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The development of an automatic method of identifying microplastic particles within live cells and organisms is crucial for high-throughput analysis of their biodistribution in toxicity studies. State-of-the-art technique in the data analysis tasks is the application of deep learning algorithms. Here, we propose the approach of polystyrene microparticle classification differing only in pigmentation using enhanced dark-field microscopy and a residual neural network (ResNet). The dataset consisting of 11,528 particle images has been collected to train and evaluate the neural network model. Human skin fibroblasts treated with microplastics were used as a model to study the ability of ResNet for classifying particles in a realistic biological experiment. As a result, the accuracy of the obtained classification algorithm achieved up to 93% in cell samples, indicating that the technique proposed will be a potent alternative to time-consuming spectral-based methods in microplastic toxicity research.

Dark field scattering microscopy can create large hyperspectral data sets that contain a wealth of information on the properties and the molecular environment of noble metal nanoparticles. For a quick screening of samples of microscopic dimensions that contain many different types of plasmonic nanostructures, we propose a multivariate analysis of data sets of thousands to several hundreds of thousands of scattering spectra. By using non-negative matrix factorization for decomposing the spectra, components are identified that represent individual plasmon resonances and relative contributions of these resonances to particular microscopic focal volumes in the mapping data sets. Using data from silver and gold nanoparticles in the presence of different molecules, including gold nanoparticle-protein agglomerates or silver nanoparticles forming aggregates in the presence of acrylamide, plasmonic properties are observed that differ from those of the original nanoparticles. For the case of acrylamide, we show that the plasmon resonances of the silver nanoparticles are ideally suited to support surface enhanced Raman scattering (SERS) and the two-photon excited process of surface enhanced hyper Raman scattering (SEHRS). Both vibrational tools give complementary information on the in situ formed polyacrylamide and the molecular composition at the nanoparticle surface. Caroline von Humboldt Professorship of HU

Adrenaline (AD) is an important hormone in central nervous system of mammals, and also works as a medium to transmit signal. The abnormal level of AD in nerve tissues and biological fluids can cause a variety of diseases. In this work, we developed a simple and sensitive dark-field microscopy (DFM)-based single-particle detection (SPD) method for quantitative detection of AD in aqueous solution. MnO2 layer of GNP@MnO2 core-shell nanoparticles could be readily etched due to the reducibility of AD. Since the localized surface plasmon resonance (LSPR) of GNPs was sensitive to the dielectric change of local environment, the etching process resulted in an evident color change of the probe and could be easily observed in real time under dark-field microscope. The AD was thus sensitively detected in a wide linear concentration range (0.02–5 μmol/L) with a limit of detection (LOD) of 7.17 nmol/L. Moreover, this method was used for the analysis of AD content in commercial injection and satisfactory recoveries were acquired. As a consequence, the sensing assay demonstrated herein based on GNP@MnO2 NPs showed great application prospects in molecular recognition and drug quality assay.

Hypothesis While dynamics of particles in slurries is usually evaluated by dynamic light scattering measurements, this technique had only been applicable to particles in the bulk slurries. Because this limitation is mainly owing to strong reflection of light, the dynamics of particles in slurries spreading/drying on solid substrates is to be obtained by spatially separating the reflection light from scattering (signal) light. This may allow us to track the particles in practical samples such as cosmetics or inks spreading on solid surfaces. Experiment We developed novel “dark-field dynamic light scattering microscopy”. The system was evaluated with test samples of polystyrene beads dispersed in several viscosities of bulk glycerol aqueous solutions. This setup was then applied to slurries spreading/drying on planar and nonplanar substrates. Findings The results for planar surface indicate that origin of coffee-ring are the particles flowing into the edge of the droplet just before complete drying. On a skin-modelled nonplanar substrate, the slurry on bumps was found to maintain semi-dry condition longer than that at dents. This suggests that the dispersive medium was supplied to bumps from dents. This unique flow was explained as effective drying from the bumps increased surface tension at the bumps to pull up the liquid around.

The CytoViva Enhanced Darkfield (EDF) Illuminator enables direct optical observation of nano-scale entities in a wide range of transparent and translucent sample environments, without requiring the use of labels or other markers. The improved performance is a result of a uniquely designed light path, which focuses maximum photon density on the sample, thus enabling superior signal-to-noise imaging performance. This high-performance capability not only provides users with previously unobtainable images, but it also allows established techniques such as hyperspectral and Raman microscopy to be used in new and exciting ways.

Linking the chemical composition of bitumen to its microstructure is challenge due to its complexity, viscoelasticity and sensitivity to high energy input. Therefore, fluorescence spectroscopy comb...

Optical microscopes have emerged as powerful tools towards electrochemical analysis of single nanoparticles, owing to their sufficient spatial and temporal resolution, high throughput, excellent sensitivity and non-contact nature. This review summarized recent progress on studying the nanochemistry at single-particle level by using four types of advanced optical techniques, such as fluorescence microscopy, surface plasmon resonance microscopy, dark-field microscopy, and electrogenerated chemiluminescence. Finally, we provided our opinions on the remaining challenges and prospects for future work in this field.

Residual strain gradients and crystallographic mosaicity across embedded ceramic particles within metal-matrix composites have remained experimentally inaccessible. In this work, we use synchrotron dark-field X-ray microscopy to characterize strain and orientation gradients across ~20 µm large TiC and TiN particles embedded in a steel matrix annealed at 480 and 600 °C. The results document gradual crystallographic orientation changes across the particles and the presence of sub-grains, in contrast to electron backscatter diffraction data. These gradients appear, however, not directly correlated with the observed strain gradients, which are attributed mainly to the different strain states within particles’ interiors and envelopes.

Thearticledealswith the comparative application of optical and raster microscopy for non-metallic objects and non-conducting surfaces. It is noted that this issue is not covered much in the special literature. There are practically no publications that compare and describe photos of the structure of materials obtained using fundamentally different microscopes, in particular, metallographic and raster. The causes of image distortion in a raster electron microscope in the study of dielectrics are considered. Comparative images of the oxidized surface, fabrics and natural leather obtained using raster and optical microscopy are presented. The advantages of optical microscopy in the study of non-conducting surfaces are shown.

Sidestream dark field (SDF) imaging enables direct visualisation of the microvasculature from which quantification of key variables is possible. The new MicroScan USB3 (MS-U) video-microscope is a hand-held SDF device that has undergone significant technical upgrades from its predecessor, the MicroScan Analogue (MS-A). The MS-U claims superior quality of sublingual microcirculatory image acquisition over the MS-A, however, this has yet to be robustly confirmed. In this manuscript, we therefore compare the quality of image acquisition between these two devices. The microcirculation of healthy volunteers was visualised to generate thirty video images for each device. Two independent raters, blinded to the device type, graded the quality of the images according to the six different traits in the Microcirculation Image Quality Score (MIQS) system. Chi-squared tests and Kappa statistics were used to compare not only the distribution of scores between the devices, but also agreement between raters. MS-U showed superior image quality over MS-A in three of out six MIQS traits; MS-U had significantly more optimal images by illumination (MS-U 95% optimal images, MS-A 70% optimal images (p-value 0.003)), by focus (MS-U 70% optimal images, MS-A 35% optimal images (p-value 0.002)) and by pressure (MS-U 72.5% optimal images, MS-A 47.5% optimal images (p-value 0.02)). For each trait, there was at least 85% agreement between the raters, and all the scores for each trait were independent of the rater (all p-values > 0.05). These results show that the new MS-U provides a superior quality of sublingual microcirculatory image acquisition when compared to old MS-A

Chlamydia pneumoniae is a spherical zoonotic pathogen with a diameter of ∼200 nm, which can lead to a wide range of acute and chronic diseases in human body. Early and reliable on-site detection of C. pneumoniae is the key step to control the spread of the pathogen. However, the lack of a current technology with advantages of rapidity, ultrasensitivity, and convenience limits the implementation of traditional techniques for on-site detection of C. pneumoniae. Herein, we developed a naked-eye counting of C. pneumoniae based on the light scattering properties of gold nanoparticle (GNP) under dark-field microscopy (termed “GNP-labeled dark-field counting strategy”). The recognition of single C. pneumoniae by anti-C. pneumoniae antibodies-functionalized GNP probes with size of 15 nm leads to the formation of wreath-like structure due to the strong scattered light resulted from hundreds of GNP probes binding on one C. pneumoniae under dark-field microscopy. Hundreds of GNP probes can bind to the surface of C. pneumoniae due to the high stability and specificity of the nucleic acid immuno-GNP probes, which generates by the hybridization of DNA-modified GNP with DNA-functionalized antibodies. The limit of detection (LOD) of the GNP-labeled dark-field counting strategy for C. pneumoniae detection in spiked samples or real samples is down to four C. pneumoniae per microliter, which is about 4 times more sensitive than that of quantitative polymerase chain reaction (qPCR). Together with the advantages of the strong light scattering characteristic of aggregated GNPs under dark-field microscopy and the specific identification of functionalized GNP probes, we can detect C. pneumoniae in less than 30 min using a cheap and portable microscope even if the sample contains only a few targets of interest and other species at high concentration. The GNP-labeled dark-field counting strategy meets the demands of rapid detection, low cost, easy to operate, and on-site detection, which paves the way for early and on-site detection of infectious pathogens. usc Refereed/Peer-reviewed

Dark-field microscopy is a standard imaging technique widely employed in biology that provides high image contrast for a broad range of unstained specimens1. Unlike bright-field microscopy, it accentuates high spatial frequencies and can therefore be used to emphasize and resolve small features. However, the use of dark-field microscopy for reliable analysis of blood cells, bacteria, algae and other marine organisms often requires specialized, bulky microscope systems, as well as expensive additional components, such as dark-field-compatible objectives or condensers2,3. Here, we propose to simplify and downsize dark-field microscopy equipment by generating the high-angle illumination cone required for dark-field microscopy directly within the sample substrate. We introduce a luminescent photonic substrate with a controlled angular emission profile and demonstrate its ability to generate high-contrast dark-field images of micrometre-sized living organisms using standard optical microscopy equipment. This new type of substrate forms the basis for miniaturized lab-on-chip dark-field imaging devices that are compatible with simple and compact light microscopes. A luminescent photonic substrate with a controlled angular emission profile is introduced and its ability to generate high-contrast dark-field images of micrometre-sized living organisms is demonstrated using standard optical microscopy equipment.

Fusarium oxysporum f. sp. cubense is a soil-borne fungi that has become a major threat to the current banana industry. The presence of this fungi can destroy entire plantations if not detected and stopped early enough. The purpose of this study is to create a Convolutional Neural Network (CNN) that can detect hyphae in microscopic images. By detecting hyphae, the presence of fungi in the soil can be confirmed. To create a model that can detect hyphae, a dataset of various microscopic images of fungi was sorted into hyphae images and non-hyphae images (labeled as others). Four subsequent datasets were created from this, namely: (1) bright field, (2) dark field, (3) fluorescent, and (4) all microscopy techniques. Pretrained ResNet34 and ResNet152 models were used for each of the datasets and the use of heatmaps on these models was done to analyze how the models looked for hyphae. The ResNet34 model achieved accuracies of 86.38% for bright field, 87.31% for dark field, 88.37% for fluorescent, and 87.60% for all microscopy techniques. The ResNet152 model achieved accuracies of 87.97% for bright field, 86.79% for dark field, 89.37% for fluorescent, and 87.69% for all microscopy techniques. Additionally, to improve the accuracy even further, automated cropping using edge detection and contour detection was done on the datasets to create cropped photos of hyphae. This resulted in average test accuracies of 87.17% for bright field, 86.90% for dark field, 91.22% for fluorescent, and 89.99% for all microscopy techniques. Generally, fluorescent consistently performed the best, but the heatmaps generated from each model show that hyphae can also be detected using the other microscopy techniques. This study can act as a steppingstone for future studies involving the classification of fungi through hyphae and other features.

The design of dynamic, reconfigurable devices is crucial for the bottom-up construction of artificial biological systems. DNA can be used as an engineering material for the de-novo design of such dynamic devices. A self-assembled DNA origami switch is presented that uses the transition from double- to single-stranded DNA and vice versa to create and annihilate an entropic force that drives a reversible conformational change inside the switch. It is distinctively demonstrated that a DNA single-strand that is extended with 0.34 nm per nucleotide - the extension this very strand has in the double-stranded configuration - exerts a contractive force on its ends leading to large-scale motion. The operation of this type of switch is demonstrated via transmission electron microscopy, DNA-PAINT super-resolution microscopy and darkfield microscopy. The work illustrates the intricate and sometimes counter-intuitive forces that act in nanoscale physical systems that operate in fluids.

Electron microscopy (EM) is the most versatile tool for the study of matter at scales ranging from subatomic to visible. The high vacuum environment and the charged irradiation require careful stabilization of many specimens of interest. Biological samples are particularly sensitive due to their composition of light elements suspended in an aqueous medium. Early investigators developed techniques of embedding and staining with heavy metal salts for contrast enhancement. Indeed, the Nobel Prize in 1974 recognized Claude, de Duve, and Palade for establishment of the field of cell biology, largely due to their developments in separation and preservation of cellular components for electron microscopy. A decade later, cryogenic fixation was introduced. Vitrification of the water avoids the need for dehydration and provides an ideal matrix in which the organic macromolecules are suspended; the specimen represents a native state, suddenly frozen in time at temperatures below -150 °C. The low temperature maintains a low vapor pressure for the electron microscope, and the amorphous nature of the medium avoids diffraction contrast from crystalline ice. Such samples are extremely delicate, however, and cryo-EM imaging is a race for information in the face of ongoing damage by electron irradiation. Through this journey, cryo-EM enhanced the resolution scale from membranes to molecules and most recently to atoms. Cryo-EM pioneers, Dubochet, Frank, and Henderson, were awarded the Nobel Prize in 2017 for high resolution structure determination of biological macromolecules.A relatively untapped feature of cryo-EM is its preservation of composition. Nothing is added and nothing removed. Analytical spectroscopies based on electron energy loss or X-ray emission can be applied, but the very small interaction cross sections conflict with the weak exposures required to preserve sample integrity. To what extent can we interpret quantitatively the pixel intensities in images themselves? Conventional cryo-transmission electron microscopy (TEM) is limited in this respect, due to the strong dependence of the contrast transfer on defocus and the absence of contrast at low spatial frequencies.Inspiration comes largely from a different modality for cryo-tomography, using soft X-rays. Contrast depends on the difference in atomic absorption between carbon and oxygen in a region of the spectrum between their core level ionization energies, the so-called water window. Three dimensional (3D) reconstruction provides a map of the local X-ray absorption coefficient. The quantitative contrast enables the visualization of organic materials without stain and measurement of their concentration quantitatively. We asked, what aspects of the quantitative contrast might be transferred to cryo-electron microscopy?Compositional contrast is accessible in scanning transmission EM (STEM) via incoherent elastic scattering, which is sensitive to the atomic number Z. STEM can be regarded as a high energy, low angle diffraction measurement performed pixel by pixel with a weakly convergent beam. When coherent diffraction effects are absent, that is, in amorphous materials, a dark field signal measures quantitatively the flux scattered from the specimen integrated over the detector area. Learning to interpret these signals will open a new dimension in cryo-EM. This Account describes our efforts so far to introduce STEM for cryo-EM and tomography of biological specimens. We conclude with some thoughts on further developments.

Melissopalynology is an important analytical method to identify botanical origin of honey. Pollen grain recognition is commonly performed by visual inspection by a trained person. An alternative method for visual inspection is automated pollen analysis based on the image analysis technique. Image analysis transfers visual information to mathematical descriptions. In this work, the suitability of three microscopic techniques for automatic analysis of pollen grains was studied. 2D and 3D morphological characteristics, textural and colour features, and extended depth of focus characteristics were used for the pollen discrimination. In this study, 7 botanical taxa and a total of 2482 pollen grains were evaluated. The highest correct classification rate of 93.05% was achieved using the phase contrast microscopy, followed by the dark field microscopy reaching 91.02%, and finally by the light field microscopy reaching 88.88%. The most significant discriminant characteristics were morphological (2D and 3D) and colour characteristics. Our results confirm the potential of using automatic pollen analysis to discriminate pollen taxa in honey. This work provides the basis for further research where the taxa dataset will be increased, and new descriptors will be studied.

Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an "open top" for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans.

Dark-field microscopy (DFM) based on localized surface plasmon resonance (LSPR) was used for observation of experimental phenomena, which is a hopeful nondamaging and non-photobleaching biological imaging technique. In this strategy, plasma nanoaggregates with stronger scattering efficiency were formed in the presence of the target, causing a "turn-on" phenomenon, when asymmetry modified AuNPs were introduced as probes with zero LSPR background. First, Au1-N3 probe and Au2-C≡C probe were designed for the cycloaddition between azide and alkyne to form AuNP dimers under catalytic action by Cu+, which was obtained from the reduction of Cu2+ by sodium ascorbate. The two kinds of probes were successfully used for the detection of Cu2+ in rat serum. Then, to apply this concept to protein on cells, DNA and antibody were modified on the probes. DNA1/Au1-N3 probe and anti-HER2/Au2-C≡C probe were proposed for HER2 protein DFM on cells. By designing an aptamer sequence in primer, the rolling circle amplification (RCA) was introduced in HER2 DFM on cells, and the image signal was much brighter than that from no-RCA. The unique design made it easier to discriminate the target signal from background noise in cell DFM. This method might be used in the fields of molecular diagnostics and cell imaging.

Rational: p53 is suppressing tumor protein correlated with the cell cycle factors and apoptosis. Here, a gold nanoparticle bouquet is designed for an ultrasensitive dark-field imaging approach for cancer cell analysis. Methods: AuNP60/APBA is functionalized by a gold nanoparticle bouquet-plasmonic 60 nm gold nanoparticles. And consistent APBA can be held on the plasma membrane. After 13 nm gold nanoparticles are functionalized with mannose (AuNP13/MN), the AuNP60/APBA gold nanoparticles are captured. The absorption spectrum of aggregation gold nanoparticles (AuNPs) shifts to near-infrared (NIR) region which can be observed under dark-field microscopy (DFM) and is treated the subsequent with photothermal therapy. Results: The results that MCF-7 cells were successfully destroyed under the near-infrared (NIR) irradiation and the intracellular WTp53 increased while the MTp53 decreased. These results indicated that p53 is the key molecule in the apoptosis signaling pathway. Photothermal therapy can stimulate the MTp53 in the cell signal conductive pathway. Conclusion: This work offers a new method for intracellular p53 analysis and a potential targeted cancer treatment.

Cells and cell like particles detection and segmentation are of significant interest to many biological and clinical studies. Traditionally, these tasks are usually performed by visual inspection, which is time consuming, labor intensity and prone to induce subjective bias between different people. These make automatic cell analysis protocols essential for large-scale and objective studies. In recent years, imaging technical has been significantly advanced following the great success by computer vision. In addition, these technologies enable the cross module microscopy analysis, and make the task of cell analysis extremely challenging. Over these decades, computer aided cell detection, counting and segmentation have evolved from earlier filter based methods to the state-of-art deep learning protocols. However, there are still few suitable frameworks that can process multiple source cell images at the same time. In this paper, we seek a different route and propose a novel efficient framework for robust cell analysis based on Long Short Term Memory Channeled Fully Convolution Neural Networks (LSTM-CFCN). The results demonstrates that our framework is able to perform most of cell detection, counting and segmentation tasks from different cell type, and it can also cover most kinds of microscopy images scenarios including dark field, bright field, pathological and electron images. We have perform substantial experiments on several benchmark datasets, the LSTM-CFCN achieves the highest or at least top-2 performance in terms of F1-score, compared with other state-of-the-art methods.

The redox reaction between Ag and I2 was investigated by the localized surface plasmon resonance (LSPR) technique. The different color ratio and scattering intensity of Au@Ag nanocubes (Au@Ag NCs) probe were analyzed under the dark-field microscopy (DFM) after addition of different concentrations of I–/I2. Furthermore, transmission electron microscopy (TEM) and UV-Visible (UV-Vis) absorption spectra showed that Au@Ag NCs were continuously oxidized from the edges, became spherical gradually, and finally quasi-spherical. Finally, Au@Ag NCs were used as probes to test I2 and the detection range was from 0.1 μM to 50.0 μM with detection limit of 17 nM. Moreover, this method was applied to detect I2 in rat brain dialysate and explore the redox reaction process between Au@Ag NCs and I–/I2. This work provided a new highly sensitive method to detect I2 in brain rat dialysate with recover method.

Electrochemistry has been developed toward high spatial resolution and throughput during the past decades to match the growing demands for studying heterogeneous electrodes and nanomaterials that are widely used in electrocatalysis, electroanalysis and energy conversion. Combining optical methods with electrochemistry enables us to obtain local electrochemical information via a noninvasive way. Optical methods also possess high throughput, which are beneficial to investigate large amounts of nanoelectrodes or nanocatalysts simultaneously, and to develop electrochemical multiplex sensors. This article critically reviews the optical methods that have been developed with spatial resolution for imaging electrochemical reactions and processes in the nanometer-scale. Recent development on these methods, such as plasmonics-based electrochemical current microscopy (PECM), dark field microscopy (DFM), Raman spectroscopy, fluorescence microscopy (FLM) and electrogenerated chemiluminescence (ECL), and research progress in the relevant fields, particularly nanocatalysis and single particle/molecule electrochemistry, will be briefly overviewed. Finally, current limitations and trends of these optical methods and potential strategies to further improve the spatial resolution of electrochemistry are discussed.

The evolution of radiation-induced defects in Ni and the single-phase concentrated solid solution alloy, NiCoCr, were investigated during in situ 2.8 MeV Au ion irradiation and post-irradiation analysis using transmission electron microscopy. Compared to Ni, both the size and area density of defect clusters decreased in NiCoCr under the same irradiation conditions, suggesting that the chemical complexity, i.e., randomness of lattice site occupations, of NiCoCr suppressed radiation-induced damage. One-dimensional glide of defect clusters was observed in Ni but not in the NiCoCr alloy. The structural nature of small defect clusters in NiCoCr were further investigated using high-angle annular dark field scanning transmission electron microscopy.

Assessing tissues’ inhomogeneous optical properties is helpful for diagnosis, but high-cost measurement and experimental setups limit its development, data collecting and applications. In this paper, a portable microscope is proposed to assess the inhomogeneous optical properties of the sample. In the single LED illumination mode, accurate quantitative phase (QP) map can be recovered from 5 intensity images captured at different axial positions. Then based on the scattering-phase theorem and statistical dispersion relation (SDR), the inhomogeneous optical properties of the sample can be quantitatively assessed from these QP maps. In the incoherent dark field illumination mode, strong scattering structures can be observed easily without post processing. An improved resolution comparing with bright field imaging and qualitative optical properties would be obtained. In contrast to DHM and SLIM, our setup is cost-effective, use-flexible, and with a small amount of data acquisition, thus having the potential to promote the development of assessing tissues’ inhomogeneous optical properties, especially in resource-limited areas.

Here, we report an ultrasensitive colorimetric method for determination of Ag+ based on gold nanoparticle (AuNP) oligomers with dark-field microscopy. In a proof-of-concept trial, two cytosine-rich (C-rich) aptamers as the sensing elements and AuNPs as the transducer to produce the detectable signal are designed for Ag+ determination. Obviously, the C bases in the two aptamers play a crucial role in the coordination of highly specific C–Ag+–C. Through a specific C–Ag+–C coordination, one aptamer immobilized on the surface of AuNPs sitting on a glass slide hybridizes with the other aptamer in the solution, which triggers the formation of AuNP oligomers. The formation of plasmonic oligomers results in an obvious increase in the number of yellow and red spots in dark-field images, further leading to a significantly enhanced scattering intensity. By measuring the intensity change of AuNP oligomers in the dark-field images, the concentration of Ag+ can be readily determined. The limits of detection (LODs) of 0.246 pM and 0.388 pM in solution and river water sample were obtained, respectively. This ultrasensitive colorimetric approach could be extended to probe Hg2+ via T–Hg2+–T coordination in environmental samples.

A new microscopic principle based on radiometric stereo microscopy is presented, which is designed for investigating macro-dispersion of filler in rubber. The image acquisition is combined with a stereological method of estimating the volume-weighted size distribution of the filler particles. Experimental results for carbon black filler in rubber obtained by radiometric stereo microscopy are compared with those from microtomography using synchrotron radiation, and, furthermore, a simulation study is used for evaluation. It turns out that using the new three-dimensional microscopic method, the size distribution of the filler particles can be estimated from fresh cuts of rubber with high accuracy, and thus it is an interesting alternative to well-established dark field microscopy. LAY DESCRIPTION: Macro-dispersion of globular filler particles in a rubber matrix is an important quantity that depends on manufacturing parameters and influences various rubber properties. Therefore, it must be carefully adjusted during the incorporation process and investigated by industrial quality control (ASTM D7723-18). Quality control is usually based on freshly made planar sections so-called fresh cuts through rubber specimen. After stress retention of the rubber one obtains a rough cutting surface in which the filler particles appear as imprints or bumps, called nodges. These nodges can be made visible by classical light microscopy under dark field (DFM) illumination. The systems disperGRADER+ or the disperGRADER Alpha View were specifically designed for rubber inspection. However, it has proved to be very difficult estimating the size distribution of the filler particles from the observed white spots in the DFM image. In any case it is still necessary to compute the size distribution of the filler particles from an estimated size distribution of the section profiles. The latter is numerically unstable, i.e. small errors of the estimated size distribution of the section profiles lead to large errors of the computed filler size distribution. Applying DFM combined with filler dispersion estimation as described in ASTM D7723-18 appears to be a fingerprint method only. For this reason, the new microscope nSPEC 3D was applied for rubber inspection. The principle used for surface imaging is based on radiometric stereo allowing for perfect three-dimensional reconstruction of curved surfaces of fresh cuts. From this reconstruction it is possible to measure the height of particle nodges as well as their volumes. Furthermore, we present a new stereological method for estimating the filler size distribution from samples of the height and the volume of the nodges. Finally, microtomography with synchrotron radiation and computer simulation are applied to evaluate accuracy of the presented method.

A significant hurdle in conducting effective health and safety hazard analysis and risk assessment for the nanotechnology workforce is the lack of a rapid method for the direct visualization and analysis of filter media used to sample nanomaterials from work environments that represent potential worker exposure. Current best-known methods include transmission electron microscopy (TEM) coupled with energy dispersive x-ray spectroscopy (EDS) for elemental identification. TEM-EDS is considerably time-, cost-, and resource-intensive, which may prevent timely health and safety recommendations and corrective actions. A rapid screening method is currently being explored using enhanced darkfield microscopy with hyperspectral imaging (EDFM-HSI). For this approach to be effective, rapid, and easy, sample preparation that is amenable to the analytical technique is needed. Here, we compare the sample preparation steps for mixed cellulose ester (MCE) filter media specified in NIOSH Method 7400-Asbestos and Other Fibers by Phase Contrast Microscopy (PCM)-against a new method, which involves saturation of the filter media with acetone. NIOSH Method 7400 was chosen as a starting point since it is an established technique for preparing transparent MCE filters for optical microscopy. Limitations in this method led to the development and comparison of a new method. The new method was faster, easier, and rendered filters more transparent, resulting in improved visualization and analysis of nanomaterials via EDFM-HSI. This new method is suitable for a rapid screening protocol due to its speed, ease of use, and the improvement in image acquisition and analysis.

Cell staining techniques are well established in cell biology and associated with a broad range of dedicated dyes; however, they are accompanied by non-negligible costs, preparation time and unavoidable alterations of the sample with foreign molecules. In this context, we point out and propose the use of darkfield microscopy (DM) combined with different fixation protocols (to be used anyway) to enhance the different cell structures and districts as a timesaving and inexpensive support to the techniques that need staining or immuno-staining protocols and products. In a first step, we have analysed the effect of different fixation protocols on DM images for various human cellular lines. The presented imaging study shows that cell morphology actually changes with the fixation protocols that enhance, through contrast and luminosity variations, different shapes and patterns and thus structures of the cells. The different chemical action of various fixations, in fact, modifies the local scattering coefficient, thus affecting in a different way the morphology shown by DM images. As a second step we have compared the observed DM morphologies to those of selective fluorescent staining being therefore able to associate them to specific cell districts (e.g. nucleus, membrane or cytoskeleton). The obtained results indicate that this common microscopy technique can give images with particular cellular structures or districts enhanced more than others depending on the choice of fixation protocol. Therefore Darkfield Microscopy can be considered as a simpler, cheaper and faster method to provide morphological indications, respect to staining techniques, even at low and medium magnifications.

Optical imaging represents one of the most essential tools in biological studies. Although with great advances, bio-optical imaging still suffers from problems such as resolution, sensitivity, speed, and penetration depth. Due to the unique optical properties of gold nanoparticles (AuNPs), i.e., surface plasmon resonance, AuNPs can be readily used to enhance optical imaging based on their absorption, scattering, fluorescence, Raman scattering, etc. Here, we include the most recent achievements and challenges associated with using AuNPs to improve resolution and sensitivity in biological imaging in vitro and in vivo. The application of AuNPs in the following three aspects were discussed: 1) Direct visualization of AuNPs inside the biosystems using i) dark field (DF) microscopy, ii) differential interference contrast (DIC) microscopy, and iii) other techniques, such as interferometric scattering (iSCAT) microscopy and photothermal imaging. Additionally, since orientation and rotational motions are closely related to various biological processes, we also summarized the recent advances of optical imaging methods in the rotational and orientation tracking of AuNPs. 2) Monitoring of biomolecular events and physiological processes using i) surface-enhanced Raman spectroscopy (SERS) and ii) plasmon enhanced fluorescence (PEF) for ultra-sensitive detection of biomolecules, including proteins, metabolites, DNA, RNA, etc. 3) In vivo deep tissue imaging using i) two-photon and/or multi-photon imaging, ii) optical coherence tomography (OCT), and iii) photoacoustic (PA) imaging for disease diagnoses, such as detecting tumors and other diseases in eye, brain, and bone. In conclusion, based on our literature study, AuNPs-assisted bioimaging acts as a promising tool in exploring fundamental biological questions and early diagnosis of diseases.

In clinical diagnosis, the level of biological enzymes in serum has been generally regarded as markers of human diseases. In this work, a kind of simple and sensitive plasmonic probe (indicated as Au@FeOOH) has been synthesized with the guidance of plasmonic imaging and subsequently developed for the alkaline phosphatase (ALP) level detection under dark-field microscopy (DFM). As a kind of hydrolysis enzyme, ALP can promote the hydrolysis of l-ascorbic acid 2-phosphate to ascorbic acid (AA). AA further acts as a strong reduction reagent for the decomposition of the FeOOH shell, which results in a blue shift of localized surface plasmon resonance spectra and an obvious color change under DFM. RGB analyses show that using a ΔR/G value instead of scattering wavelength or R/G value as the analytical signal, the deviation attributed to the size distribution of the initial Au NPs is greatly suppressed, and a linear range from 0.2 to 6.0 U/L (R2 = 0.99) and a limit of detection of 0.06 U/L are acquired with various concentrations of ALP during the detection. Besides, this approach exhibits excellent selectivity in complex biological serum samples, which is expected to be applied for the early diagnosis of clinical diseases by monitoring various biomarkers in the future.

Leptospirosis is a zoonosis caused by the pathogenic bacterium Leptospira. The Microscopic Agglutination Test (MAT) is widely used as the gold standard for diagnosis of leptospirosis. In this method, diluted patient serum is mixed with serotype-determined Leptospiras, and the presence or absence of aggregation is determined under a dark-field microscope to calculate the antibody titer. Problems of the current MAT method are 1) a requirement of examining many specimens per sample, and 2) a need of distinguishing contaminants from true aggregates to accurately identify positivity. Therefore, increasing efficiency and accuracy are the key to refine MAT. It is possible to achieve efficiency and standardize accuracy at the same time by automating the decision making process. In this study, we built an automatic identification algorithm of MAT using a machine learning method to determine aggregation within microscopic images. The machine learned the features from 316 positive and 230 negative MAT images created with sera of Leptospira- infected (positive) and non-infected (negative) hamsters, respectively. In addition to the acquired original images, wavelet-transformed images were also considered as features. We utilized a support vector machine (SVM) as a proposed decision method. We validated the trained SVMs with 210 positive and 154 negative images. When the features were obtained from original or wavelet-transformed images, all negative images were misjudged as positive, and the classification performance was very low with sensitivity of 1 and specificity of 0. In contrast, when the histograms of wavelet coefficients were used as features, the performance was greatly improved with sensitivity of 0.99 and specificity of 0.99. We confirmed that the current algorithm judges the positive or negative of agglutinations in MAT images and gives the further possibility of automatizing MAT procedure.

We present the computational technique for automatic numerical refocusing in coherent lensless Gabor microscopy (digital in-line holographic microscopy). It is based on the adaptive filtering of the recorded on-axis Gabor hologram to eliminate its incoherent background term and extract interference fringes determined by the light scattered on the sample. Numerical propagation of such filtered hologram, based on the angular spectrum method, yields the computationally generated dark-field imaging realized in amplitude channel of the complex propagated f ield. As the focus metric we calculate the variance of the dark-field gradient - it attains maximum value in the focal planes for all types of objects (phase, amplitude and mixed phase-amplitude). Demonstrated autofocusing technique is positively validated using experimental data exhibiting significant variation of the confluence for double focal plane scenarios (two closely located sample planes filled with microbeads). Described technique compares favorably with other well-established automatic numerical refocusing methods (e.g., based on the high-pass filtered complex amplitude and edge sparsity) mainly in terms of higher axial resolution and better robustness to hologram low signal-to-noise ratio and object non-uniformity.

12 pags., 6 figs., 4 tabs., The diatoms have intricate silica-based cell walls with multi-scale patterns. High dynamic range (HDR) imaging is widely used to examine the three-dimensional structure of diatoms for recovering the wide range of contrast and brightness. In order to construct a HDR image of a diatom, multiple images of the specimen are taken at different exposure settings with bright or dark field microscopy. In the proposed method, multi-scale decomposition based on nonsubsampled contourlet transform is adopted to separate the structured and detailed information of the HDR image. And then, by processing all layers independently, the tone-mapped image is reconstructed to retain details present in the dark and light regions. Quantitative and qualitative analysis is performed in order to assess the performance of the proposed and seven existing tone-mapping operators. In analysis, the study indicates that the proposed method enhances the diatom frustules to extract more details., Spanish Government, Grant/Award Number:CTM2014-51907-C2-2- R-MINECO

In light transmission microscopy, axial scanning does not directly provide tomographic reconstruction of specimen. Phase deconvolution microscopy can convert a raw intensity image stack into a refractive index tomogram, the intrinsic sample contrast which can be exploited for quantitative morphological analysis. However, this technique is limited by reconstruction artifacts due to unoptimized optical conditions, which leads to a sparse and non-uniform optical transfer function. Here, we propose an optimization method based on simulated annealing to systematically obtain optimal illumination schemes that enable artifact-free deconvolution. The proposed method showed precise tomographic reconstruction of unlabeled biological samples.

Single particle tracking has found broad applications in the life and physical sciences, enabling the observation and characterization of nano- and microscopic motion. Fluorescence-based approaches are ideally suited for high-background environments, such as tracking lipids or proteins in or on cells, due to superior background rejection. Scattering-based detection is preferable when localization precision and imaging speed are paramount due to the in principle infinite photon budget. Here, we show that micromirror-based total internal reflection dark field microscopy enables background suppression previously only reported for interferometric scattering microscopy, resulting in nanometer localization precision at 6 μs exposure time for 20 nm gold nanoparticles with a 25 × 25 μm2 field of view. We demonstrate the capabilities of our implementation by characterizing sub-nanometer deterministic flows of 20 nm gold nanoparticles at liquid–liquid interfaces. Our results approach the optimal combination of background suppression, localization precision, and temporal resolution achievable with pure scattering-based imaging and tracking of nanoparticles at interfaces., ACS Photonics, 8 (10), ISSN:2330-4022

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

This proof-of-concept study shows that dark-field microscopy provides sufficient contrast for cell visualization in fingerprints with high sebum content. Although the application is limited to smooth surfaces that do not scatter light, such as polyethylene terephthalate (PET), it was able to measure the number of cells deposited within a fingerprint residue and the reduction in cell transfer with repeated skin contact. On a PET surface, at roughly 5 N of contact force, a typical finger transfers several hundred cells onto the surface. Over subsequent finger contacts onto a clean PET surface, this number decreased exponentially until a steady state was reached, which is characterized by the transfer of (78 ± 36) cells or (0.46 ± 0.21) cells/mm2 when normalized for fingerprint area. High uncertainty in cell transfer was due to: the highly variable nature of a human finger (where the number of loose cells varies from person to person and from day to day depending on what they touch) and difficulties in controlling the contact force and finger movement such as twisting during deposition (where twisting of the finger can expose a new patch of skin to the substrate, increasing the number of cell transfer). Plasma etching was also explored as an effective way to validate dark-field microscopy for cell counting. Although limited to inorganic substrates due to etching effects, exposing the fingerprint for less than 10 min can remove a majority of the sebum while keeping the cells intact for a before-and-after comparison using light microscopy.

Materials 14(16), 4531 (2021). doi:10.3390/ma14164531, Published by MDPI, Basel

Interferometric imaging is an emerging technique for particle tracking and mass photometry. Mass or position are estimated from weak signals, coherently scattered from nanoparticles or single molecules, and interfered with a co-propagating reference. In this work, we perform a statistical analysis and derive lower bounds on the measurement precision of the parameters of interest from shot-noise limited images. This is done by computing the classical Cramér–Rao bound (CRB) for localization and mass estimation, using a precise vectorial model of interferometric imaging techniques. We then derive fundamental bounds valid for any imaging system, based on the quantum Cramér–Rao formalism. This approach enables a rigorous and quantitative comparison of common techniques such as interferometric scattering microscopy (iSCAT), coherent brightfield microscopy, and dark-field microscopy. In particular, we demonstrate that the light collection geometry in iSCAT greatly increases the axial position sensitivity, and that the Quantum CRB for mass estimation yields a minimum relative estimation error of σ m / m = 1 / ( 2 N ) , where N is the number of collected scattered photons.

Significance: Currently, tissue biopsies are sectioned into 3- to 5-μm-thick slices that are used for conventional pathology analysis. Previous work by confocal microscopy and light-sheet microscopy has shown that analyzing biopsies intact in three-dimensions (3D) is possible and may lead to a better understanding of cancer growth patterns. Although accurate, these methods require fluorescent staining of the tissue, in addition to tissue clearing. If the 3D biopsy analysis could be done sufficiently swiftly, this approach may be used for on-site assessment of the adequacy of a biopsy taken. Aim: We aim to show that, by transmission microscopy of optically cleared tissue punches, the tissue architecture can be determined without the need for fluorescent staining. Approach: Transmission microscopy is used by combining bright field microscopy with dark field and epifluorescent microscopy to compare samples that have also been analyzed by fluorescent confocal microscopy. Results: With increasing distance to the focal plane, the higher-frequency part of the spatial frequency spectrum of transmitted light is attenuated increasingly. This property is exploited for tissue segmentation, detecting whether tissue is present at a certain position in the focal plane image. Using this approach, we show that a 3D rendering of the internal cavity or tubules structure of punch biopsies, which are up to 1-mm thick, can be acquired in ≈1 min scan time per imaging modality. The images of the overall tissue architecture that are obtained are similar to those from the confocal microscopy benchmark, without requiring fluorescent staining. Conclusions: Images of the overall tissue architecture can be obtained from transmission microcopy; they are similar to those from the confocal microscopy benchmark without requiring fluorescent staining. Tissue clearing is still needed. The total scan time of the present method is significantly shorter at a fraction of the device costs.

We present for the first time a lens-free, oblique illumination imaging platform for on-sensor dark- field microscopy and shadow-based 3D object measurements. It consists of an LED point source that illuminates a 5-megapixel, 1.4 µm pixel size, back-illuminated CMOS sensor at angles between 0° and 90°. Analytes (polystyrene beads, microorganisms, and cells) were placed and imaged directly onto the sensor. The spatial resolution of this imaging system is limited by the pixel size (∼1.4 µm) over the whole area of the sensor (3.6×2.73 mm). We demonstrated two imaging modalities: (i) shadow imaging for estimation of 3D object dimensions (on polystyrene beads and microorganisms) when the illumination angle is between 0° and 85°, and (ii) dark-field imaging, at >85° illumination angles. In dark-field mode, a 3-4 times drop in background intensity and contrast reversal similar to traditional dark-field imaging was observed, due to larger reflection intensities at those angles. With this modality, we were able to detect and analyze morphological features of bacteria and single-celled algae clusters.

Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen. Monitoring sortase activity and screening its inhibitors are of great significance to fundamental understanding of the infection mechanism and pharmaceutical development. Herein, we developed a digital single-particle imaging method to quantify sortase A (SrtA) activity based on transpeptidation-mediated assembly and enumeration of gold nanoparticles (GNPs). The assay utilizes two peptide stands, in which one has the SrtA recognition sequence LPXTG motif while the other carries an oligoglycine nucleophile at the one end and a biotin group at the other. The presence of SrtA enables the ligation of two peptides and allows for the immobilization of streptavidin-functionalized GNPs. Thus, SrtA activity can be quantified by imaging and enumeration of the surface-assembled GNPs at the single-particle level via dark-field microscopy. The single-particle method was highly sensitive to SrtA activity with a low detection limit of 7.9 pM and a wide linear dynamic range from 0.05 to 50 nM. Besides detection of SrtA in complex biological samples such as Gram-positive pathogen lysates, the proposed method was also successfully applied to estimate the half-maximal inhibitory concentration (IC50) values of SrtA inhibitors (curcumin, berberine hydrochloride and quercetin). The present method, combining single-GNP counting and dark-field imaging, provides a facile and novel analytical tool for SrtA activity and its inhibitor screening.