Dark-field Microscopy Images Research Articles

Illumination characteristics of vortex beams in dark-field microscopic systems.

A Laguerre-Gaussian (LG) vortex beam is employed as an illumination source for a dark-field microscopy imaging system. To discover the influences of beam characteristics on the imaging quality, an analysis model has been established to show the light-field change rule on both object and image planes. The analytic expressions of the light field on the two planes are deduced. When a rectangular defect is simulated, the light distributions on the object and image planes with different parameters are calculated. The results show that the size of the beam spot on the object plane can be changed by adjusting the topological charge of the vortex beam to obtain the best imaging effect for defects of different scales.

Orientation-independent reaction activity monitoring with single particle and data analytics

Single-particle analysis is the most powerful method to obtain accurate local information for understanding and monitoring chemical reactions. However, investigations about obtaining comprehensive information at the single-particle level to overcome individual errors and sampling randomness have not been reported to date. Plasmonic nanorods, which have excellent anisotropic optical and chemical properties, make us in situ acquisition of conformation and dynamics of the biological information. On the basis of their anisotropic optical properties of the plasmonic nanorods such as Au nanorods (AuNRs) and data analytics, herein we developed a high-throughput resonance scattering imaging method of AuNRs under dark-field microscopy (DFM) to monitor orientation-independent reaction activity of AuNRs. Data analytics are introduced to determine a large number of AuNRs orientation obtained from a series of polarized DFM images, allowing us to real-time monitor reaction activity of AuNRs at all orientations, and also makes it possible to study the global and local reaction processes of AuNRs at single-particle level. Our method is expected to provide a new strategy for analytical study and single-particle sensing in chemistry.

Assessment of Glial Fibrillary Acidic Protein Binding to the Surface of Leukocytes with Dark‐Field Imaging and Computational Analysis

AbstractThe glial fibrillary acidic protein (GFAP) is widely established as a traumatic brain injury (TBI) biomarker and can be used in early diagnosis. As essential primary human immune cells, leukocytes are recruited to injured cerebral sites during TBI response, where they can interact with and potentially bind to TBI biomarkers. To date, no studies have demonstrated ultra‐low GFAP binding enumeration on leukocytes. Herein, a dark‐field imaging technique coupled with computational analysis is introduced to quantify GFAP bound to peripheral blood mononuclear cells (PBMCs). Dark‐field microscopy (DFM) with a custom‐written image acquisition software is developed for rapid 3D PBMC imaging by utilizing specific antiGFAP monoclonal antibody functionalized gold nanoparticles (antiGFAP‐AuNPs) as contrast‐generating probes. Subsequently, the developed algorithm is utilized in processing thousands of acquired images for rapid visualization and enumeration of bound antiGFAP‐AuNP on each leukocyte. The proposed method is demonstrates the specific binding of GFAP to the surface of PBMCs on a healthy donor blood. Thereafter, subpopulations of PBMCs with antiGFAP‐AuNP binding are identified with the assistance of fluorescence imaging and DFM imaging, paving a new way to understanding the relationship between TBI and leukocyte classes. Hence, this study offers a rapid and ultra‐sensitive strategy for biomarker assessment following TBI.

Automated Plasmonic Resonance Scattering Imaging Analysis via Deep Learning.

Plasmonic nanoparticles, which have excellent local surface plasmon resonance (LSPR) optical and chemical properties, have been widely used in biology, chemistry, and photonics. The single-particle light scattering dark-field microscopy (DFM) imaging technique based on a color-coded analytical method is a promising approach for high-throughput plasmonic nanoparticle scatterometry. Due to the interference of high noise levels, accurately extracting real scattering light of plasmonic nanoparticles in living cells is still a challenging task, which hinders its application for intracellular analysis. Herein, we propose an automatic and high-throughput LSPR scatterometry technique using a U-Net convolutional deep learning neural network. We use the deep neural networks to recognize the scattering light of nanoparticles from background interference signals in living cells, which have a dynamic and complicated environment, and construct a DFM image semantic analytical model based on the U-Net convolutional neural network. Compared with traditional methods, this method can achieve higher accuracy, stronger generalization ability, and robustness. As a proof of concept, the change of intracellular cytochrome c in MCF-7 cells under UV light-induced apoptosis was monitored through the fast and high-throughput analysis of the plasmonic nanoparticle scattering light, providing a new strategy for scatterometry study and imaging analysis in chemistry.

Active colorimetric lipid-coated polyaniline nanoparticles for redox state sensing in cancer cells.

Herein, lipid-coated polyaniline (LiPAni) nanoparticles were fabricated to monitor the redox state of cancer cells. To confirm the characteristics of LiPAni, we firstly analyzed the size and chemical structures of the LiPAni nanoparticles. The absorbance properties of the LiPAni nanoparticles were observed to vary with the pH conditions. Furthermore, cell viability tests conducted with breast cancer cell lines showed that the cell viability of the cells with LiPAni nanoparticles was dramatically increased compared to those with the Tween80-coated polyaniline nanoparticles (TPAni) as a control. Subsequently, the colors of the LiPAni nanoparticles were observed and analyzed using spectroscopic methods. Finally, in order to investigate the more accurate sensing of the redox state using the color changes of the LiPAni nanoparticles with cancer cell lines, dark field microscopic images and scattering spectra were recorded at the single nanoparticle scale. For the TPAni nanoparticles, there was only a change in brightness and no change in color, but for the LiPAni nanoparticles, there was a change of color from yellow to pink in the dark field images.

Tracking endocytosis and intracellular distribution of spherical nucleic acids with correlative single-cell imaging.

A comprehensive understanding of interactions between nanoparticles (NPs) and biological components is critical to the clinical application of NPs and nanomedicine. Here we provide a step-by-step correlative imaging approach to investigate plasmonic NPs of different aggregation states at the single-cell level. Traceable spherical nucleic acids (SNAs) are fabricated by decorating 50-nm spherical gold NPs with fluorophore-labeled DNA, serving as dually emissive (fluorescent and plasmonic) NPs. The in situ correlative imaging with dark-field microscopy (DFM) and fluorescence microscopy (FM) reveals intracellular distribution of SNAs, whereas DFM combined with scanning electron microscopy (SEM) allows semi-quantification of SNA clustering states in solution. The imaging data are analyzed by ImageJ and a colorimetry-based algorithm written in Python. The clustering states of SNAs in a single cell can be efficiently distinguished within 20 s. This method can be readily installed to monitor real-time endocytosis and cellular distribution of plasmonic NPs of different aggregation states and to quantitatively image targets of interest (e.g., specific DNA, messenger RNA, peptides or proteins) in living cells. The entire procedure can be completed in 3-5 d and requires standard DFM, FM and SEM imaging and data analysis skills and equipment.

Biodistribution, cardiac and neurobehavioral assessments, and neurotransmitter quantification in juvenile rats following oral administration of aluminum oxide nanoparticles.

Little is known about the uptake, biodistribution, and biological responses of nanoparticles (NPs) and their toxicity in developing animals. Here, male and female juvenile Sprague-Dawley rats received four consecutive daily doses of 10mg/kg Al2 O3 NP (diameter: 24nm [transmission electron microscope], hydrodynamic diameter: 148nm) or vehicle control (water) by gavage between postnatal days (PNDs) 17-20. Basic neurobehavioral and cardiac assessments were performed on PND 20. Animals were sacrificed on PND 21, and selected tissues were collected, weighed, and processed for histopathology or neurotransmitter analysis. The biodistribution of Al2 O3 NP in tissue sections of the intestine, liver, spleen, kidney, and lymph nodes were evaluated using enhanced dark-field microscopy (EDM) and hyperspectral imaging (HSI). Liver-to-body weight ratio was significantly increased for male pups administered Al2 O3 NP compared with control. HSI suggested that Al2 O3 NP was more abundant in the duodenum and ileum tissue of the female pups compared with the male pups, whereas the abundance of NP was similar for males and females in the other tissues. The abundance of NP was higher in the liver compared with spleen, lymph nodes, and kidney. Homovanillic acid and norepinephrine concentrations in brain were significantly decreased following Al2 O3 NP administration in female and male pups, whereas 5-hydroxyindoleacetic acid was significantly increased in male pups. EDM/HSI indicates intestinal uptake of Al2 O3 NP following oral administration. Al2 O3 NP altered neurotransmitter/metabolite concentrations in juvenile rats' brain tissues. Together, these data suggest that orally administered Al2 O3 NP interferes with the brain biochemistry in both female and male pups.

Biomolecule-tailored assembly and morphology of gold nanoparticles for LSPR applications

Gold nanoparticles (GNPs) have attracted the particular attention of nanoscience and nanoengineering due to their unique localized surface plasmonic resonance (LSPR) derived optical, electronic and magnetic properties. These LSPR-based properties are highly dependent on the nanostructures of GNPs which could be precisely tuned via strategies of assembly and morphological regulation. Biomolecules (DNA, amino acids and peptides) serving as linking and capping agents are capable to program the assembling and tailor the morphological evolution of GNPs to achieve varying nanostructures and plasmonic properties as well as biofunctionalities due to their specific recognition capabilities and unique interface affinity with GNPs. Versatile applications based on tunable LSPR properties of plasmonic nanoprobes composed of GNPs and biomolecules, therefore, have been successfully demonstrated in various fields, including metal-enhanced fluorescence, dark-field microscopy imaging, colorimetric assay, plasmonic circular dichroism-based assay, surface-enhanced Raman scattering-based bioprobes and LSPR shift-based biosensing, etc. This review introduces the recent advances in the design, structural tuning, property characterization and biosensing and bioimaging applications of these plasmonic nanoprobes, with detailed examples in previous applications and prospective discussions for their developments in future.

Automatic evaluation system for bulk defects in optics

In the high frequency laser system, the internal defects of optics will affect their damage threshold and downstream modulation, and even lead to subsequent surface damage. In order to efficiently and reliably evaluate the internal defects in optics, an automatic evaluation method of scattering defects in optics is proposed, and the corresponding three-dimensional (3D) precision measurement system is established. The detection principle, image acquisition, image processing and bulk defects (BDs) information extraction methods of the system are studied. Based on a 3D dark-field microscopic imaging (DFMI) technology, samples are scanned with multi-dimensional motion devices. Meanwhile, the scattered beam from the BD was collected by a detector, and finally combined with the digital image processing technology, the acquired images are processed in real time. Through real-time focusing and improved 3D reconstruction algorithm, the images with full aperture BD features are obtained, and the position and size information of defects are extracted. The system has detected the BD inside the fused silica samples. The results show that our system can distinguish the 612.7 lp/mm. Furthermore, the 3D distributions of the BD were also reconstructed.

High-Resolution Vertical Polarization Excited Dark-Field Microscopic Imaging of Anisotropic Gold Nanorods for the Sensitive Detection and Spatial Imaging of Intracellular microRNA-21.

As an important biomarker for early diagnosis of cancers, sensitive detection and high-resolution imaging of microRNA-21 in cancer cells have become important and challengeable. In this work, highly sensitive detection and spatial imaging of intracellular microRNA-21 were realized by the reduced signal background through vertical polarization excitation with a polarizer. The lateral local surface plasmon resonance property of gold nanorods (AuNRs) displayed a pronounced green color with low scattering intensity, which was adjusted to red color with strong scattering intensity when the core-satellite gold nanoparticle (AuNP) assembly was constructed on the side of AuNRs through a catalyzed hairpin assembly (CHA) circuit in the presence of microRNA-21. This unique approach allows for effectively reducing the strong background signal to improve the sensitivity of detection. Additionally, the proposed strategy can not only realize the sensitive detection of microRNA-21 with the limit of detection as low as 2 pM (3σ) but also achieve the high spatial imaging of cancer cells, which provided a specific strategy for the construction and imaging of intracellular imaging probes. It is believed that the simple and sensitive approach on the basis of lateral local surface plasmon resonance property of anisotropic AuNRs with excellent sensitivity combined with high spatial imaging holds promising potentials to visualize intracellular microRNAs with low abundance.

ZnO micron rods as single dielectric resonator for optical sensing

Light scattering properties of dielectric nanostructures (DNSs) have been recently applied for biosensing. It is worth noting that, however, the application of dielectric materials as light scattering probes in the field of analysis and detection has rarely been reported, especially at the single particle level. Herein, for the first time, we use ZnO micron rods (ZnOMRs) as dark field microscope (DFM) imaging probes for single particle level optical sensing. It was found that a drastic reduction of scattering intensity of the ZnOMRs with increase of the surrounding medium’s refractive index (RI), especially when illuminated by transverse electric (TE) polarized light, showing polarization-dependent RI sensitivity properties. In addition, the scattering color of the ZnOMRs gradually blue-shifted with the increase of solvents’ RI, this can avoid the problem of low sensitivity of the probe with a red scattering color in the previous reports. The characteristic scattering properties and polarization-dependent RI sensitivity properties of ZnOMRs provide more possibilities for the development of new single-particle optical analytical methods.

Gold nanoparticles enumeration with dark-field optical microscope for the sensitive glycoprotein sandwich assay

Protein glycosylation is an important post-translational modification and glycoproteins are associated with many crucial metabolic progresses of life. In order to detect glycoproteins sensitively, we propose a gold nanoparticles (GNPs) enumeration method based on boronate affinity sandwich system, which is constructed between the boronic acid polymer functionalized magnetic nanoparticles (Fe3O4@MPS@VPBA NPs) and 4-mercaptophenylboronic acid modified GNPs (GNPs-MPBA) by the targeted glycoproteins as the linker. Therefore, the sandwich complex is formed, resulting in the decrease of GNPs-MPBA counts in the solution. Based on the dark-field microscope (DFM) imaging technique, the sensitive GNPs enumeration assay is developed for glycoproteins quantitation. Immunoglobulin (IgG), as one of the important glycoproteins, is introduced to evaluate the proposed method. A low detection limit of 1.22 ng mL−1 for IgG analysis is obtained. The result indicates that the proposed GNPs enumeration method offers a simple, effective, label-free and highly sensitive strategy without signal amplification. It also possesses great potential for various target molecules determination at the single-particle level in the future.

Growth and fluorination of CaSi2 thin film

We fabricated bilayer silicene (BLSi) from an epitaxial CaSi2 thin film grown on a Si (111) substrate followed by treatment with BF−4-based ionic liquid. First, a 6R-CaSi2 thin film was grown from simultaneously supplied Ca and Si sources. After reacting the CaSi2 thin film with BF−4-based ionic liquid, the BLSi was formed. The reaction proceeded laterally along the CaSi2 layer. The rearrangement of the Si and Ca atomic layers in the CaSi2 thin film was investigated by high-angle annular dark-field scanning transmission electron microscope images.

Automated Nanoplasmonic Analysis of Spherical Nucleic Acids Clusters in Single Cells.

Spherical nucleic acids (SNAs) have been extensively used in the field of biosensing, drug delivery, and theranostics. Precise engineering of SNAs and their clinical application require better understanding of their cellular internalization process. We demonstrate a colorimetry-based algorithm that can analyze the aggregation states of SNAs clusters on the basis of the changes of plasmonic colors of SNAs. The dark-field microscopy (DFM) images of cytoplasmic region of single cells are imported as raw data. All the image spots are analyzed in the interference reduction process, and the clustering states of target image spots are assigned on the basis of the distribution of coordinates of all the pixels in the CIE map. This method provides faster analysis on clustering states of extracellular and intracellular SNAs with good accuracy. Moreover, the clustering states of SNAs in 20 single cells (generally >1000) can be efficiently distinguished within 200 s. Therefore, our method provides an automatic, quantitative, objective, and repeatable way to analyze SNAs aggregations, and shows good application potential in robust and quantitative nanoplasmonic analysis in single cells.

Magnetic bead-gold nanoparticle hybrids probe based on optically countable gold nanoparticles with dark-field microscope for T4 polynucleotide kinase activity assay

T4 polynucleotide kinase (T4 PNK) plays an essential role in DNA phosphorylation during the DNA repair process. Therefore, the sensitive, selective and convenient detection of T4 PNK activity is of great significance. In this work, we proposed a sensitive non-amplification strategy for the sensing of T4 PNK activity via dark field microscope (DFM) based on magnetic bead (MB)-gold nanoparticle (AuNP) hybrids probe, MB-dsDNA-AuNP (MDA). In the presence of T4 PNK, the 5'-OH termini of DNA are phosphorylated and cleaved into oligonucleotides by lambda exonuclease (λexo), resulting in the destruction of the MDA probe and the separation of AuNP from the MB. Through automatic counting of AuNPs from DFM images, T4 PNK activity can be quantitatively measured. This strategy revealed a limit of detection (LOD) as low as 0.0058 U/mL and exhibited a dynamic range from 0.01 to 1 U/mL. The strategy presents an excellent ability to discriminate T4 PNK from the other proteins and enzymes. Notably, this strategy was applied to screen the T4 PNK inhibitors and test the activity in cell lysates, showing great potential for the discovery of new anticancer drugs and other related research field.

Revealing Lectin-Sugar Interactions with a Single Au@Ag Nanocube.

An individual nanoparticle-based plasmonic nanotechnology was used for real-time monitoring of lectin-sugar interactions, which could be designed as novel plasmonic nanobiosensors for the detection of trace concanavalin A (ConA) with high sensitivity and selectivity. The localized surface plasmon resonance (LSPR) spectra of Au@Ag nanocubes (NCs) are linearly shifted to a long wavelength with an increasing concentration of ConA. In fact, each Au@Ag NC can act as a nanobiosensor for the quantified detection of trace ConA, which enables the miniaturization of the biosensor system to nanoscale. Furthermore, the results demonstrated the perfect biosensing ability with the dual channel of dark-field microscopy images and LSPR spectra. We expect that this nanobiosensor system can provide an alternative important method for monitoring the specific binding of lectin-sugar at a single nanoparticle surface.

Intracellular dark-field imaging of ATP and photothermal therapy using a colorimetric assay based on gold nanoparticle aggregation via tetrazine/trans-cyclooctene cycloaddition.

In this study, we developed a colorimetric ATP assay based on the ATP-induced aggregation of Au nanoparticles (AuNPs). This aggregation modified the local surface plasmon resonance (LSPR) of the AuNPs, which was used to detect and localize ATP in cells via dark-field imaging. The AuNP aggregation process involved the reaction of two types of functionalized AuNPs with each other: tetrazine-modified AuNPs (Au3-N4) and asymmetrically functionalized trans-cyclooctene-modified AuNPs (Au1-(E)-cyclooctene). This cycloaddition reaction occurs without the need for a catalyst such as the Cu ions that are used in the "click" reactions often employed in assays of this type. Initially, we asymmetrically functionalized both types of AuNPs and let them dimerize, which permitted us to explore the resulting wavelength shift in the LSPR of the AuNPs. Then, to facilitate the specific recognition of ATP, a designed DNA (DNA1) containing an ATP aptamer sequence was attached to carboxyl polystyrene microbeads (MBs). After attaching a different DNA (DNA2, which hybridizes with DNA1) to Au1-(E)-cyclooctene, the assay probe MB/DNA1/DNA2/Au1-(E)-cyclooctene (MB/Au1) was generated. While bound to MB/DNA1, the DNA2/Au1-(E)-cyclooctene cannot react with Au3-N4 due to steric hindrance from the MB. However, in the presence of ATP, the probe MB/Au1 dissociates, and the resulting free DNA2/Au1-(E)-cyclooctene can then react with the Au3-N4, leading to the formation of AuNP aggregates. Dark-field microscopy (DFM) images showed that the LSPR of the AuNPs shifted from the green region (AuNP monomers) to the orange-red region (AuNP aggregates) in the presence of intracellular ATP. Moreover, the AuNP aggregates were found to exhibit significant photothermal effects under 808-nm laser irradiation. Upon introducing the probe MB/Au1 and Au3-N4 into HeLa cells in vitro and in vivo, and then irradiating the cells with a 808-nm NIR laser, the resulting AuNP aggregates showed promising photothermal cancer therapy performance. This assay therefore has the potential to be widely used for the identification and determination of nanoparticles in biological DFM and in tumor theranostics. Graphical abstract.

Generation of Multi-Wavelength Light Pulses by Femtosecond Bessel Laser Beam in Silica Glass

When the near-infrared femtosecond Bessel laser beam (fs-BLB) is obliquely incident on the silica glass, annular beams (ABs) with bandwidths of several tens of nanometers and different peak wavelengths discretely distributed in a visible and near-infrared range are generated simultaneously. It is considered that four-wave mixing (FWM) and the corresponding cascaded parametric processes are responsible for the generation of multi-wavelength ABs. It is also found that the broken symmetry of the optical setup due to the obliquely oriented silica glass and the absorption band of the sample significantly enhance the generation efficiency of ABs. Since the diffraction angles of these colorful ABs mainly depend on their wavelengths and are insensitive to the input laser energy, this technique may find important applications where multi-wavelength ultrashort pulses are needed. For example, these multi-wavelength ABs can be used as light sources in nonlinear dark-field microscopy and multiphoton imaging to further suppress the background noise and improve resolution. They can also be used in multidimensional multiplexing optical communications.

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes.

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Biomedical Image Processing with Containers and Deep Learning: An Automated Analysis Pipeline: Data architecture, artificial intelligence, automated processing, containerization, and clusters orchestration ease the transition from data acquisition to insights in medium-to-large datasets.

Here, a streamlined, scalable, laboratory approach is discussed that enables medium-to-large dataset analysis. The presented approach combines data management, artificial intelligence, containerization, cluster orchestration, and quality control in a unified analytic pipeline. The unique combination of these individual building blocks creates a new and powerful analysis approach that can readily be applied to medium-to-large datasets by researchers to accelerate the pace of research. The proposed framework is applied to a project that counts the number of plasmonic nanoparticles bound to peripheral blood mononuclear cells in dark-field microscopy images. By using the techniques presented in this article, the images are automatically processed overnight, without user interaction, streamlining the path from experiment to conclusions.