Conventional Optical Microscopy Research Articles

All black: a microplastic extraction combined with colour-based analysis allows identification and characterisation of tire wear particles (TWP) in soils

While tire wear particles (TWP) have been estimated to represent more than 90% of the total microplastic (MP) emitted in European countries and may have environmental health effects, only few data about TWP concentrations and characteristics are available today. The lack of data stems from the fact that no standardized, cost efficient or accessible extraction and identification method is available yet. We present a method allowing the extraction of TWP from soil, performing analysis with a conventional optical microscope and a machine learning approach to identify TWP in soil based on their colour. The lowest size of TWP which could be measured reliably with an acceptable recovery using our experimental set-up was 35 µm. Further improvements would be possible given more advanced technical infrastructure (higher optical magnification and image quality). Our method showed a mean recovery of 85% in the 35–2000 µm particle size range and no blank contamination. We tested for possible interference from charcoal (as another black soil component with similar properties) in the soils and found a reduction of the interference from charcoal by 92% during extraction. We applied our method to a highway adjacent soil at 1 m, 2 m, 5 m, and 10 m and detected TWP in all samples with a tendency to higher concentrations at 1 m and 2 m from the road compared to 10 m from the road. The observed TWP concentrations were in the same order of magnitude as what was previously reported in literature in highway adjacent soils. These results demonstrate the potential of the method to provide quantitative data on the occurrence and characteristics of TWP in the environment. The method can be easily implemented in many labs, and help to address our knowledge gap regarding TWP concentrations in soils.

Dual-spherical-wave optical scanning holographic microscopy with resolution enhancement and an extended depth of field

Conventional optical scanning holographic microscopy (OSHM) is realized by using a plane wave and a spherical wave as the illumination. The resolution of the conventional OSHM is limited by the numerical aperture of the spherical wave and cannot exceed the Rayleigh limit. In this paper, the OSHM by using dual spherical waves as the illumination is studied. The model of a Gaussian wave, together with the constraints on the signal strength and the visibility, are considered in the analysis of the dual-spherical-wave (DS) OSHM. For an object located at the symmetry plane of the DS-OSHM, the resolution can slightly exceed the Rayleigh limit, and the depth of field (DoF) is extended significantly. For a transparent object, the noise in the DS-OSHM is less than that of the conventional OSHM, thanks to the highly divergent scanning beam. If the object is off the symmetry plane, the resolution is still enhanced, but the extension of the DoF is limited. In addition, a clear reconstructed image will be observed not only at the original object plane, but also at its mirror plane. To the best of our knowledge, this phenomenon is reported for the first time.

Three-color single-molecule localization microscopy in chromatin.

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-label studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multi-plexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

Multimodal laser opto-ultrasonic spectroscopy (LOUS) Approach to investigate the impact of elemental composition on the engineering properties of high-density tungsten alloys'

The impact of elemental composition on the engineering properties of high-density tungsten alloy is crucial, particularly in relation to grain size, hardness, elastic modulus, and surface degradation. This study introduces a new multimodal laser opto-ultrasonic spectroscopy (LOUS) technique, which simultaneously integrates the benefits of laser-induced breakdown spectroscopy (LIBS) and laser ultrasonic testing to evaluate the engineering properties of tungsten alloys. The findings indicate that the increasing tungsten concentration significantly enhances the samples' hardness, grain size and elastic modulus. The composition of elements and hardness were assessed using a calibration curve derived from the emission intensity ratio (W-II/W-I) and plasma electron temperature (Te) in optical emission. The correlation results of (W-II/W-I) and Te showed significant enhancement with the increase of hardness with a regression coefficient (R2 ≥ 0.996), validating the Saha-Eggert relation and underscoring model effectiveness. Additionally, the correlation of the laser ultrasonic testing parameters (attenuation coefficient and velocities) in assessing grain size and elastic modulus showed good reliability (R2≥0.993) when compared to the results obtained from conventional optical microscopy and tensile testing. The results underscore the accuracy and predictive ability of the LOUS method for in-situ characterization.

Comprehensive study of optical contrast, reflectance, and Raman spectroscopy of multilayer graphene

Graphene research has developed quite rapidly partially because even a monatomic layer can be visualized with a conventional optical microscope. Although optical properties of multilayer graphene such as optical contrast, reflectance (R), and Raman scattering have been well studied, they are studied independently and the thickness dependence is limited to a rather thin region. In this paper, the evolution of optical properties by thickness from monolayer to multilayer graphene up to 107 nm thick is studied comprehensively. The empirically known change of color of multilayer graphene is confirmed from the R, G and B intensities extracted from the optical images. It is also found that, as far as R for visible light is concerned, multilayer graphene is not necessarily considered as a layered material, and the refractive index for monolayer graphene is applicable even for the thickest multilayer graphene flake in this study. On the other hand, the layered structure and Raman scattering at each layer are essential to reproduce the G-band intensity of Raman scattering (I(G)). Not only the multiple reflection but also the interference of scattered Raman light should be considered for I(G) of multilayer graphene thicker than ∼30 nm.

A comparison of super-resolution microscopy techniques for imaging tightly packed microcolonies of an obligate intracellular bacterium.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyze their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

Development of the Interosseous Muscles of the Human Hand: Morphological and Functional Aspects of the Terminal Insertion.

To date, there have been no studies conducted on the development of interosseous muscles (IO) in the human hand. This study aimed to investigate the development of these muscles in order to clarify their terminal insertions and their relationship with the metacarpophalangeal joints. Serial sections of 25 human specimens (9 embryos and 16 fetuses) between the 7th and 14th weeks of development, sourced from the Collection of the Department of Anatomy and Embryology at UCM Faculty of Medicine, were analyzed bilaterally using a conventional optical microscope. Our findings revealed that, during the 7th week of development, the metacarpophalangeal interzone mesenchyme extended into the extensor apparatus of the fingers. Furthermore, we observed that the joint capsule and the tendon of the IO derive from the articular interzone mesenchyme. By the end of the 7th week, corresponding to Carnegie stage 21, the myotendinous junction appeared, initiating cavitation of the metacarpophalangeal joint. During the fetal period, the terminal insertions of the IO were identified: both the dorsal interosseous (DI) and palmar interosseous (PI) muscles insert into the metacarpophalangeal joint capsule and establish a connection with the volar plate located at the base of the proximal phalanx and the extensor apparatus. Some muscle fibers also attach to the joint capsule at the level of the proximal synovial cul-de-sac. The functional implications of these findings are discussed within this work. This study provides the first detailed description of the development of the interosseous muscles in the human hand.

Scanning Near-Field Optical Microscopy: Recent Advances in Disordered and Correlated Disordered Photonics

Disordered and correlated disordered photonic materials have emerged in the past few decades and have been rapidly proposed as a complementary alternative to ordered photonics. These materials have thrived in the field of photonics, revealing the considerable impact of disorder with and without structural correlations on the scattering, transport, and localization of light in matter. Scanning near-field optical microscopy (SNOM) has proven to be a fundamental tool for the study of the interaction between light and matter at the nanoscale in such systems, allowing for the investigation of optical properties and local electromagnetic fields with extremely high spatial resolution, surpassing the diffraction limit of conventional optical microscopy. In this review, the most important and recent advances obtained for disordered and correlated disordered luminescent structures by means of the aperture SNOM technique are addressed, showing how it allows the tailoring of local density of states (LDOS), as well as providing access to statistical analysis for multi-resonance disordered and hyperuniform disordered structures at telecom wavelengths.

Waveguide-based microscope slide for label-free high-resolution imaging

Waveguide-based total internal reflection fluorescence microscopy has been widely adopted due to its excellent signal-to-noise ratio over a large field of view. However, with the increasing demand for label-free imaging, waveguide-based evanescent light scattering microscopy (ESM) has also garnered significant attention. Here, we present a low-cost waveguide-based microscope slide that offers easier integration with conventional optical microscopy. This microscope slide uses an incoherent light source coupled to a lithium tantalate (LT) planar waveguide to generate an evanescent light that illuminates samples located within a few hundred nanometers of the waveguide surface. We perform its application for imaging chromium nanoholes and polystyrene nanospheres, demonstrating its label-free, high-resolution, high-contrast imaging performance. LT waveguide microscope slides provide a simple and effective solution for ESM.

Deep learning and defocus imaging for determination of three-dimensional position and orientation of microscopic objects

We present a method to determine the three-dimensional position and orientation of microscopic, non-spherical objects in microfluidic and laboratory-on-a-chip systems observed through conventional optical microscopes. The method is based on the combination of the General Defocusing Particle Tracking technique [Barnkob et al., “General defocusing particle tracking,” Lab Chip 15, 3556–3560 (2015)] and deep learning. It requires minimal input from the user, is suitable for real-time applications, and can be applied to any microscopic object with an approximately ellipsoidal shape, such as unicellular swimming organisms, red blood cells, or spheroidal colloids. The main challenge is linked to the construction of suitable training datasets for the neural network. We provide a procedure generally valid for active microswimmers and discuss possible strategies for other types of objects. An implementation using the Visual Geometry Group convolutional neural network (VGG-16) is presented and tested on synthetic images with different backgrounds and noise levels. The same implementation is used to track the position and orientation of different specimens of the heterotrophic ciliate Euplotes Vannus in free-swimming motion. The measurements were performed with a 10 × objective over a depth of 800 μm with an average estimated uncertainty in the orientation angles of 9.0%.

Converting evanescent waves into propagating waves by hyper-hemi-microsphere.

Hyper-hemi-microspheres (HHMS) have shown promise in enhancing super-resolution imaging when combined with conventional optical microscopy. To offer actionable guidance for optimizing HHMS and hold broad applicability in the field of super-resolution imaging, the mechanism underpinning the enhanced imaging facilitated by HHMS is revealed by deriving the conversion and transmission conditions for evanescent waves. This is achieved by elucidating the intricate interplay between evanescent wave conversion and factors including refractive index, thickness, and surroundings of HHMS. Using the finite-difference time-domain (FDTD) method, influences of various HHMS properties on the conversion and transmission process are analyzed in detail. To fully harness the potential of HHMS in super-resolution imaging, the immersion conditions are elucidated.

Extremely high spatiotemporal resolution microscopy for live cell imaging by single photon counting, noise elimination, and a novel restoration algorithm based on probability calculation.

Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this "diffraction limit" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.

Large-scale microscope with improved resolution using SRGAN

Due to the limited Space-Bandwidth Product (SBP) of optical microscopy system, the Numerical Aperture (NA) and Field of View (FOV)/ Depth of Field (DOF) of conventional optical microscopes are mutually restricted. In this paper, we propose a 20× large-scale (large FOV&DOF) microscope objective lens which is enhanced by deep neural network to achieve the improved resolution close to a 40× Olympus objective lens. Based on training a Super-resolution Generative Adversarial Network (SRGAN) model to transform low-resolution images into super-resolved ones, the resolution of input images captured by low magnification objective is greatly improved without any loss of FOV. And the network output images have better performance on DOF. We also compared SRGAN with two other deep-learning based methods to prove its superiority. Such large-scale microscope with improved resolution could serve to democratize super-resolution imaging.

Multimodal 2D and 3D microscopic mapping of growth cartilage by computational imaging techniques – a short review including new research

Being able to image the microstructure of growth cartilage is important for understanding the onset and progression of diseases such as osteochondrosis and osteoarthritis, as well as for developing new treatments and implants. Studies of cartilage using conventional optical brightfield microscopy rely heavily on histological staining, where the added chemicals provide tissue-specific colours. Other microscopy contrast mechanisms include polarization, phase- and scattering contrast, enabling non-stained or ‘label-free’ imaging that significantly simplifies the sample preparation, thereby also reducing the risk of artefacts. Traditional high-performance microscopes tend to be both bulky and expensive. Computational imaging denotes a range of techniques where computers with dedicated algorithms are used as an integral part of the image formation process. Computational imaging offers many advantages like 3D measurements, aberration correction and quantitative phase contrast, often combined with comparably cheap and compact hardware. X-ray microscopy is also progressing rapidly, in certain ways trailing the development of optical microscopy. In this study, we first briefly review the structures of growth cartilage and relevant microscopy characterization techniques, with an emphasis on Fourier ptychographic microscopy (FPM) and advanced x-ray microscopies. We next demonstrate with our own results computational imaging through FPM and compare the images with hematoxylin eosin and saffron (HES)-stained histology. Zernike phase contrast, and the nonlinear optical microscopy techniques of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are explored. Furthermore, X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies and in vivo possibilities conclude the article.

Metasurface‐Based Mueller Matrix Microscope

AbstractIn conventional optical microscopes, image contrast of objects mainly results from the differences in light intensity and/or color. Muller matrix optical microscopes (MMMs), on the other hand, can provide significantly enhanced image contrast and rich information about objects by analyzing their interactions with polarized light. However, state‐of‐the‐art MMMs are fundamentally limited by bulky and slow polarization state generators and analyzers. Here, the study demonstrates a metasurface‐based MMM, i.e., Meta‐MMM, which is equipped with a chip‐integrated, single‐shot metasurface polarization state analyzer (Meta‐PSA). The Meta‐MMM is featured with high‐speed measurement (≈2s per Muller matrix (MM) image), superior operation stability, dual‐color operation, and high measurement accuracy (measurement error 1–2%) for MM imaging. The Meta‐MMM is applied to nanostructure characterization, surface morphology analysis, and discovering birefringent structures in honeybee wings. The Meta‐MMMs hold the promise to revolutionize various applications from biological imaging, medical diagnosis, and material characterization to industry inspection and space exploration.

In Situ Structural Characterization of Cardiomyocyte Microenvironment by Multimodal STED Microscopy.

Within the myocardium, cardiomyocytes reside in a complex and dynamic extracellular matrix (ECM) consisting of a basement membrane (BM) and interstitial matrix. The interactions between cardiomyocytes and the myocardial ECM play a critical role in maintaining cardiac geometry and function throughout cardiac development and in adult hearts. Understanding how the structural changes of the myocardial ECM affect cardiomyocyte function requires knowledge of pericellular structures. These structures are of a size beyond the resolution of conventional optical microscopy. Here, we demonstrated multi-scale and multi-aspect characterization of the cardiomyocyte microenvironment in myocardial tissue sections using multimodal stimulated emission depletion (STED) microscopy. Second harmonic generation and autofluorescence facilitated multiplexed imaging, enabling the interpretation of protein distribution in 3D. STED imaging modality revealed BM structures of cardiomyocytes and myocardial capillaries at the subdiffractional level. Moreover, meaningful measurements retrieved from acquired images, such as sarcomere length and capillary density, enabled quantitative assessment of myocardial structures.

Far-Field Super-Resolution Microscopy Using Evanescent Illumination: A Review

The resolution of conventional optical microscopy is restricted by the diffraction limit. Light waves containing higher-frequency information about the sample are bound to the sample surface and cannot be collected by far-field optical microscopy. To break the resolution limit, researchers have proposed various far-field super-resolution (SR) microscopy imaging methods using evanescent waves to transfer the high-frequency information of samples to the low-frequency passband of optical microscopy. Optimization algorithms are developed to reconstruct a SR image of the sample by utilizing the high-frequency information. These techniques can be collectively referred to as spatial-frequency-shift (SFS) SR microscopy. This review aims to summarize the basic principle of SR microscopy using evanescent illumination and introduce the advances in this research area. Some current challenges and possible directions are also discussed.

Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives.

Modern biomedical research often requires a three-dimensional microscopic analysis of the ultrastructure of biological objects and materials. Conceptual technical and methodological solutions for three-dimensional structure reconstruction are needed to improve the conventional optical, electron, and probe microscopy methods, which to begin with allow one to obtain two-dimensional images and data. This review discusses the principles and potential applications of such techniques as serial section transmission electron microscopy; techniques based on scanning electron microscopy (SEM) (array tomography, focused ion beam SEM, and serial block-face SEM). 3D analysis techniques based on modern super-resolution optical microscopy methods are described (stochastic optical reconstruction microscopy and stimulated emission depletion microscopy), as well as ultrastructural 3D microscopy methods based on scanning probe microscopy and the feasibility of combining them with optical techniques. A comparative analysis of the advantages and shortcomings of the discussed approaches is performed.

A Review of Microsphere Super-Resolution Imaging Techniques.

Conventional optical microscopes are only able to resolve objects down to a size of approximately 200 nm due to optical diffraction limits. The rapid development of nanotechnology has increased the demand for greater imaging resolution, with a need to break through those diffraction limits. Among super-resolution techniques, microsphere imaging has emerged as a strong contender, offering low cost, simple operation, and high resolution, especially in the fields of nanodevices, biomedicine, and semiconductors. However, this technology is still in its infancy, with an inadequate understanding of the underlying principles and the technology's limited field of view. This paper comprehensively summarizes the status of current research, the advantages and disadvantages of the basic principles and methods of microsphere imaging, the materials and preparation processes, microsphere manipulation methods, and applications. The paper also summarizes future development trends.

Spatiotemporal Visualization of Photogenerated Carriers on an Avalanche Photodiode Surface Using Ultrafast Scanning Electron Microscopy

The spatiotemporal evolution of photogenerated charge carriers on surfaces and at interfaces of photoactive materials is an important issue for understanding fundamental physical processes in optoelectronic devices and advanced materials. Conventional optical probe-based microscopes that provide indirect information about the dynamic behavior of photogenerated carriers are inherently limited by their poor spatial resolution and large penetration depth. Herein, we develop an ultrafast scanning electron microscope (USEM) with a planar emitter. The photoelectrons per pulse in this USEM can be two orders of magnitude higher than that of a tip emitter, allowing the capture of high-resolution spatiotemporal images. We used the contrast change of the USEM to examine the dynamic nature of surface carriers in an InGaAs/InP avalanche photodiode (APD) after femtosecond laser excitation. It was observed that the photogenerated carriers showed notable longitudinal drift, lateral diffusion, and carrier recombination associated with the presence of photovoltaic potential at the surface. This work demonstrates an in situ multiphysics USEM platform with the capability to stroboscopically record carrier dynamics in space and time.