared to the staining protocol. The fluorescent images showed the distinction between adjacent leaf tissues with their own naturally reflective colors. In addition, the anatomical parameters, including the xylem area, phloem area, bundle sheath area, and palisade/spongy width ratio, were easily measured in good-quality images. These parameters were used in discriminative analysis by the Principal Component Analysis (PCA). The PCA diagram demonstrated the separation of six species, thus suggesting that these anatomical parameters can be used for taxonomy. In conclusion, our study showed a helpful technique in histological analysis that significantly contributes to the taxonomy of golden camellias species and can be applied in other plant varieties.

Efficient Superoscillation Measurement for Incoherent Optical Imaging

I propose a superoscillation measurement method for subdiffraction incoherent optical sources, with potential applications in astronomy, remote sensing, fluorescence microscopy, and spectroscopy. The proposal, based on coherent optical processing, can capture all the light on the aperture in principle, perform better than direct imaging on statistical terms, and approach the fundamental quantum limit.

Semiconductive and Biocompatible Nanofibrils from the Self-Assembly of Amyloid π-Conjugated Peptides.

Owing to their capacity to self-assemble into organized nanostructures, amyloid polypeptides can serve as scaffolds for the design of biocompatible semiconductive materials. Herein, symmetric and asymmetric amyloid π-conjugated peptides were prepared through condensation of perylene diimide (PDI) with a natural amyloidogenic sequence derived from the islet amyloid polypeptide. These PDI-bioconjugates assembled into long and linear nanofilaments in aqueous solution, which were characterized by a cross-β-sheet quaternary organization. Current-voltage curves exhibited a clear signature of semiconductors, whereas the cellular assays revealed cytocompatibility and potential application in fluorescence microscopy. Although the incorporation of a single amyloid peptide appeared sufficient to drive the self-assembly into organized fibrils, the incorporation of two peptide sequences at the PDI's imide positions significantly enhanced the conductivity of nanofibril-based films. Overall, this study exposes a novel strategy based on amyloidogenic peptide to guide the self-assembly of π-conjugated systems into robust, biocompatible, and optoelectronic nanofilaments.

Two-photon absorption of FAPbBr3 perovskite nanocrystals

Perovskite nanocrystals (NCs) with high two-photon absorption (TPA) cross-section are of great interest due to their potential applications in three-dimensional optical data storage and multiphoton fluorescence microscopy. Among various perovskite materials, FAPbBr3 NCs show a better development prospect due to their excellent stability. However, there are few reports on their nonlinear optical properties. In this work, the nonlinear optical behavior of FAPbBr3 NCs is studied. The methods of multiphoton absorption photoluminescence saturation and open aperture Z-scan technique were applied to determine the TPA cross-section of FAPbBr3 NCs, which was around 2.76 × 10−45 cm4⋅s⋅photon−1 at 800 nm. In addition, temperature-dependent photoluminescence induced by TPA was investigated, and the small longitudinal optical phonon energy and electron–phonon coupling strength was obtained, which confirm the weak Pb–Br interaction. Meanwhile, it is found that the exciton binding energy in FAPbBr3 NCs was 69.668 meV, which may be ascribed to the strong hydrogen bond interaction. It is expected that our findings will promote the application of FAPbBr3 NCs in optoelectronic devices.

A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and trans-cyclooctene

Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy.

Fast and simple super-resolution with single images

We present a fast and simple algorithm for super-resolution with single images. It is based on penalized least squares regression and exploits the tensor structure of two-dimensional convolution. A ridge penalty and a difference penalty are combined; the former removes singularities, while the latter eliminates ringing. We exploit the conjugate gradient algorithm to avoid explicit matrix inversion. Large images are handled with ease: zooming a 100 by 100 pixel image to 800 by 800 pixels takes less than a second on an average PC. Several examples, from applications in wide-field fluorescence microscopy, illustrate performance.

A highly photostable and bright green fluorescent protein

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

Real-time image denoising of mixed Poisson–Gaussian noise in fluorescence microscopy images using ImageJ

Fluorescence microscopy imaging speed is fundamentally limited by the measurement signal-to-noise ratio (SNR). To improve image SNR for a given image acquisition rate, computational denoising techniques can be used to suppress noise. However, common techniques to estimate a denoised image from a single frame either are computationally expensive or rely on simple noise statistical models. These models assume Poisson or Gaussian noise statistics, which are not appropriate for many fluorescence microscopy applications that contain quantum shot noise and electronic Johnson–Nyquist noise, therefore a mixture of Poisson and Gaussian noise. In this paper, we show convolutional neural networks (CNNs) trained on mixed Poisson and Gaussian noise images to overcome the limitations of existing image denoising methods. The trained CNN is presented as an open-source ImageJ plugin that performs real-time image denoising (within tens of milliseconds) with superior performance (SNR improvement) compared to conventional fluorescence microscopy denoising methods. The method is validated on external datasets with out-of-distribution noise, contrast, structure, and imaging modalities from the training data and consistently achieves high-performance ( > --> 8 d B ) denoising in less time than other fluorescence microscopy denoising methods.

Application of super-resolution fluorescence microscopy in hematologic malignancies

Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells. The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type, treatment and prognosis of hematologic malignancies. Super-resolution fluorescence microscopy (SRM) is a special kind of optical microscopy technology, which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014. With the development of SRM, many related technologies have been applied to the diagnosis and treatment of clinical diseases. It was reported that a major type of SRM technique, single molecule localization microscopy (SMLM), is more sensitive than flow cytometry (FC) in detecting cell membrane antigens’ expression, thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools. Furthermore, SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases. In this paper, we mainly discuss the application of SRM in clone hematological malignancies.

Single-shot mid-infrared incoherent holography using Lucy-Richardson-Rosen algorithm

In recent years, there has been a significant transformation in the field of incoherent imaging with new possibilities of compressing three-dimensional (3D) information into a two-dimensional intensity distribution without two-beam interference (TBI). Most of the incoherent 3D imagers without TBI are based on scattering by a random phase mask exhibiting sharp autocorrelation and low cross-correlation along the depth. Consequently, during reconstruction, high lateral and axial resolutions are obtained. Imaging based on scattering requires an astronomical photon budget and is therefore precluded in many power-sensitive applications. In this study, a proof-of-concept 3D imaging method without TBI using deterministic fields has been demonstrated. A new reconstruction method called the Lucy-Richardson-Rosen algorithm has been developed for this imaging concept. We believe that the proposed approach will cause a paradigm-shift in the current state-of-the-art incoherent imaging, fluorescence microscopy, mid-infrared fingerprinting, astronomical imaging, and fast object recognition applications.