Conventional Optical Microscopy Research Articles

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 FPM and advanced X-ray microscopies. We next demonstrate with our own results computational imaging through Fourier ptychographic microscopy (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 reported. X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are also presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies and in vivo possibilities conclude the article.

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.

Digital Microscopy Augmented by Artificial Intelligence to Interpret Bone Marrow Samples for Hematological Diseases.

Analysis of bone marrow aspirates (BMAs) is an essential step in the diagnosis of hematological disorders. This analysis is usually performed based on a visual examination of samples under a conventional optical microscope, which involves a labor-intensive process, limited by clinical experience and subject to high observer variability. In this work, we present a comprehensive digital microscopy system that enables BMA analysis for cell type counting and differentiation in an efficient and objective manner. This system not only provides an accessible and simple method to digitize, store, and analyze BMA samples remotely but is also supported by an Artificial Intelligence (AI) pipeline that accelerates the differential cell counting process and reduces interobserver variability. It has been designed to integrate AI algorithms with the daily clinical routine and can be used in any regular hospital workflow.

Subphase Exchange Cell for Studying Fluid-Fluid Interfaces with Optical Microscopy.

A subphase exchange cell was designed to observe fluid-fluid interfaces with a conventional optical microscope while simultaneously changing the subphase chemistry. Materials including phospholipids, asphaltenes, and nanoparticles at fluid-fluid interfaces exhibit unique morphological changes as a function of the bulk-phase chemistry. These changes can affect their interfacial material properties and, ultimately, the emergent bulk material properties of the films, foams, and emulsions produced from such interfacial systems. In this work, we combine experiments, computational fluid dynamics simulations, and modeling to establish the operating parameters for a subphase exchange cell of this type to reach a desired concentration. We used the experimental setup to investigate changes to a graphene film during a common wet-etching transfer process. Observations reveal that capillary interactions can induce defects and deformations in the graphene film during the wet-etching process, an important finding that must be considered for any wet-etching transfer technique for 2D materials. More generally, conventional optical microscopy was shown to be able to image the dynamics of interfacial systems during a bulk-phase chemistry change. Potential applications for this equipment and technique include observing morphological dynamics of phospholipid film structure with subphase salinity, asphaltene film structure with subphase pH, and particle film synthesis with subphase chemistry.

Wavelength-scanning pixel-super-resolved lens-free on-chip quantitative phase microscopy with a color image sensor

We report a wavelength-scanning-based lens-free on-chip microscope using a color CMOS sensor and a matching modified phase retrieval algorithm for pixel super-resolution. Compared to traditional monochrome industrial cameras, color sensors favored by the consumer electronics industry have smaller pixel sizes, higher performance, and lower costs. However, the color filtering array (CFA) introduces inherent modulation to the holograms acquired under quasi-monochromatic illumination, which complicates the data processing in lens-free on-chip microscopy. Without physically removing the CFA positioned on the sensor chip, we demonstrate quantitative phase imaging (QPI) with a lateral half-width resolution of 615 nm over a wide field-of-view of 51.88 mm2 by exploiting the green-channel data from Bayer-masked holograms. The resulting spatial bandwidth product is 137.2 megapixels, over 10 times that of a conventional optical microscope. The rationale for using only green-channel data is that the information from each sampling point is not lost during propagation but rather distributed to all pixels in the image. Therefore, the missing data in other channels can be recovered by exploiting the sufficient differences among the raw images captured at different wavelengths. Compared to the scheme with monochrome sensors, this method requires the acquisition of several more images to guarantee the convergence of the algorithm. Experimental results show that we can achieve high-quality QPI performance, thus demonstrating the applicability of cost-effective color sensors in the field of lens-free holographic microscopy.

Label-Retention Expansion Microscopy (LR-ExM) for Enhanced Fluorescent Signals using Trifunctional Probes.

Expansion microscopy (ExM) is a super-resolution imaging technique that bypasses the diffraction limit of conventional optical microscopy (∼250 nm) by enlarging samples with a swellable hydrogel. Combined with various light microscopes, ExM enables an effective resolution ranging from 5 to 70 nm. ExM has now been successfully applied to cell, tissue, and whole-organism samples, providing biologists with a low-cost strategy to visualize samples at the molecular level. However, fluorescence signal loss easily happens for beginners and with early versions of ExM protocols. Here, we describe a protocol called label-retention expansion microscopy (LR-ExM), which can preserve and enhance the signal of ExM imaging via a series of trifunctional probes. These trifunctional probes are antibody-based and easy to prepare, and thus suit the needs of most laboratories. © 2024 Wiley Periodicals LLC. Basic Protocol: LR-ExM using trifunctional probes for enhanced fluorescent signals.

Deep-learning-augmented microscopy for super-resolution imaging of nanoparticles.

Conventional optical microscopes generally provide blurry and indistinguishable images for subwavelength nanostructures. However, a wealth of intensity and phase information is hidden in the corresponding diffraction-limited optical patterns and can be used for the recognition of structural features, such as size, shape, and spatial arrangement. Here, we apply a deep-learning framework to improve the spatial resolution of optical imaging for metal nanostructures with regular shapes yet varied arrangement. A convolutional neural network (CNN) is constructed and pre-trained by the optical images of randomly distributed gold nanoparticles as input and the corresponding scanning-electron microscopy images as ground truth. The CNN is then learned to recover reversely the non-diffracted super-resolution images of both regularly arranged nanoparticle dimers and randomly clustered nanoparticle multimers from their blurry optical images. The profiles and orientations of these structures can also be reconstructed accurately. Moreover, the same network is extended to deblur the optical images of randomly cross-linked silver nanowires. Most sections of these intricate nanowire nets are recovered well with a slight discrepancy near their intersections. This deep-learning augmented framework opens new opportunities for computational super-resolution optical microscopy with many potential applications in the fields of bioimaging and nanoscale fabrication and characterization. It could also be applied to significantly enhance the resolving capability of low-magnification scanning-electron microscopy.

Imaging Strain-Localized Single-Photon Emitters in Layered GaSe below the Diffraction Limit.

Nanoscale strain control of exciton funneling is an increasingly critical tool for the scalable production of single photon emitters (SPEs) in two-dimensional materials. However, conventional far-field optical microscopies remain constrained in spatial resolution by the diffraction limit and thus can provide only a limited description of nanoscale strain localization of SPEs. Here, we quantify the effects of nanoscale heterogeneous strain on the energy and brightness of GaSe SPEs on nanopillars with correlative cathodoluminescence, photoluminescence, and atomic force microscopy, supported by density functional theory simulations. We report the strain-localized SPEs have a broad range of emission wavelengths from 620 to 900 nm. We reveal substantial strain-controlled SPE wavelength tunability over a ∼100 nm spectral range and 2 orders of magnitude enhancement in the SPE brightness at the pillar center due to Type-I exciton funneling. In addition, we show that radiative biexciton cascade processes contribute to observed CL photon superbunching. Also, the GaSe SPEs show excellent stability, where their properties remain unchanged after electron beam exposure. We anticipate that this comprehensive study on the nanoscale strain control of two-dimensional SPEs will provide key insights to guide the development of truly deterministic quantum photonics.

Seeing cells without a lens: Compact 3D digital lensless holographic microscopy for wide-field imaging

Optical microscopy is an essential tool for biomedical discoveries and cell diagnosis at micro- to nano-scales. However, conventional microscopes rely on lenses to record 2-D images of samples, which limits in-depth inspection of large volumes of cells. This research project implements a novel 3-D lensless microscopic imaging system that achieves a wide field of view, high resolution, and an extremely compact, cost-effective design: the Digital Lensless Holographic Microscope (DLHM).A lensless holographic microscope is built with only a light source, a sample, and an imaging chip (with other non-essential supporting structures). The entire setup costs $500 to $600. A series of MATLAB-based algorithms were designed to reconstruct phase information of samples simultaneously from the recorded hologram with built-in high-resolution and phase unwrapping functions. This produces 3-D images of cell samples. The 3-D cell reconstruction of biological samples maintained a comparable resolution with conventional optical microscopes while covering a field of view of 36.2 mm2, which is 20-30 times larger. While most microscopes are extremely time-consuming and require professional expertise, the lensless holographic microscope is portable, low-cost, high-stability, and extremely simple. This makes it accessible for point-of-care testing (POCT) to a broader coverage, including developing regions with limited medical facilities.

Universal Molecular Retention with 11-Fold Expansion Microscopy.

The nanoscale imaging of biological specimens can improve the understanding of disease pathogenesis. In recent years, expansion microscopy (ExM) has been demonstrated to be an effective and low-cost alternative to optical super-resolution microscopy. However, it has been limited by the need for specific and often custom anchoring agents to retain different biomolecule classes within the gel and by difficulties with expanding standard clinical sample formats, such as formalin-fixed paraffin-embedded tissue, especially if larger expansion factors or preserved protein epitopes are desired. Here, we describe Magnify, a new ExM method for robust expansion up to 11-fold in a wide array of tissue types. By using methacrolein as the chemical anchor between the tissue and gel, Magnify retains multiple biomolecules, such as proteins, lipids, and nucleic acids, within the gel, thus allowing the broad nanoscale imaging of tissues on conventional optical microscopes. This protocol describes best practices to ensure robust and crack-free tissue expansion, as well as tips for handling and imaging highly expanded gels.

Fully optic characterization of acoustic impedance implementable in conventional optical microscope

The intrinsic acoustic impedance (Z) changes with inflammation and canceration. This paper proposes an optically Z measurement method using a common optical microscope for simultaneous measurement of optical-acoustical observation. Photoacoustic (PA) waves were generated by the PA effect of the pulsed laser (a 527 nm nanosecond laser light) at the black ink and resin pasted area on the outside of the bottom of the petri dish (surface A). The generated PA waves propagated to the inner boundary of the petri dish and reflected, with a reflection coefficient of R. The reflected waves propagated to surface A and vibrated it. The surface vibration was measured with a self-built Sagnac interferometer using a contentious light laser and a 10x objective lens. Measurements were made air-filled and water-filled petri dishes. The observed times of echo signal corresponded to the sound speed of longitudinal and transverse propagation. The reflected wave amplitude of air was larger than in a water-filled petri dish. They related that R water and R air are 0.24 and 1.00, respectively. There corresponds to the Z ratio of the polystyrene petri dish and the water or air inside. Thus, this result showed that sample Z was measured optically.

Investigating the impact of plasma nitriding on Ti6Al4V surface, structural, and mechanical properties and their simultaneous evaluation via laser opto-ultrasonic dual detection (LOUD) approach

Titanium alloys possess exceptional properties, but due to their poor surface characteristics, several engineering processes have been developed to modify surface properties. Surface modification methodologies such as plasma nitriding exhibit a favourable pathway to improve these properties. This study investigates the impact of plasma nitriding on surface properties and their evaluation via the laser opto-ultrasonic dual detection (LOUD) technique. The scanning electron microscope, X-ray diffraction, and tensile testing results showed that with the increase in nitride plasma power density from 1.42 to 10.0 × 102 W/cm2, a substantial variation in microstructure and phase transformation occurs, eventually refining the grain size, increasing the hardness from 310.83-HV5.0 to 745.50-HV5.0 and elastic modulus (E) from 115.26 to 128.35 GPa, respectively. Furthermore, these characteristics were assessed concurrently through ultrasonic and optical signal processing for LOUD detection. The results of G.S and E from the acoustic attenuation coefficient and longitudinal and shear wave velocities are reliable with the results of conventional optical microscopy and tensile testing with (R2 ≥ 0.996). Meanwhile, the optical spectral data were analyzed to determine the hardness. The results showed that the calibration curve of the intensity ratios (Ti-II/Ti-I) and plasma electron temperature exhibited a linear relationship with hardness (R2 ≥ 0.989), which showed a good approximation.

Microlens-Assisted Light-Scattering Imaging of Plasmonic Nanoparticles at the Single Particle Level.

We present a microlens-assisted imaging approach to record the scattering light of plasmonic nanoparticles at the single particle level. The microlens can significantly enhance the backscattering of visible light from individual plasmonic nanoparticles by several dozen folds, and single gold nanoparticles with a diameter as low as 60 nm can be imaged under a conventional optical microscope. This can benefit from a significant increase in the scattering intensity afforded by the microlens, meaning that the imaging of gold nanoparticles at a high temporal resolution (up to 5000 Hz) can be achieved, which is fast enough to record single particle adhesion events on the substrate. This research presents a fast and efficient means of acquiring scattering light from plasmonic nanoparticles, which has great potential to develop plasmonic nanoparticle-based biosensors and investigate a wide range of plasmonic nanoparticle-based fast interaction processes.

Three-dimensional oxygen concentration monitoring in hydrogels using low-cost phosphorescence lifetime imaging for tissue engineering.

Oxygen concentration measurement in 3D hydrogels is vital in 3D cell culture and tissue engineering. However, standard 3D imaging systems capable of measuring oxygen concentration with adequate precision are based on advanced microscopy platforms, which are not accessible in many laboratories due to the system's complexity and the high price. In this work, we present a fast and low-cost phosphorescence lifetime imaging design for measuring the lifetime of oxygen-quenched phosphorescence emission with 0.25 µs temporal precision and sub-millimeter spatial resolution in 3D. By combining light-sheet illumination and the frequency-domain lifetime measurement using a commercial rolling-shutter CMOS camera in the structure of a conventional optical microscope, this design is highly customizable to accommodate application-specific research needs while also being low-cost as compared to advanced instruments. As a demonstration, we made a fluidic device with a gas-permeable film to create an artificial oxygen gradient in the hydrogel sample. Dye-embedded beads were distributed in the hydrogel to conduct continuous emission lifetime monitoring when nitrogen was pumped through the fluidic channel and changed oxygen distribution in the sample. The dynamics of the changes in lifetime co-related with their location in the gel of size 0.5 mm×1.5 mm×700 µm demonstrate the ability of this design to measure the oxygen concentration stably and precisely in 3D samples.

Visualization of invisible cell-death sign by electric-double-layer modulation

Leakage and self-spreading of a cytoplasmic lipid droplet in association with cell death, which was totally invisible in a conventional optical microscope, was discovered by using the recently developed electric-double-layer modulation imaging (EDLMI). The method allowed real-time imaging of the leaked lipid droplet with the thickness of a few nanometers. We demonstrate that the discovered phenomenon is useful as a new cellular sign for diagnosis of cell death.

Image quality enhancement of 4D light field microscopy via reference impge propagation-based one-shot learning

Four-dimensional (4D) light-field (LF) microscopes can acquire 3D information about target objects using a microlens array (MLA). However, the resolution and quality of sub-images in the LF images are reduced because of the spatial multiplexing of rays by the element lenses of the MLA. To overcome these limitations, this study proposes an LF one-shot learning technique that can convert LF sub-images into high-quality images similar to the 2D images of conventional optical microscopes obtained without any external training datasets for image enhancement. The proposed convolutional neural network model was trained using only one training dataset comprising a high-resolution reference image captured without an MLA as the ground truth. Further, its input was the central view of the LF image. After LF one-shot learning, the trained model should be able to convert well the other LF sub-images of various directional views that were not used in the main training process. Therefore, novel learning techniques were designed for LF one-shot learning. These novel techniques include an autoencoder-based model initialization method, a feature map-based learning algorithm to prevent the overfitting of the model, and cut loss to prevent saturation. The experimental results verified that the proposed technique effectively enhances the LF image quality and resolution using a reference image. Moreover, this method enhances the resolution by up to 13 times, decreases the noise amplification effect, and restores the lost details of microscopic objects. The proposed technique is stable and yields superior experimental results compared with those of the existing resolution-enhancing methods.

Ultrastructural skin alterations of healthy subjects with anti-desmoglein 1 antibodies in endemic areas to pemphigus foliaceus: A case series

BackgroundEndemic pemphigus foliaceus and endemic pemphigus vulgaris are autoimmune dermatologic disorders endemic to the Peruvian Amazon. ObjectiveTo determine the ultrastructural skin alterations of three healthy subjects with anti DSG-1 antibodies in areas endemic to pemphigus foliaceus and pemphigus vulgaris in the Peruvian Amazon. Patients and methodsCase series carried out from data of three clinically healthy subjects positive to anti DSG-1 antibodies, from Peru. This study consists of a sub-analysis of data gathered in a previous study. ResultsUltrastructural results are presented from the skin biopsies of three clinically healthy patients positive to anti-desmoglein 1 (DSG-1) antibodies. High Resolution Optical Microscopy (HROM) showed the absence of acantholysis. Transmission Electron Microscopy (TEM) showed the widening of intercellular space between keratinocytes, the presence of vacuoles in intercellular space with granular material and cytoplasmic vacuolization, loss of desmosome structure, loss of normal distribution among tonofilaments and lateral separation among cells in the stratum basale. ConclusionAccording to our results, healthy subjects that present anti-desmoglein 1 antibodies can develop ultrastructural alterations that are visible through transmission electron microscopy but not through conventional optical microscopy.

Chiral diketopyrrolo[3,4-c]pyrrole-based oligothiophenes: Synthesis and characterization of aggregated states in solution and thin films.

In this work, we synthesized a family of three structurally related chiral oligothiophenes containing a 1,4-diketo-3,6-diarylpyrrolo[3,4-c]pyrrole (DPP) unit as the central core; functionalized with the same (S)-3,7-dimethyl-1-octyl chains on the nitrogen atoms of lactam moieties, they only differ in the number of lateral thiophene units. The aggregation modes of these π-conjugated chiral systems were evaluated by means of UV-Vis absorption and ECD spectroscopies in conditions of solution aggregation (CHCl3 /MeOH mixtures) and as thin films, describing in particular the impact of the π-conjugation length on the chiroptical properties. Interestingly, we found that the variable number of thiophene units attached to the DPP core affects not only the propensity to aggregation but also the aggregates' helicity. ECD revealed information about the supramolecular arrangement of these molecules, that one would not obtain by using conventional optical spectroscopy and microscopy techniques. Thin film samples revealed very different aggregation modes with respect to solution aggregates, casting doubts on the common assumption that these latter may serve as simple models of the former ones.