Conventional Optical Microscopy Research Articles

Evaluation of elastic change during the mitotic phase of murine breast cancer cells using scanning acoustic microscopy

Scanning acoustic microscopy (SAM) is a useful observational tool in cellular study as non-invasive living observation is feasible, unlike in conventional optical microscopy. In a previous study, cell morphological changes were successfully visualized using acoustic impedance measurements. These acoustic impedance changes correspond to cell elasticity, mainly reflecting changes in the cytoskeleton. In this study, we evaluate the elastic changes in murine breast cancer cell C127I during mitosis. C127I cells were cultured to ∼75% confluency before measurement, using a transducer with a central frequency of 320 MHz. Dynamic changes during mitosis were successfully mapped using SAM and confirmed by laser confocal microscopy. Cells in prometaphase, anaphase, and telophase, which could previously only be confirmed through immunostaining, were successfully visualized using SAM. This suggests that SAM is capable of distinguishing cells in different mitotic phases based on the changes in acoustic impedance.

Resolution and Contrast Enhancement for Lensless Digital Holographic Microscopy and Its Application in Biomedicine

An important imaging technique in biomedicine, the conventional optical microscopy relies on relatively complicated and bulky lens and alignment mechanics. Based on the Gabor holography, the lensless digital holographic microscopy has the advantages of light weight and low cost. It has developed rapidly and received attention in many fields. However, the finite pixel size at the sensor plane limits the spatial resolution. In this study, we first review the principle of lensless digital holography, then go over some methods to improve image contrast and discuss the methods to enhance the image resolution of the lensless holographic image. Moreover, the applications of lensless digital holographic microscopy in biomedicine are reviewed. Finally, we look forward to the future development and prospect of lensless digital holographic technology.

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

High-Speed Ultraviolet Photoacoustic Microscopy for Histological Imaging with Virtual-Staining assisted by Deep Learning.

Surgical margin analysis (SMA), an essential procedure to confirm the complete excision of cancerous tissue in tumor resection surgery, requires intraoperative diagnostic tools to avoid repeated surgeries due to a positive surgical margin. Recently, by taking the advantage of the high intrinsic optical absorption of DNA/RNA at 266 nm wavelength, ultraviolet photoacoustic microscopy (UV-PAM) has been developed to provide high-resolution histological images without labeling, showing great promise as an intraoperative tool for SMA. To enable the development of UV-PAM for SMA, here, a high-speed and open-top UV-PAM system is presented, which can be operated similarly to conventional optical microscopies. The UV-PAM system provides a high lateral resolution of 1.2 µm, and a high imaging speed of 55 kHz A-line rate with one-axis galvanometer mirror scanning. Moreover, to ensure UV-PAM images can be easily interpreted by pathologists without additional training, the original grayscale UV-PAM images are virtually stained by a deep-learning algorithm to mimic the standard hematoxylin- and eosin-stained images, enabling training-free histological analysis. Mouse brain slice imaging is performed to demonstrate the high performance of the open-top UV-PAM system, illustrating its great potential for SMA applications.

Feasibility of control of particle assembly by dielectrophoresis in liquid-cell transmission electron microscopy.

Liquid-cell transmission electron microscopy (LC-TEM) is a useful technique for observing phenomena in liquid samples with spatial and temporal resolutions similar to those of conventional transmission electron microscopy (TEM). This method is therefore expected to permit the visualization of phenomena previously inaccessible to conventional optical microscopy. However, dynamic processes such as nucleation are difficult to observe by this method because of difficulties in controlling the condition of the sample liquid in the observation area. To approach this problem, we focused on dielectrophoresis, in which electrodes are used to assemble particles, and we investigated the phenomena that occurred when an alternating-current signal was applied to an electrode in an existing liquid cell by using a phase-contrast optical microscope (PCM) and TEM. In PCM, we observed that colloidal particles in a solution were attracted to the electrodes to form assemblies, that the particles aligned along the electric field to form pearl chains and that the pearl chains accumulated to form colloidal crystals. However, these phenomena were not observed in the TEM study because of differences in the design of the relevant holders. The results of our study imply that the particle assembly by using dielectrophoretic forces in LC-TEM should be possible, but further studies, including electric device development, will be required to realize this in practice.

Blood Cell Analysis: From Traditional Methods to Super-Resolution Microscopy

Blood cell analysis is essential for the diagnosis and identification of hematological malignancies. The use of digital microscopy systems has been extended in clinical laboratories. Super-resolution microscopy (SRM) has attracted wide attention in the medical field due to its nanoscale spatial resolution and high sensitivity. It is considered to be a potential method of blood cell analysis that may have more advantages than traditional approaches such as conventional optical microscopy and hematology analyzers in certain examination projects. In this review, we firstly summarize several common blood cell analysis technologies in the clinic, and analyze the advantages and disadvantages of these technologies. Then, we focus on the basic principles and characteristics of three representative SRM techniques, as well as the latest advances in these techniques for blood cell analysis. Finally, we discuss the developmental trend and possible research directions of SRM, and provide some discussions on further development of technologies for blood cell analysis.

Gires-Tournois Immunoassay Platform for Label-Free Bright-Field Imaging and Facile Quantification of Bioparticles.

Bright-field imaging of nanoscale bioparticles is a challenging task for optical microscopy because the light-matter interactions of bioparticles are weak on conventional surfaces due to their low refractive index and small size. Alternatively, advanced imaging techniques, including near-field microscopy and phase microscopy, have enabled visualization and quantification of the bioparticles, but they require assistance of sophisticated/customized systems and post-processing with complex established algorithms. Here, a simple and fast immunoassay device, Gires-Tournois immunoassay platform (GTIP) is presented, which provides unique color dynamics in response to optical environment changes and thus enables the label-free bright-field imaging and facile quantification of bioparticles using conventional optical microscopy. Bioparticles on GTIP slow down the velocity of reflected light, leading to vivid color change according to the local particle density and maximizing chromatic contrast for high spatial distinguishability. The particle distribution and density on the surface of the resonator are readily analyzed through 2D raster-scanning-based chromaticity analysis. GTIP offers multiscale sensing capability for target analytes that possess different refractive indices and sizes.

Peroxyl radical induced membrane instability of giant unilamellar vesicles and anti-lipooxidation protection

The present work is intended to investigate the morphological instability of lipid membrane induced by peroxyl radical (ROO•) and the underlying mechanism. To this end, the giant unilamellar vesicle (GUV) made from phosphatidylcholine was employed as a membrane model, and the azo compounds 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) were used as the precursors of ROO•. Upon mild pyrolysis, the GUV immobilized in agarose gel was followed by conventional optical microscopy in real time, and the morphological variation was quantified by the image heterogeneity, perimeter and area all as a function of time for up to an hour. Lipid oxidation initiated from lipid phase with AMVN and from aqueous phase with AAPH led to different types of morphological changes, i.e. membrane coarsening and vesicle deformation/budding, respectively. Based on the compositional analysis of lipid oxidation products, we propose that ROO• as the primary radical initiator is responsible for the morphological changes of the GUV-AMVN while both ROO• and RO• are responsible for the morphological changes of the GUV-AAPH system. Lipophilic β-carotene and amphipathic plant phenols as antioxidants are found to be able to stabilize the membrane integrity effectively, in corroboration with the proposed mechanisms for membrane destruction.

3D Volume Rendering of Invertebrates Using Light-Sheet Fluorescence Microscopy

Light-Sheet Fluorescence Microscopy has recently emerged as the technique of choice for obtaining high quality three-dimensional (3D) images of whole organisms, with low photo-damage and fast acquisition rates. Unlike conventional optical and confocal microscopy or scanning electron microscopy systems, it offers the possibility of obtaining multiple views of the sample by rotating it. We show that the use of light-sheet fluorescence microscopy, for the analysis of invertebrates, provides a fair compromise compared to scanning electron microscopy in terms of resolution, but avoids some of its drawbacks, such as sample preparation or limited three-dimensional perspectives. In this paper, we will show how LSFM techniques can provide a cheap, high quality, multicolor, 3D alternative to classic microscopes, for the study of the morphological structure of insects and invertebrates in morphogenesis studies of the whole animal.

Circular polarized light microscopy to investigate the crystal orientation of aluminium

The link between microstructure, crystallographic texture and mechanical properties is of substantial importance to the understanding of materials behaviour of aluminium alloys. The favoured approach to determine these relations has been electron backscatter diffraction (EBSD) and has received extensive prior attention. However, compared to conventional optical microscopy, EBSD is both cost and potentially time intensive limiting its viability as a widespread method in industrial quality assurance. Following early successes by the authors and other investigators with optically birefringent metals, this investigation assesses the suitability of reflected circular polarized light microscopy (CPLM) as a rapid low-cost alternative to EBSD for cubic systems. Direct comparisons between EBSD crystal orientation maps (COMs) and a quantitative determination of the character of reflected polarized light images is presented to demonstrate that this technique can provide quantitative texture information for aluminium. Further, the inherently surface sensitive nature of the technique allowed further verification of orientation relationships between the substrate and surface oxide only previously made possible by high resolution transmission electron microscopy. • A novel approach that links polarized light microscopy with electron backscattered crystallographic analysis. • A new rapid crystallographic analysis method suitable for routine quality control. • Crystallographic analysis of anodized Aluminium using polarized light. • Insights into the symmetry and data recording of crystallographic data using commercially available software.

Abstract P2-11-18: Weekly evaluation of α estrogen and progesterone receptors in women´s breast epithelium after oral combined hormonal contraceptive use for one month

Abstract Introduction: The increased risk of breast cancer is the most controversial and most feared adverse effect of combined oral contraceptives (COC) by women. The mechanisms by which COC would act on the breast parenchyma leading to its increased incidence, are not yet well known. Objectives: To calculate and compare α estrogen receptor (ERα) and progesterone receptor (PR) expression in the mammary epithelium in the first, second, third and fourth weeks of patients who do not use COC (natural cycle) and those who use one cycle of COC composed of 30 µg ethinyl estradiol (EE) and 150 µg levonorgestrel (LNG). Casuistic and Methods: A retrospective study with paraffin blocks of 118 women from February 2001 to February 2004. At the time, women were included in the pre-menopause who had a breast lump with the triple diagnosis of positive benignity and eumenorrheic during the last six months. The study project was analyzed and approved by the Research Ethics Committee of UNIFESP-EPM, under number CEP 1182/06. The patients underwent excision of the breast lumps and adjacent normal breast tissue fragments which were fixed in 10% buffered formalin. 31 patients were excluded and 87 patients were analyzed. The patients were divided into two groups: group A (with COC) consisted of 42 women who used an COC cycle composed of 30μg of EE and 150μg of LNG, group B (without COC) consisted of 45 women with natural cycles. The immunohistochemical reaction was performed in an automation device and the following antibodies were used: ERα antibody clone SP1 titer 1:500 and PR antibody clone PgR636 titer 1:400. The histological slides were read using conventional optical microscopy. After identifying the epithelial areas, 7 fields with 40X magnification were evaluated. Statistical analysis for parity was performed using Fisher's exact test. At age the Students t-Test was applied. ERα and PR counts were evaluated in the Generalized Estimation Equation (EEG) model. In order to compare the total ERα and PR counts by evaluation and group moments, we used the Analysis of Variance (ANOVA) and the Kolmogorov-Smirnov test. Statistical analyzes were performed using the programs SPSS 20.0 and STATA 12. For all statistical tests the significance level of 5% (p ≤ 0.05) was adopted. Results: The control group (without COC) had a higher mean age of 23.7 ± 5.9 years compared to the study group (with COC) with a mean age of 20.5 ± 5.1 years. Regarding parity, the study group (with COC) had 83.9% of nulliparous versus 73.3% in the control group (without COC). From the EEG model a statistically significant mean difference in ERα expression was observed in the fourth week in the study group (with COC) compared to the control group (without COC) with p <0.001. The comparison of PR expressions in the control group (without COC) in the fourth week or in the late luteal phase (LLP) with the ERα expressions, also in the fourth week (LLP) of the control group (without COC) the mean PR expression was higher and statistically significant than ERα expression with p <0.001. The group of women using COC showed higher average percentages of ERα (p <0.001) and PR (p <0.001) when compared to women in the control group (without COC). Conclusions: The average of expressions of the ERα and PR in users of an COC cycle composed of 30 µg EE and 150 µg LNG higher percentages of ERα (p <0.001) and PR (p <0.001) when compared to women from the control group (without COC). The ERα expression in the study group (with COC) in the fourth week (pause) was higher than the ERα expression in the fourth week (LLP) of the control group (without COC), a statistically significant difference (p <0.001). The expression PR in the fourth week of the control group (without COC) or LLP was higher than the ERα and this difference was statistically significant (p <0.001). Citation Format: Weekly evaluation of α estrogen and progesterone receptors in women´s breast epithelium after oral combined hormonal contraceptive use for one month [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-18.

A Coloration Biochip for Optical Virus Detection Based on Printed Single Nanoparticle Array

AbstractDirect and ultrasensitive detection of nanoscale objects is of great importance for materials science and biomedical application. Here, a printed single nanoparticle array‐based coloration biochip is developed for direct visualizing nanoscale objects with the microscopy image. Arising from the scattering cross section induced Mie scattering enhancement, the color change of nanoparticles with/without viruses can be observed with a conventional optical microscope. The scattering light from single nanoparticle has the size‐dependent characteristics of wavelength and intensity variation, allowing for optically detecting nanoscale virus. By comparing optical images before and after attaching viruses, quantitative detection can be achieved with the virus concentrations from 1.0 × 102 to 1.0 × 106 PFU mL–1 and a detection limit of 1.0 × 102 PFU mL–1. The antibody‐modified array can specifically recognize influenza viruses in 15 min. The result provides a promising approach to detect diverse biological samples, including protein, exosomes, and platelet microparticle.

Advances in MALDI Mass Spectrometry Imaging Single Cell and Tissues

Compared with conventional optical microscopy techniques, mass spectrometry imaging (MSI) or imaging mass spectrometry (IMS) is a powerful, label-free analytical technique, which can sensitively and simultaneously detect, quantify, and map hundreds of biomolecules, such as peptides, proteins, lipid, and other organic compounds in cells and tissues. So far, although several soft ionization techniques, such as desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS) have been used for imaging biomolecules, matrix-assisted laser desorption/ionization (MALDI) is still the most widespread MSI scanning method. Here, we aim to provide a comprehensive review of MALDI-MSI with an emphasis on its advances of the instrumentation, methods, application, and future directions in single cell and biological tissues.

Enhanced Measurement Accuracy for Nanostructures Using Hybrid Metrology

Light-matter interplay is widely used for analyzing the topology of surfaces on small scales for use in areas such as nanotechnology, nanoelectronics, photonics, and advanced materials. Conventional optical microscope imaging methods are limited in resolution to a value comparable to the wavelength, the so-called Abbe limit, and cannot be used to measure nano-sized structures. Scatterometry and Mueller ellipsometry are spectroscopic optical methods that can measure structures smaller than the wavelength. However, the relative uncertainties of the structure dimensions measured with scatterometry increase with decreasing structure size, and the industry is therefore replacing simple intensity based scatterometry with Mueller ellipsometry for the most demanding measurements. The accuracy of Mueller ellipsometry and scatterometry are closely related to the ability of the employed regression and regularization algorithms to extract the structural dimension. In this work, we demonstrate how the measurement accuracy on three-dimensional periodic structures may be increased by measuring the same periodic structure with multiple techniques and applying a χ2-regression method that finds the best solution based on the input from all the instruments. We furthermore report on a new and improved calibration method for Mueller ellipsometry and demonstrate how the Mueller matrix may be used to find the geometrical anisotropy of the structure.

Gold-viral particle identification by deep learning in wide-field photon scattering parametric images.

The ability to identify virus particles is important for research and clinical applications. Because of the optical diffraction limit, conventional optical microscopes are generally not suitable for virus particle detection, and higher resolution instruments such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are required. In this paper, we propose a new method for identifying virus particles based on polarization parametric indirect microscopic imaging (PIMI) and deep learning techniques. By introducing an abrupt change of refractivity at the virus particle using antibody-conjugated gold nanoparticles (AuNPs), the strength of the photon scattering signal can be magnified. After acquiring the PIMI images, a deep learning method was applied to identify discriminating features and classify the virus particles, using electron microscopy (EM) images as the ground truth. Experimental results confirm that gold-virus particles can be identified in PIMI images with a high level of confidence.

Preservation of immunoexpression of type I collagen, BSP and BMP4 in the dentin-pulp complex of head and neck cancer patients after radiotherapy.

This study tested the hypothesis that head and neck radiotherapy (HNRT) impacts the immunoexpression of type I collagen, bone sialoprotein (BSP) and bone morphogenetic protein 4 (BMP4), thereby leading to micromorphological changes in the dentin-pulp complex (DPC), and promoting the onset and progression of radiation caries (RC). Twenty-two demineralized sections of carious teeth (a group of 11 irradiated teeth and a control group of 11 non-irradiated teeth) extracted from 19 head and neck cancer patients were analyzed by conventional optical microscopy and immunohistochemistry to investigate the micromorphology (cellular layer hierarchy, blood vessels, odontoblasts, fibroblasts, extracellular matrix, calcification, necrosis, reactionary dentin formation, and chronic inflammation), and the patterns of staining/immunolocalization of type I collagen, BSP and BMP4 in the dental pulp of irradiated and control samples. No significant differences attributable to the direct impact of radiotherapy were detected in DPC micromorphology between the groups. In addition, the patterns of immunohistochemical staining and immunolocalization of the proteins studied did not differ between the irradiated and the control samples for type I collagen, BSP or BMP4. This study rejected the hypothesis that HNRT directly damages dentition by changing the organic components and the microstructure of the DPC, ultimately leading to RC.

A virtual microscope for simulation of Nanostructures

Light-matter interplay is widely used for analyzing the topology of surfaces on small scales for use in areas such as nanotechnology, nanoelectronics, photonics, and advanced materials. Conventional optical microscope imaging methods are limited in resolution to a value comparable to the wavelength, the so-called Abbe limit, and cannot be used to measure nano-sized structures. Scatterometry is an optical method that can measure structures smaller than the wavelength. However, the relative uncertainties of the structure dimensions measured with scatterometry increase with decreasing structure size, and the industry is therefore looking for replacing simple intensity based scatterometry with a phase-sensitive measurement method such as coherent Mueller ellipsometry. In this work, we present a virtual microscope capable of simulating the coherent Mueller ellipsometry and scatterometry response from one-dimensional and two-dimensional periodic structures. Furthermore, we argue that coherent nonnormalized Mueller ellipsometry gives results with less uncertainties than standard normalized Mueller ellipsometry.

Morphometry and Stiffness of Red Blood Cells-Signatures of Neurodegenerative Diseases and Aging.

Human red blood cells (RBCs) are unique cells with the remarkable ability to deform, which is crucial for their oxygen transport function, and which can be significantly altered under pathophysiological conditions. Here we performed ultrastructural analysis of RBCs as a peripheral cell model, looking for specific signatures of the neurodegenerative pathologies (NDDs)—Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD), utilizing atomic force (AFM) and conventional optical (OM) microscopy. We found significant differences in the morphology and stiffness of RBCs isolated from patients with the selected NDDs and those from healthy individuals. Neurodegenerative pathologies’ RBCs are characterized by a reduced abundance of biconcave discoid shape, lower surface roughness and a higher Young’s modulus, compared to healthy cells. Although reduced, the biconcave is still the predominant shape in ALS and AD cells, while the morphology of PD is dominated by crenate cells. The features of RBCs underwent a marked aging-induced transformation, which followed different aging pathways for NDDs and normal healthy states. It was found that the diameter, height and volume of the different cell shape types have different values for NDDs and healthy cells. Common and specific morphological signatures of the NDDs were identified.

Direct Observation of Axial Dynamics of Particle Manipulation With Weber Self-Accelerating Beams

Optical manipulation of micro-particles with nondiffracting and self-accelerating beams has been successfully applied in many research fields such as chemicophysics, material sciences and biomedicine. Such operation mainly focuses on the particle transport and control in the beam propagation direction. However, the conventional optical microscopy is specifically designed for obtaining the sample information located in the lateral plane, which is perpendicular to the optical axis of the detecting objective lens, making the real-time observation of particle dynamics in axial plane a challenge. In this work, we propose and demonstrate a technique which integrates a special beam optical tweezer with a direct axial plane imaging system. Here, particles are transported in aqueous solution along a parabolic trajectory by a designed nonparaxial Weber self-accelerating beam, and the particle motion dynamics both in lateral and axial plane are monitored in real-time by the axial plane imaging technique.

Fast In-Situ Optical Visualization of Carbon Nanotubes Assisted by Smoke.

The fast visualization and manipulation of individual carbon nanotubes (CNTs) has always been a significant technology for their fundamental research. Here, a fast and facile approach is proposed to realize the optical observation of individual suspended CNTs and CNT networks under conventional optical microscopes with the assistance of tar nanodroplets from smoke. The nanodroplets deposited on CNTs render them with strong light scattering to visible light, thus making the CNTs visible under optical microscopes. This visualization method is controllable, environmentally friendly, low-cost, and can be completed in just a few seconds in ambient conditions. Besides, the tar nanodroplets can be easily removed from the CNTs by laser irradiation. More importantly, the smoke sources are widely available and there are no strict requirements for operating conditions. This smoke-assisted visualization method shows great potential in the fundamental research of CNTs.