Traditional Optical Microscopy Research Articles

Achieving more accurate microindentation hardness measurements of shale rocks – Insight into the effect of sink-in and pile-up

Shale rock has become a major source of oil and natural gas production. Hence, understanding the mechanical properties of shales is important for field applications, such as drilling and fracking. In this work, a workflow was developed for assessing the hardness of shale rocks, with the kerogen-rich and calcite-rich shales as the model systems, using Vickers microindentation coupled with confocal microscopy. The use of confocal microscopy facilitates the direct observation of indent impressions, overcoming the challenges posed by the complex contrasts of shales seen in traditional optical microscopy. Sink-in and substantial elastic recovery were observed in kerogen-rich shales due to the large amount of organic content. However, sink-in did not affect the hardness calculation. Apparent pile-up was observed in calcite-rich shales due to its granular nature, which could lead to overestimation of hardness if not accounted for. The hardness measurement in calcite-rich shale is further complicated by spallation. Therefore, only hardness ranges, instead of individual hardness values, can be obtained in calcite-rich shales. This research not only refines the methodology for determining the hardness of shales but also provides insight into indentation artifacts, such as sink-in and pile-up, to improve the accuracy in measuring and analyzing the microhardness of complex geological materials.

Miniaturized quantitative detection of particles in transformer oil based on lensless holographic microscopy

The insulation effectiveness of transformer oil has a substantial impact on the safety of transformers. In the presence of particles within the transformer oil, they are able to adversely affect its performance, potentially originating from electrical arcs or severe carbonization processes. These particles are commonly capable of reducing the insulating capability of the transformer oil, thereby enhancing the risk of transformer failure. To overcome this dilemma, we propose a miniaturized quantitative particle detection approach in transformer oil based on a lensless digital holographic microscope. In general, methods based on traditional high-resolution optical microscopy are essentially limited by their fairly huge cost and importability. However, the proposed approach utilizes a homemade miniaturized model combined with a CMOS image sensor to achieve microscopic imaging. The deployed device here weighs ∼ 50 g, making it portable and affordable for on-the-go detection. By simplifying the microscope system, we can quickly and accurately detect tiny particles in transformer oils. This enables us to efficiently assess the status of impurities in power transformers and to identify potential issues in advance, thereby enhancing the reliability of transformer oil. This detection method not only features simplicity of operation, convenient on-the-go detection, low cost, and high precision but also provides a convenient approach for accurately assessing the condition of impurity particles in power transformers and preemptively identifying potential issues.

DHM/SERS reveals cellular morphology and molecular changes during iPSCs-derived activation of astrocytes.

The activation of astrocytes derived from induced pluripotent stem cells (iPSCs) is of great significance in neuroscience research, and it is crucial to obtain both cellular morphology and biomolecular information non-destructively in situ, which is still complicated by the traditional optical microscopy and biochemical methods such as immunofluorescence and western blot. In this study, we combined digital holographic microscopy (DHM) and surface-enhanced Raman scattering (SERS) to investigate the activation characteristics of iPSCs-derived astrocytes. It was found that the projected area of activated astrocytes decreased by 67%, while the cell dry mass increased by 23%, and the cells changed from a flat polygonal shape to an elongated star-shaped morphology. SERS analysis further revealed an increase in the intensities of protein spectral peaks (phenylalanine 1001 cm-1, proline 1043 cm-1, etc.) and lipid-related peaks (phosphatidylserine 524 cm-1, triglycerides 1264 cm-1, etc.) decreased in intensity. Principal component analysis-linear discriminant analysis (PCA-LDA) modeling based on spectral data distinguished resting and reactive astrocytes with a high accuracy of 96.5%. The increase in dry mass correlated with the increase in protein content, while the decrease in projected area indicated the adjustment of lipid composition and cell membrane remodeling. Importantly, the results not only reveal the cellular morphology and molecular changes during iPSCs-derived astrocytes activation but also reflect their mapping relationship, thereby providing new insights into diagnosing and treating neurodegenerative diseases.

Super-Resolution Imaging of Sub-diffraction-Limited Pattern with Superlens Based on Deep Learning

The development of super-resolution imaging techniques has revolutionized our ability to study the nano-scale world, where objects are often smaller than the diffraction limit of traditional optical microscopes. Super-resolution superlenses have been proposed to solve this problem by manipulating the light wave in the near field. A superlens is a kind of metamaterial-based lens that can enhance the evanescent waves generated by nano-scale objects, utilizing the surface plasmon phenomenon. The superlens allows for the imaging of nano-scale objects that would otherwise be impossible to resolve using traditional lenses. Previous research has shown that nanostructures can be imaged using superlenses, but the exact shape of the superlens must be known in advance, and an analytical calculation is needed to reconstruct the image. Localized plasmon structured illumination microscopy is an approach to achieve super-resolution by imaging the superlens-enhanced evanescent wave with illumination shifts. This study proposes a new approach utilizing a conditional generative adversarial network to obtain super-resolution images of arbitrary nano-scale patterns. To test the efficacy of this approach, finite-difference time-domain simulation was utilized to obtain superlens imaging results. The data from the simulation were then used for deep learning to develop the model. With the help of deep learning, the inverse calculation of complex sub-diffraction-limited patterns can be achieved. The super-resolution feature of the superlens based on deep learning is investigated. The findings of this study have significant implications for the field of nano-scale imaging, where the ability to resolve arbitrary nano-scale patterns will be crucial for advances in nanotechnology and materials science.

Direct Observation of Carbonate Chemisorption on Barium Titanate Surfaces by Tip‐Enhanced Raman Spectroscopy

AbstractSurface chemistry significantly influences the physicochemical and functional properties of nanoparticles, thus necessitating the investigation of surfaces, especially when subjected to chemisorption. Seeking a method that delivers both high‐resolution imaging and precise chemical information, tip‐enhanced Raman spectroscopy (TERS) emerges as a critical technique. By the combination of scanning probe microscopy with Raman spectroscopy, TERS effectively addresses the limitations of traditional optical microscopes in the study of nanoparticles. The results provide detailed nanoscale structural insights, shed light on surface chemistry, and enable the detection of Raman‐forbidden modes caused by the substantial electric field at the tip apex (≈3 × 108 V m−1). This electric field plays a crucial role in altering selection rules, thereby broadening the scope of TERS beyond merely detecting Raman modes. By combining topographical with TERS mapping, the presence of a carbonate monolayer on the barium titanate (BaTiO3) nanoparticle surface is unveiled – an observation elusive to common characterization techniques.

Demonstration of computer vision for void characterisation of 3D-printed continuous carbon fibre composites

Microstructural void formation during the processing of continuous fibre-reinforced polymer composites are a significant limitation of processes such as 3D printing, as voids inhibit resultant mechanical properties. Traditional optical microscopy approaches to void characterisation are tedious and are prone to errors due to non-ideal but realistic image conditions, including low contrast typical of microscopic images. This paper proposes a novel application of automated computer vision image processing techniques (i.e. a contrast-limited adaptive histogram equalisation (CLAHE) algorithm and Otsu's method) for detailed void characterisation of continuous fibre-reinforced composites. Microstructural void fraction was determined for both unidirectional and bi-axial laminates of a variety of thicknesses. The void characterisation by computer vision was shown to produce comparable results with manual contrast/brightness control, but with significantly less standard deviation. The computer vision software used in void characterisation is made freely available online at https://github.com/Xiao-ying/VoidDetector.

A simplified version of rapid susceptibility testing of bacteria and yeasts using optical nanomotion detection.

We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or antifungals on the nanometric scale displacements of bacteria or yeasts to assess their sensitivity or resistance to drugs. The technique relies on a traditional optical microscope, a video camera, and custom-made image analysis software. It provides reliable results in a time frame of 2-4 h and can be applied to motile, non-motile, fast, and slowly growing microorganisms. Due to its extreme simplicity and low cost, the technique can be easily implemented in laboratories and medical centers in developing countries.

Automating parasite egg detection: insights from the first AI-KFM challenge.

In the field of veterinary medicine, the detection of parasite eggs in the fecal samples of livestock animals represents one of the most challenging tasks, since their spread and diffusion may lead to severe clinical disease. Nowadays, the scanning procedure is typically performed by physicians with professional microscopes and requires a significant amount of time, domain knowledge, and resources. The Kubic FLOTAC Microscope (KFM) is a compact, low-cost, portable digital microscope that can autonomously analyze fecal specimens for parasites and hosts in both field and laboratory settings. It has been shown to acquire images that are comparable to those obtained with traditional optical microscopes, and it can complete the scanning and imaging process in just a few minutes, freeing up the operator's time for other tasks. To promote research in this area, the first AI-KFM challenge was organized, which focused on the detection of gastrointestinal nematodes (GINs) in cattle using RGB images. The challenge aimed to provide a standardized experimental protocol with a large number of samples collected in a well-known environment and a set of scores for the approaches submitted by the competitors. This paper describes the process of generating and structuring the challenge dataset and the approaches submitted by the competitors, as well as the lessons learned throughout this journey.

Initial institutional experience using a robotic arm-enabled 4K 3D exoscope in neurosurgical operations.

The extracorporeal telescope (exoscope) presents a novel digital camera system as a versatile alternative to traditional optical microscopy for microsurgery and minimally invasive neurosurgical operations. Recent innovations in exoscope technology offer 4K-definition multiscreen outputs, pneumatic robot arms, 3-dimensional depth perception, and greater illumination, focus, and magnification powers for enhanced intraoperative visualization. The authors present their initial institutional experience using a robotic arm-enabled 4K 3D exoscope in a variety of cranial and spinal neurosurgical operations, namely Chiari decompression, microvascular decompression for trigeminal neuralgia, anterior cervical discectomy, and lumbar decompressions. The video can be found here: https://stream.cadmore.media/r10.3171/2023.10.FOCVID23150.

Label‐Free Imaging of Low‐Index Samples Using Substrate‐Enhanced Subwavelength‐Resolution Scattering Nanoscopy

AbstractThere is an increasing demand for label‐free imaging of subwavelength low‐index dielectric samples with high contrast and high sensitivity using a dark‐field optical microscope. However, subwavelength low‐index dielectric samples usually do not show strong Mie scattering resonance characteristics due to the weak light confining ability, which makes them difficult to clearly resolve under a traditional optical microscope. In this work, a fast, simple, and effective approach is proposed to enhance the optical response of subwavelength low‐index dielectric samples by using metallic substrate‐enhanced scattering nanoscopy. It is found that the high reflectance of a substrate can significantly enhance the electric dipole resonance of a SiO2 nanoparticle and the substrate‐nanoparticle interaction, which amplifies the scattering signal of the sample and improves the imaging contrast. Compared with the silica substrate, the Ag‐coated substrate can enhance the scattering intensity of a SiO2 nanoparticle up to 3.7 times under 540 nm wavelength illumination. Furthermore, label‐free stand‐alone 50‐nm‐radius SiO2 nanoparticles and ≈100‐nm‐thick bacterial capsules can be clearly observed when placed on Ag‐coated substrates under a conventional dark‐field optical microscope. The substrate‐enhanced subwavelength‐resolution scattering nanoscopy can enhance the scattering of low‐index subwavelength samples and benefit a significant application in biology, medical testing, and optical detection.

Role of Phase Information Propagation in the Realisation of Super-Resolution Based on Speckle Interferometry

Super-resolution technology is important not only in bio-related fields but also in nanotechnology, particularly in the semiconductor industry, where fine patterning is required and super-resolution is essential. However, observing microstructures beyond the diffraction limit proposed by Abbe and Rayleigh is considered impossible because of diffraction in traditional optical microscopy observation techniques. However, in recent years, it has been possible to observe microstructures beyond the Rayleigh criterion by analysing the phase distribution of light. This study investigated the physical reasons why phase analysis makes this new analysis technique possible using simulations. The results confirmed that the phase component of the zero-order diffracted light reflected from the microstructure and able to pass through the lens system contained phase information related to the shape of the measured object. Analysis of this information demonstrates the possibility of realising super-resolution based on speckle interferometry.

Candida albicans Adhesion Measured by Optical Nanomotion Detection

Cellular adhesion plays an important role in numerous fundamental physiological and pathological processes. Its measurement is relatively complex, requires sophisticated equipment, and, in most cases, cannot be carried out without breaking the links between the studied cell and its target. In this contribution, we propose a novel, nanomotion-based, technique that overcomes these drawbacks. The applied force is generated by the studied cell itself (nanomotion), whereas cellular movements are detected by traditional optical microscopy and dedicated software. The measurement is non-destructive, single-cell sensitive, and permits following the evolution of the adhesion as a function of time. We applied the technique on different strains of the fungal pathogen Candida albicans on a fibronectin-coated surface. We demonstrated that this novel approach can significantly simplify, accelerate, and make more affordable living cells–substrate adhesion measurements.

ACHIEVEMENTS IN PHYSICAL CHEMISTRY IN THE FIELD OF MICROSCOPY AND VISUALIZATION OF NANOSYSTEMS

The review presents modern views and the history of the development of microscopic studies of nanosystems which heve been started 2014, after the Nobel Prize in Chemistry was awarded to Eric Betzig, William Mörner, and Stefan Gell "for the development of super-resolved fluorescence microscopy". Their work ushered in a new era of optical microscopy, enabling the precise examination of individual molecules and molecular clusters by using optical microscopes. By circumventing the diffraction limitations that had constrained traditional optical microscopes, scientists gained access to the nanoscale realm, investigating structures within the 1–100 nanometer range. Special attention is paid to the use of carbon quantum dots and plasmon resonance to enhance fluorescence when obtaining the effect of super-resolution images, which allow the use of optical microscopes in the estimation of the sizes of cluster and single molecules. This breakthrough in removing the diffraction li­mitation allowed scientists to use the working range of 1–100 nm and obtain 3D images of nanosystems and images of living cells. Particular attention is paid to the achievements and prospects of high-resolution fluorescent nanoscopy SRM, which is successfully deve­lo­ping and studying the nanoworld in the range of 1–100 nm at the level of scanning electron microscopy. In cell biology, nanomedicine, etc. are developing roadmaps for scientific breakthroughs in super-resolution visualization me­thods for "live" images. Prospects of Immuno-­SERS microscopy and medicine of indivi­dual diagnosis are considered
 Key Findings:
 
 This article highlights the achievements and future prospects of super-resolution fluorescence microscopy SRM.
 High-resolution fluorescence microscopy has proven instrumental in advancing our understanding of the living world within the 1–100 nanometer range, which is akin to the capabilities of scanning electron microscopy.
 Within the domains of cell biology and nanomedicine, roadmaps for scientific breakthroughs are emerging, fueled by super-re­so­lution imaging techniques, providing "live" insights into cellular processes.
 The horizons of Immuno-SERS Microscopy and Personalized Diagnostics Medicine are expanding, promising exciting prospects in the field of medical diagnostics.

Evaluating Sand Particle Surface Smoothness Using a New Computer-Based Approach to Improve the Characterization of Macroscale Parameters

The analysis of sands, and the foundation systems with which they interact, are largely dependent on macroscale behavioral parameters that represent the aggregated response of several microscale characteristics. This research paper examines the influence of surface texture, or smoothness, on the behavior of sands. The challenge of estimating or measuring smoothness, due to its microscale feature domain, is addressed through an examination of six artificially graded sand specimens. These specimens are evaluated both visually and numerically to characterize their surface smoothness. The first approach described is a simple visual method that uses a smoothness scale consistent with those of roundness and sphericity. This method, which can be performed with a tool as simple as a hand lens, evaluates a group of representative particles collectively. The second approach is also a visual evaluation, but it utilizes images obtained via scanning electronic microscopy, traditional optical microscopy, and newer low-cost digital microscopes that can be rapidly connected to a smartphone or laptop. To validate these visual estimates, a novel third approach is introduced. This approach is a more objective numerical analysis measurement technique that enables rapid and economic quantification of smoothness. This technique may assist both practitioners and academics in their understanding of the macroscale response of coarse-grained soils. In addition to the visual methods, this research also conducted several laboratory index tests to observe the mechanical behavior of the specimens, considering their particle shape and surface smoothness properties. The results indicate that angular sands have greater minimum and maximum void ratios, a larger difference between the minimum and maximum void ratios, greater critical state friction angles, and greater flow rates through an orifice of fixed size. When adjusted for surface smoothness using the proposed approach, the behavior of the sands—particularly the limit void ratio results—appears to be more predictable in some cases. These results provide additional evidence of particle smoothness contributing to the strength behavior of sand, which may be particularly useful in the domains of slope stability, land reclamation, soil–structure interaction, and soil dynamics.

The Bioimaging Story of AIEgens.

Observations of the micro world, especially the structures of organelles, have been attractive topics since the 17th century. As a powerful detection tool, the fluorescence technique has played a significant role in bioimaging to provide more details and enhance the signal-to-noise ratio compared to that of traditional optical microscopes. The boom of aggregate-induced emission luminogens (AIEgens) in the last two decades has revolutionized the design strategy of luminescent materials for biological applications. This Review summarizes the advantages and recent progress of AIEgens in imaging and tracking. Different imaging strategies of AIEgens including turn-on imaging, stimuli-response sensing, and long-term tracking are presented. NIR AIEgens used for in-depth bioimaging via different methods are also discussed. Finally, we propose several potential development directions for AIEgens in bioimaging.

Minimally Invasive Glaucoma Surgery (MIGS) using a 3D Digital Surgical Ophthalmic Exoscope

Intraoperative positioning required for direct gonioscopy during minimally invasive glaucoma surgery (MIGS) can pose a challenge for the patient, the surgeon, and the surgical trainee. With traditional optical microscopy, the patient's head, the microscope, and the surgeon's oculars must all be adjusted to achieve an adequate view of the iridocorneal angle. Even then, the surgical view is often inferior for observing or assisting trainees. This video demonstrates several benefits of digital microscopy using the Beyeonics One™ Surgical Exoscope (Beyeonics Surgical, Israel) and an augmented reality (AR) 3D immersive headset. The digital exoscope can be triggered to auto tilt to facilitate direct gonioscopy. The degree to which the exoscope and the patient's head are adjusted may be less than what one traditionally requires with optical microscopy. Surgeon working distance should also be less affected. A digital recenter feature maintains a centered view without having to physically move the camera. The surgeon, who is wearing an immersive headset, does not have to move or adjust the oculars. The foot pedal or headset can be used for fine focus or zoom. The gonioscopic view is near identical to optical microscopy, but the digital technology and automation allow for improved ergonomics and enhanced visualization. A trainee can also don a headset for an identical view to the primary surgeon.

Engineered Cancer Cells as Signal Probes for Fluorescence-Assisted Digital Counting Analysis.

Fluorescence-assisted digital counting analysis allowed sensitive quantification of targets by measuring individual fluorescent labels. However, traditional fluorescent labels suffered from low brightness, small size, and sophisticated preparation procedures. Herein, engineering fluorescent dye-stained cancer cells with magnetic nanoparticles were proposed to construct single-cell probes for fluorescence-assisted digital counting analysis by quantifying the target-dependent binding or cleaving events. Various engineering strategies of cancer cells including biological recognition and chemical modification were developed for rationally designing single-cell probes. Introduction of suitable recognition elements into single-cell probes allowed digital quantification of each target-dependent event via counting the colored single-cell probes in the representative image taken using a confocal microscope. The reliability of the proposed digital counting strategy was corroborated by traditional optical microscopy- and flow cytometry-dependent counting technologies. The advantages of single-cell probes, including high brightness, big size, ease of preparation, and magnetic separation, contributed to the sensitive and selective analysis of targets of interest. As proof-to-concept assays, indirect analysis of exonuclease III (Exo III) activity, as well as direct quantitation of cancer cells, were investigated, and the potential in biological sample analysis was also assessed. This sensing strategy will open a new avenue for the development of biosensors.

High-fidelity deep functional photoacoustic tomography enhanced by virtual point sources

Photoacoustic tomography (PAT), a hybrid imaging modality that acoustically detects the optical absorption contrast, is a promising technology for imaging hemodynamic functions in deep tissues far beyond the traditional optical microscopy. However, the most clinically compatible PAT often suffers from the poor image fidelity, mostly due to the limited detection view of the linear ultrasound transducer array. PAT can be improved by employing highly-absorbing contrast agents such as droplets and nanoparticles, which, however, have low clinical translation potential due to safety concerns and regulatory hurdles imposed by these agents. In this work, we have developed a new methodology that can fundamentally improve PAT’s image fidelity without hampering any of its functional capability or clinical translation potential. By using clinically-approved microbubbles as virtual point sources that strongly and isotropically scatter the local pressure waves generated by surrounding hemoglobin, we can overcome the limited-detection-view problem and achieve high-fidelity functional PAT in deep tissues, a technology referred to as virtual-point-source PAT (VPS-PAT). We have thoroughly investigated the working principle of VPS-PAT by numerical simulations and in vitro phantom experiments, clearly showing the signal origin of VPSs and the resultant superior image fidelity over traditional PAT. We have also demonstrated in vivo applications of VPT-PAT for functional small-animal studies with physiological challenges. We expect that VPS-PAT can find broad applications in biomedical research and accelerated translation to clinical impact.

A rapid multiplex assay of human malaria parasites by digital PCR

BackgroundBlood smear examination through traditional optical microscopy is the gold standard for malaria diagnosis. However, it imposes strict requirements for operational staff and its sensitivity cannot perfectly satisfy the needs of clinical requirements. More sensitive and accurate modern technologies should be applied to this field. Digital PCR (dPCR), as an absolute quantification detection method, can serve as an effective tool to facilitate the diagnosis and classification of different malaria species. ObjectiveWe aimed to establish a new multiplex dPCR detection system for four main Plasmodium species: P. vivax, P. falciparum, P. ovale and P. malariae, which can distinguish exact species of malaria by one PCR reaction. MethodsA total of 39 patients were identified as malaria-positive by microscopic examination in Huashan Hospital from 2016 to 2021; seventy blood samples from these patients were collected. Additionally, 20 healthy individuals, 20 patients with fever and 6 patients with other types of blood parasites infection were also included in this study. Each blood sample was subjected to examination by both blood smears and dPCR. By optimizing four different fluorescence-labeled probes in one reaction system, dPCR permitted the performance of accurate quantitation and working out the exact number of copies of malaria DNA per microliter in whole blood. Rapid diagnostic tests were also conducted to verify part of the results obtained by dPCR. ResultsThe dPCR system was able to make rapid diagnosis and quantification of malaria DNA samples. The analytical sensitivity of multiplex dPCR was as low as 0.557 copies/μL (95% CI 0.521 to 0.607), and it had a sensitivity of 98.0% and a specificity of 100% in clinical samples. Additionally, three multiple malaria co-infection samples have been detected by this dPCR system, including one triple malaria infection case. By testing consecutive daily blood samples of Patient 39, dPCR facilitated monitoring the efficacy of drug treatment. It showed that the DNA concentrations of P. falciparum ranged from 5474 copies/μL to 0 copies/μL, which can reflect the efficacy of antimalarials in real time. This study also found that haemocyte samples (plasma removed) rather than whole blood had higher malaria detection capability and an enhanced positive rate. ConclusionThe multiplex dPCR system newly established here made a substantial contribution in detecting malaria infection at low concentrations. It is suitable for mixed-infection diagnosis and multi-sample continuous monitoring, and presents a promising candidate as an absolute quantitative tool in clinical practice.

Far-field sub-diffraction optical imaging using near-field illumination by optoplasmonic hybrid materials

Here, we propose a method to fabricate large-scale optoplasmonic hybrid materials (OHMs) as a near-field illumination source for sub-diffraction imaging through a traditional optical microscope. An OHM is comprised of a metal/dielectric multilayer coated substrate and a microcylinder (MC) placed on top of the multilayer. Under the dark-field illumination of a traditional optical microscope, the photonic nanojet (PNJ) generated by the MC and the electric field produced on the substrate surface by the photonic–plasmonic interaction work as the near-field illumination source for an object placed on the substrate near the PNJ. High spatial frequencies of the object can be shifted to the passband of the optical microscope and weak scattering signals of the object can be enhanced upon the near-field illumination. Low-contrast objects, such as a 300-nm-diameter SiO2 nanoparticle array, two 100-nm-diameter polystyrene nanoparticles spaced ∼86 nm apart, and Escherichia coli can be resolved using the optical microscope. In addition, the field of view (FOV) is rectangular, the length of the FOV depends on the length of the MC, and the width relates to the diameter of the MC. The proposed method is wide-field, label-free, direct, noninvasive, and far-field, and it presents the potential of super-resolution imaging of unlabeled biological samples using a traditional optical microscope.