Dark-field Microscopy Images Research Articles

Deep Learning Assisted Plasmonic Dark-Field Microscopy for Super-Resolution Label-Free Imaging.

Dark-field microscopy (DFM) is a widely used imaging tool, due to its high-contrast capability in imaging label-free specimens. Traditional DFM requires optical alignment to block the oblique illumination, and the resolution is diffraction-limited to the wavelength scale. In this work, we present deep-learning assisted plasmonic dark-field microscopy (DAPD), which is a single-frame super-resolution method using plasmonic dark-field (PDF) microscopy and deep-learning assisted image reconstruction. Specifically, we fabricated a designed PDF substrate with surface plasmon polaritons (SPPs) illuminating specimens on the substrate. Dark field images formed by scattered light from the specimen are further processed by a pretrained convolutional neural network (CNN) using a simulation dataset based on the designed substrate and parameters of the detection optics. We demonstrated a resolution enhancement of 2.8 times on various label-free objects with a large potential for future improvement. We highlight our technique as a compact alternative to traditional DFM with a significantly enhanced spatial resolution.

Aggregation-induced color-coded imaging for HOCl detection in living cells with dark-field microscopy

In this work, we develop a nanoprobe for the detection of hypochlorous acid (HOCl), a key reactive oxygen species, utilizing dark-field microscopy (DFM). The nanoprobe is constructed by covalently attaching PEG-SH and an HOCl-sensitive molecule, FD, to gold nanoparticles (GNPs-FD-PEG). This probe detects HOCl by observing a color shift from green to red due to the aggregation of GNPs, which is triggered by HOCl-induced the cleavage of FD molecule. This aggregation occurs primarily due to electrostatic interactions between the differently charged particles following the cleavage. By recording the scattering signal changes of the GNPs-FD-PEG nanoprobe, we have achieved quantitative detection of HOCl, with a linear response range of 0.5–50 μM and a detection limit of 0.16 μM. By integrating the high-resolution capabilities of DFM imaging with the distinct color change due to electrostatic-induced nanoparticle aggregation, the constructed nanoprobe effectively monitors HOCl in live cells.

Highly Sensitive Determination of Copper Ions as MnO2 Etching Inhibitor in Single-Particle Nanoplasmonic Imaging.

Dark-field microscopy (DFM) imaging based on plasmonic metal nanoparticles has garnered significant attention. Here, we exploit the susceptibility of MnO2 to reduction to modulate the local dielectric environment of an Au nanoparticle core through the etching/antietching effects of specific targets on the encapsulated MnO2 shell. The presence of d-penicillamine promotes MnO2 etching, while the chelation of d-penicillamine with Cu2+ effectively inhibits this etching. By recording the Cu2+-induced color shift of scattered light from orange to bright green at the single-particle level and performing the statistical analysis of the green-to-red (G/R) values in DFM images, we achieved quantitative determination of Cu2+ with a wide linear range (0.1-10 μM) and a low limit of detection (4.55 nM). With the facile and reliable Cu2+ assay in real-world samples exemplifying the practicality of the single-particle nanoplasmonic imaging method, this work may inspire future DFM-based investigations of nanoshell etching inhibition processes.

Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag–Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag–Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.

Real-Time Imaging of Interfacial Copper(II) Ion-Initiated Selective Etching in the Core Region of Single Cuprous Oxide-Bismoclite Core-Shell Microcrystals.

The core-shell microstructures are attracting much interest, most notably for their superior performance compared with their pure counterparts because of the interfacial effect. Comprehensively understanding the mechanism of the interfacial effect is critical but still elusive. Here, we report real-time dark-field optical microscopy (DFM) imaging of the selective etching in the core region of single cuprous oxide-bismoclite (Cu2O@BiOCl) core-shell microcrystals by I-. In situ DFM observations reveal that the reaction activity of Cu2O is significantly improved after coating the BiOCl shell layer, and the I- diffuses through the BiOCl shell and approaches the interface region, followed by etching the Cu2O core. During the etching process, two distinct reaction pathways, such as interfacial Cu2+-driven redox etching and confinement-governed dissolution, are identified. The interfacial Cu2+ is generated due to the coordination number difference at the core-shell interface. Moreover, according to the in situ DFM single-crystal imaging results, the ensemble adsorption capacity improvement for I- is also demonstrated in Cu2O@BiOCl core-shell microcrystals. These findings provide deep insights into the interfacial effect of core-shell microcrystals and establish a bridge between microscopic imaging and macroscopic practical application.

Advances in the Toxicity Assessment of Silver Nanoparticles Derived from a Sphagnum fallax Extract for Monolayers and Spheroids.

The production of nanomaterials through environmentally friendly methods is a top priority in the sustainable development of nanotechnology. This paper presents data on the synthesis of silver nanoparticles using an aqueous extract of Sphagnum fallax moss at room temperature. The morphology, stability, and size of the nanoparticles were analyzed using various techniques, including transmission electron microscopy, Doppler laser velocimetry, and UV-vis spectroscopy. In addition, Fourier transform infrared spectroscopy was used to analyze the presence of moss metabolites on the surface of nanomaterials. The effects of different concentrations of citrate-stabilized and moss extract-stabilized silver nanoparticles on cell viability, necrosis induction, and cell impedance were compared. The internalization of silver nanoparticles into both monolayers and three-dimensional cells spheroids was evaluated using dark-field microscopy and hyperspectral imaging. An eco-friendly method for the synthesis of silver nanoparticles at room temperature is proposed, which makes it possible to obtain spherical nanoparticles of 20-30 nm in size with high bioavailability and that have potential applications in various areas of human life.

Objectification of evaluation criteria in microscopic agglutination test using deep learning

We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.

Glutamic Acid Modified Gold Nanorod Sensor for the Detection of Calcium ions in Neuronal Cells.

Calcium (Ca2+) ions play a crucial role in the functioning of neurons, governing various aspects of neuronal activity such as rapid modulation and alterations in gene expression. Ca2+ signaling has a significant impact on the development of diseases and the impairment of neuronal functions. Herein, the study reports a Ca2+ ion sensor in neuronal cells using a gold nanorod. The gold nanorod (GA-GNR) conjugated glutamic acid developed in the study was used as a nano-bio probe for the experimental and in vitro detection of calcium. The nanosensor is colloidally stable, preserves plasmonic properties, and shows good viability in neuronal cells, as well as promoting neuron cell line growth. The cytotoxicity and cell penetration of the nanosensor are studied using Raman spectroscopy, brightfield and darkfield microscopy imaging, and MTT assays. The quantification of Ca2+ ions in neuronal cells is determined by monitoring the surface plasmon resonance (SPR) of the GA-GNR. The change in the intensity profile in the presence of Ca2+ incubated neurons was effectively used to develop a portable prototype of an optical Ca2+ sensor, proposing it as a tool for neurodegenerative disease diagnosis and neuromodulation evaluation.

Imaging the Iodine Sorption-Induced Synchronous Skeleton-Pore Interactions of Single Covalent Organic Framework Particles.

Covalent organic frameworks (COFs) are promising iodine adsorbents. For improved performances, it is critical and essential to fundamentally understand the underlying mechanism. Here, using the operando dark-field optical microscopy (DFM) imaging technique, the observation of an extraordinary structure shrinkage of 2D triphenylbenzene (TPB)-dimethoxyterephthaldehyde (DMTP)-COF upon the adsorption of I2 vapor at the single-particle resolution is reported. Combining single-particle DFM imaging with other experimental and theoretical methods, it is revealed that the shrinkage mechanism of the TPB-DMTP-COF is attributed to the I2 sorption-induced synchronous skeleton-pore interactions. The redox reaction of I2 and TPB-DMTP-COF yields some cationic skeletons and I3 - species, which triggers the multi-directional halogen-bonding interactions of I2 and I3 - as well as strong cation-π interactions between neutral and cationic skeletons, accompanying the synchronous in-plane skeleton shrinking in the xy plane and compact out-of-plane layer packing in the z-direction. This understanding of the synchronous action between the skeleton and pore breaks the perspective on the structure robustness of 2D COFs with excellent stability during the I2 uptake, which offers pivotal guidance for the rational design and creation of advanced microporous adsorbents.

Dark-field imaging and fluorescence dual-mode detection of microRNA-21 in living cells by core-satellite plasmonic nanoprobes

The in situ precise quantification and simultaneous imaging of low abundance microRNAs (miRNAs) within living cells is critical for cancer diagnosis, yet it remains a significant challenge. Leveraging the excellent sensitivity and spatiotemporal resolution of dark-field microscopy (DFM) and fluorescence imaging, we have successfully devised a novel detection approach using dual-signal reporter probes (DSRPs). These probes allow for highly sensitive detection of miRNA-21 in living cells via toehold-mediated strand displacement cascades. The DSRPs were constructed by Au nanoparticles and Ag nanoclusters core-satellite nanostructures. After the recognition of miRNA-21, the strand displacement cascades were triggered, inducing the disassembly of the Au/Ag core-satellite nanostructure with apparent scattering intensity decrease and peak wavelength shifts. Additionally, the fluorescence of Ag clusters could be recovered and further enhanced when in close proximity to specific guanine-rich strands. The dual-signal response capability enables the accurate detection of miRNA-21 from 1 fM to 1 nM, with a limit of detection reached 0.75 fM. DFM and fluorescent imaging of living cells efficiently confirms the applicable detection of miRNA-21 in complex detection media. The biosensor based on DSRPs represents a promising nanoplatform for visual monitoring and imaging of biomolecules in living cells, even at the single particle level.

Enhancement of weak signals by dynamic stochastic resonance in dark-field microscopy imaging

Abstract Enhancing weak signal acquisition is pivotal for bolstering analysis and detection capabilities. Herein, a dynamic stochastic resonance (DSR) algorithm for weak signal amplification that was identified to significantly improve the imaging visibility of dark-field microscopy nanoparticles under low illumination conditions is presented. When further combined with composite filed microscopy, DSR displays signal amplification much more effectively, showing high promise for detection purposes.

Nucleic acid amplification-free biosensor for sensitive and specific cfDNA detection based on CRISPR-Cas12a and single nanoparticle dark-field microscopy (DFM) imaging

Circulating cell-free DNA (cfDNA) is known to be an attractive biomarker for liquid biopsy. In this work, a rapid and sensitive biosensor was achieved for cfDNA detection based on CRISPR-associated nuclease 12a (CRISPR-Cas12a) and single nanoparticle dark-field microscopy (DFM) imaging. The biosensor employed gold nanoparticles (AuNPs) as signal source, DFM as readout system, and Meanshift algorithms as the image processing systems, respectively. The presence or absence of cfDNA caused the different existing states (monomer or aggregated state) of AuNPs. AuNPs monomers could be effectively distinguished from the aggregated ones under DFM since the AuNPs aggregation could induced the green-to-yellow or green-to-red changing of scattering color. The monomer ratio easily obtained by Meanshift and partial least-square (PLS) algorithms was used for quantitative analysis. Using this strategy, breast cancer gene-1 (BRCA-1), a representative of cfDNA biomarker for breast cancer could be measured with high sensitivity in 40 min and has a low detection limit of 0.081 fM. It is hoped that this nucleic acid amplification-free sensor could be also utilized to detect other nucleic acid biomarkers, and thus, provides a novel strategy for biomedical image analysis.

Holistic Prediction of AuNP Aggregation in Diverse Aqueous Suspensions Based on Machine Vision and Dark-Field Scattering Imaging.

The localized surface-plasmon resonance of the AuNP in aqueous media is extremely sensitive to environmental changes. By measuring the signal of plasmon scattering light, the dark-field microscopic (DFM) imaging technique has been used to monitor the aggregation of AuNPs, which has attracted great attention because of its simplicity, low cost, high sensitivity, and universal applicability. However, it is still challenging to interpret DFM images of AuNP aggregation due to the heterogeneous characteristics of the isolated and discontinuous color distribution. Herein, we introduce machine vision algorithms for the training of DFM images of AuNPs in different saline aqueous media. A visual deep learning framework based on AlexNet is constructed for studying the aggregation patterns of AuNPs in aqueous suspensions, which allows for rapid and accurate identification of the aggregation extent of AuNPs, with a prediction accuracy higher than 0.96. With the aid of machine learning analysis, we further demonstrate the prediction ability of various aggregation phenomena induced by both cation species and the concentration of the external saline solution. Our results suggest the great potential of machine vision frameworks in the accurate recognition of subtle pattern changes in DFM images, which can help researchers build predictive analytics based on DFM imaging data.

Measurement of G-phase volume fraction and number density in duplex stainless steels using transmission electron microscopy

Duplex stainless steels (DSS) have a high toughness and strength due the presence of both austenitic and ferritic phases. These alloys have had limited use in power production applications due to thermal embrittlement caused by spinodal decomposition and development of G-phase precipitates in the ferrite. Lean grade DSS alloys (e.g., 2101, 2003) may offer improved thermal stability due to the reduction of Cr- and Ni-equivalent elements when compared to standard grade compositions (e.g., 2205, 2209). The abundance of the G-phase was measured in five duplex stainless steels, three wrought alloys (2101, 2003, 2205) and their matching filler metals (2101-w, 2209-w), after aging at 427 °C for 1000 h and 10,000 h. The G-phase volume fraction, number density, size, and precipitate spacing were found using quantitative analysis of transmission electron microscopy dark field images and the composition of G-phase precipitates on other clusters were characterized with atom probe tomography (APT). In the welded alloys, the G-phase was found to develop rapidly, relative to the wrought material. A positive correlation was found between the nickel equivalent composition of the alloy and the G-phase volume fraction. The alloys 2205, 2209, and 2101-w, which are higher in Cr and Ni, all showed significant G-phase precipitation, further strengthening the hypothesis that lean grade DSS alloys are more thermally stable against precipitation in the ferrite. Electron diffraction showed a secondary phase present in the 2101 wrought alloy at 10,000 h, but it was not crystallographically consistent with the G-phase; APT showed the presence of nanoclusters rich in both nickel and copper for this alloy. No secondary phases or clusters were found in 2003 after 10,000 h of aging, so it may be a candidate alloy for applications that require long-life times at high operating temperatures.

Core-Satellite Nanoassemblies as SPR/SERS Dual-Mode Plasmonic Sensors for Sensitively Detecting Ractopamine in Complex Media.

Highly sensitive and reliable detection of β-adrenergic agonists is especially necessary due to the illegal abuse of growth-promoting feed additives. Here, we develop a novel surface plasmon resonance/surface-enhanced Raman scattering (SPR/SERS) dual-mode plasmonic sensor based on core-satellite nanoassemblies for the highly sensitive and reliable detection of ractopamine (RAC). The addition of RAC results in the decomposition of core-satellite nanoassemblies and consequently changes the Rayleigh scattering color of dark-field microscopy (DFM) images and the Raman scattering intensity of SERS spectra. The excellent sensitivity, specificity, and uniformity of this strategy were confirmed by detecting RAC in various complex media in the farm-to-table chain, and the limit of detection (LOD) was 0.03 ng/mL in an aqueous solution. In particular, the convenient access to livestock sewage not only ensures animal welfare but also provides great convenience for the market regulation of β-agonists. The success of our on-site strategy only with a portable Raman device promises great application prospects for β-agonist detection.

Visualizing dynamic competitive adsorption processes between iodine and methyl iodide within single covalent organic framework crystals

Covalent organic frameworks (COFs) are important porous adsorbents for volatile iodine (I2) and methyl iodide (CH3I). In situ monitoring of the dynamic adsorption process of single COF crystals toward I2 and CH3I is a critical and fundamental issue for understanding the reaction mechanism and improving the sorption performance. Here, we report operando real-time dark-field optical microscopy (DFM) imaging of visually studying the dynamic adsorption behavior of LZU-111 (LZU=Lanzhou University) COFs in the I2/CH3I binary gaseous mixture at the single-crystal level. Time-lapse imaging shows that the uptake of CH3I and I2 results in the R intensity increase and B intensity decrease of the DFM images. Employing the R and B intensities as two indicators, we find an unusual blinking of R/B intensities from single LZU-111 crystals, which is attributed to the intermittent sorption-desorption processes of CH3I and I2 within the LZU-111 framework. The visualization of the dynamic reaction process provides clear evidence that the competitive adsorption between CH3I and I2 goes through a multi-time and oscillatory reaction pathway instead of a successive procedure. Combined with theoretical calculations, the difference in the migration capability and initial pressure is identified for initiating the intermittent blinking events.

Imaging Single H2 Nanobubbles Using Off-Axis Dark-Field Microscopy.

A robust and detailed physicochemical description of electrochemically generated surface nanobubbles and their effects on electrochemical systems remains at large. Herein, we report the development and utilization of an off-axis, dark-field microscopy imaging tool for probing the dynamic process of generating single H2 nanobubbles at the surface of a carbon nanoelectrode. A change in the direction of the incident light is made to significantly reduce the intensity of the background light, which enables us to image both the nanoelectrode and nanobubble on the electrode surface or the metal nanoparticles in the vicinity of the electrode. The correlated electrochemical and optical response provides novel insights regarding bubble nucleation and dissolution on a nanoelectrode previously unattainable solely from its current-voltage response.

Copper(II)-porphyrin functionalized titanium(IV) metal-organic aerogels for the visible-light driven conversion of CO2 to alcohols

This manuscript covers the synthesis and characterization of a series of titanium based metal-organic aerogels (MOAs) functionalized with copper(II)-metalated porphyrins for their application in visible-light-driven carbon dioxide (CO2) conversion to alcohols. A thorough characterization is performed using a set of spectroscopic and microstructural analysis techniques to reveal the structural and microstructural features that can aid in establishing structure-activity relationships. The parent MOAs consist of metal-organic nanoparticles (5–10 nm) crosslinked into a highly porous microstructure (surface area: 600–800 m2·g−1). The post-synthetic reaction with copper(II) enables the metalation of the tetrapyrrole ring of porphyrin, which is confirmed by analyzing the absorption and luminescence spectra. High-angle annular dark-field electron microscopy imaging demonstrates a uniform distribution of the metalation throughout the nanoparticles that compose the material. The CO2 photoreduction experiments performed in an optofluidic microreactor show that the metalation markedly invigorates the total alcohol (methanol and ethanol) production rates and apparent quantum yields (AQY), from 21 to 367 μmol·g−1·h−1 (AQY: 0.4–7%) prior to metalation to values of 356–642 μmol·g−1·h−1 (AQY: 11–20%). Additionally, the metalation inverts the selectivity towards ethanol, increasing from 0–12% in the parent MOA to 67–76% after incorporating Cu(II).

Effects of primary aberrations on a dark-field microscopy imaging system with the sharper focusing lens

The effects of primary aberrations of the sharper focusing lens on the image quality is analyzed for a dark-field microscopy imaging system, which is illuminated by a left-circularly (LC) polarized Laguerre–Gaussian (LG) vortex beam. To confirm the aberration tolerance range of the sharper focusing lens for precise measurement, the analytical expression of the field distribution on the image plane is deduced. Then the dark-field image results corresponding to a defect with a scale of 6µm×6µm are simulated. The influence of the spherical error, coma, and astigmatism on the imaging quality is compared in detail. Then the verification test has been carried out to show the effects of different primary aberrations. Finally, both the simulation and experiment results help us to identify that the tolerance range of spherical aberration, astigmatism, and coma should be less than 1.5λ, 1λ, and 1.25λ, respectively.

Plasmonic Metamaterial Ag Nanostructures on a Mirror for Colorimetric Sensing.

In this study, we demonstrate the localized surface plasmon resonance (LSPR) in the visible range by using nanostructures on mirrors. The nanohemisphere-on-mirror (NHoM) structure is based on random nanoparticles that were obtained by heat-treating silver thin films and does not require any top-down nanofabrication processes. We were able to successfully tune over a wide wavelength range and obtain full colors using the NHoM structures, which realized full coverage of the Commission Internationale de l'Eclairage (CIE) standard RGB (sRGB) color space. Additionally, we fabricated the periodic nanodisk-on-glass (NDoG) structure using electron beam lithography and compared it with the NHoM structure. Our analysis of dark-field microscopic images observed by a hyperspectral camera showed that the NHoM structure had less variation in the resonant wavelength by observation points compared with the periodic NDoG structure. In other words, the NHoM structure achieved a high color quality that is comparable to the periodic structure. Finally, we proposed colorimetric sensing as an application of the NHoM structure. We confirmed the significant improvement in performance of colorimetric sensing using the NHoM structure and succeeded in colorimetric sensing using protein drops. The ability to fabricate large areas in full color easily and inexpensively with our proposed structures makes them suitable for industrial applications, such as displays, holograms, biosensing, and security applications.