Differential Interference Microscopy Research Articles

Asbestos: detection and characterization in tissue

When asbestos fibers are inhaled, asbestos bodies can form in the lungs with the involvement of macrophages. It can take decades from the last exposure to the onset of an asbestos-related disease. The aim of this review is to present methods to detect asbestos bodies in lung tissue, the development of diagnostic criteria and to discuss pros and cons of different methods. Observations and evaluations from the German Mesothelioma Register, along with relevant literature review and expert recommendations in guidelines are presented. Assessing asbestos-related diseases requires recognition of the person's occupational history, the asbestos fiber burden in the lungs, and determining fiber types. Various methods have been developed and validated, including light microscopy techniques such as bright-field microscopy, phase-contrast microscopy, polarization microscopy, and differential interference microscopy, as well as electron microscopy techniques like field-emission-scanning electron microscopy (e.g., FE-SEM) and transmission electron microscopy (TEM). The use of asbestos has been heavily restricted worldwide, even completely banned in Europe. Thus, patients' exposure to asbestos is decreasing. However, asbestos exposure during renovations, demolitions, or through unconscious handling of asbestos-containing materials remains aconcern.

Scoliosis and kyphosis in blue-spotted and marbled flathead fish associated with a Myxobolus acanthogobii-like parasite.

Spinal deformities in finfish have the potential to impact aquaculture industries and wild populations by increasing morbidity, mortality, and reducing growth rates. Myxobolus acanthogobii has been implicated in causing scoliosis and lordosis in various aquatic species in Japan. We investigated 4 cases of spinal deformity in 2 flathead (Platycephalus) species that were submitted to the Elizabeth Macarthur Agricultural Institute (EMAI) in New South Wales (NSW), Australia, between 2015 and 2021. Flathead are commercially significant species that are popular among Australian consumers, and are also sought-after species targeted by recreational fishers. Gross deformities are concerning to the community and may impact the quality and quantity of specimens available for consumption. Three blue-spotted flathead (P. caeruleopunctatus) and one marbled flathead (P. marmoratus) were submitted, all with marked scoliosis and kyphosis; 1-2-mm cysts were present on the dorsum of the brain, most often over the optic lobe or cerebellum. Cytology and differential interference microscopy of cyst material revealed numerous oval spores, x̄ 14 ± SD 0.75 µm × x̄ 11.5 ± SD 0.70 µm, with 2 pyriform polar capsules, the morphology of which is consistent with a Myxobolus sp. PCR assay and 18S rDNA sequencing of the cyst material identified a Myxobolus sp. with 96% identity to M. acanthogobii. The identification of this Myxobolus sp. confirms the presence of parasites with the potential to cause spinal deformity in significant aquatic species in NSW waterways.

Real-time observation of the precursor film for low viscosity liquids spreading on solid substrates by a customized differential laser interference microscopy.

While static wettability is well treated with Young's equation via its static contact angle, theoretical analyses for wetting dynamics are not yet reaching consensus due to a singularity of the spreading forces worked at the vapor/liquid/solid contact line. One plausible explanation to overcome the singularity problem is that there is a so-called precursor film spreading outside the apparent contact line. After its first finding in 1919, many researchers have attempted to visualize its shape. However, because its length and thickness are as small as micrometer and nanometer-order, respectively, its visualization stillremains a challenging issue especially for low-viscosity liquids. In the present study, we developed a differential laser interference microscope, which has a thickness resolution of approximately 2nm at the best, and applied it to the wetting front of 10 cSt of silicone oil spreading on a silicon wafer with an almost constant spreading velocity. As a result, the precursor film of 14µm long and 108nm thick was clearly visualized. While the macro contact line has a finite advancing contact angle of 4.0°, the gradient of the precursor film surface gradually decreased and converged to ~ 0.1° at the micro-contact angle. The shape of the precursor film was independent of the time after the dropping for the range of 600s ± 10%, which is consistent to theoretical estimation. The present study demonstrated that our interferometer simultaneously achieved nanometer thickness resolutions, micrometer in-plane spatial resolution, and at least a millisecond temporal resolution with a simple optical setup.

Computational Portable Microscopes for Point-of-Care-Test and Tele-Diagnosis.

In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.

Subnanometer accuracy of surface characterization by reflected-light differential interference microscopy

We theorize the surface step characterization by reflected incoherent-light differential interference microscopy with consideration of the optical diffraction effect. With the integration of localization analysis, we develop a quantitative differential interference optical system, by which we demonstrate that the axial resolution of measuring surface height variation is sensitive to the shear distance between the two spatially differentiated beams. We fabricate three nanometer-size steps by photolithography, and successfully characterize their 1D height variations with 0.13 nm axial precision. Our result suggests that the optical differential interference microscopy can be used for real-time characterization of surface structure with a subnanometer accuracy and a large field of view, which is greatly beneficial to the surface characterization of micro/nano-electromechanical systems.

In situ characterization of buckling dynamics in silicon microribbon on an elastomer substrate

Buckling of rigid thin films on elastomer substrates underlies the fabrication foundation of stretchable soft electronics. Here we demonstrate an optical approach to in situ characterize the buckling of silicon microribbon driven by releasing the pre-stretched poly-dimethylsiloxane (PDMS) substrate at a controllable strain rate. The method, based on quantitative differential interference microscopy, directly captures the space–time evolution of the surface topography at a frame rate of 100 fps in a large field of view of 50 × 195μm2. The nucleation, propagation and stabilization of the buckled structure during the buckling and unbuckling processes are observed and quantified. Our experiment reveals that a sequence of partially buckled patterns are energetically stable to bridge the unbuckled and fully buckled states. This work opens a new experimental scheme for the research on stretchable soft electronics and provides new evidence for the theoretical study of the buckling dynamics.

Deep learning-based real-time detection of neurons in brain slices for in vitro physiology

A common electrophysiology technique used in neuroscience is patch clamp: a method in which a glass pipette electrode facilitates single cell electrical recordings from neurons. Typically, patch clamp is done manually in which an electrophysiologist views a brain slice under a microscope, visually selects a neuron to patch, and moves the pipette into close proximity to the cell to break through and seal its membrane. While recent advances in the field of patch clamping have enabled partial automation, the task of detecting a healthy neuronal soma in acute brain tissue slices is still a critical step that is commonly done manually, often presenting challenges for novices in electrophysiology. To overcome this obstacle and progress towards full automation of patch clamp, we combined the differential interference microscopy optical technique with an object detection-based convolutional neural network (CNN) to detect healthy neurons in acute slice. Utilizing the YOLOv3 convolutional neural network architecture, we achieved a 98% reduction in training times to 18 min, compared to previously published attempts. We also compared networks trained on unaltered and enhanced images, achieving up to 77% and 72% mean average precision, respectively. This novel, deep learning-based method accomplishes automated neuronal detection in brain slice at 18 frames per second with a small data set of 1138 annotated neurons, rapid training time, and high precision. Lastly, we verified the health of the identified neurons with a patch clamp experiment where the average access resistance was 29.25 MOmega (n = 9). The addition of this technology during live-cell imaging for patch clamp experiments can not only improve manual patch clamping by reducing the neuroscience expertise required to select healthy cells, but also help achieve full automation of patch clamping by nominating cells without human assistance.

Dual beam-shear differential interference microscopy for full-field surface deformation gradient characterization

We propose a new experimental mechanics method: dual beam-shear differential interference microscopy (DInM) for full-field surface deformation measurement. The method integrates the principles of differential interference contrast, photoelasticity, digital image correlation and the theories of continuum mechanics for a 4D quantification of the surface topography. Our first DInM prototype provides the lateral resolution of 787nm for up to 12% measurable out-of-plane strains. The resolution can be improved by using the shorter spectrum light and higher magnification objective lens. We use our system to characterize the buckling profile of Si microribbon on an elastomer substrate, which was verified by atomic force microscopy. We also demonstrate the stress-induced phase transformation in NiTi alloy by our system. The evolution of the surface topographies and the full-field deformation gradients are successfully captured and quantified. The dynamic measurement provides the information to calculate the relative transformation strains between phases of different symmetries. The establishment of dual beam-shear DInM opens a new avenue in the field of experimental meso/micromechanics.

ZPIE: an autofocusing algorithm for ptychography.

An autofocusing algorithm for ptychography is proposed. The method optimizes a sharpness metric that would be observed in a differential interference microscope and is valid for both amplitude and phase modulating specimens. We experimentally demonstrate that the algorithm, based on the extended ptychographic iterative engine (ePIE), calibrates the sample-detector distance with an accuracy within the depth of field of the ptychographic microscope. We show that the method can be used to determine slice separation in multislice ptychography, provided there are isolated regions on each slice of the specimen that do not axially overlap.

Effect of Smartphone Light Fluxes on Cornea: A Biophysical Study.

Objective:Biophysical study to investigate (a) the effects of smartphone light fluxes (SPLF) on isolated mammalian cornea and model protein (insulin), (b) to predict the possible visual interference of SPLF.Materials and Methods:Fresh goat cornea and insulin protein were used as an experimental model system. The energy of absorbed SPLF was measured using chemical dosimeter. The effect of SPLF on the aggregation of model protein was studied using fluorescence spectroscopy and dynamic light scattering (DLS). Fluorescence microscopy, scanning electron microscopy (SEM), DLS, were used for cornea imaging.Results:The spectral emission peak of SPLF was observed at 380 nm and 420 nm. Absorbed radiation of SPLF was found to be 2.82 mWm-2 and 1.92 mWm-2 for collimated (focussed) and noncollimated (nonfocussed) condition, respectively. Secondary structural changes of insulin were observed by fluorescence and zeta potential after SPLF exposure. SEM study revealed the disorganization of the epithelial cell surface, increase in intercellular space, disorganization of primary epithelium layer, and exposure of the second layer is seen in depth. Differential Interference Microscopy showed an optical gradient in images that appears to be changed in specimen structure. Fluorescence microscopy showed disorganization in epithelial cell pattern. A significant difference in bio-molecular permeation was observed in the exposed cornea. Ultraviolet UV-visible spectroscopy study indicated a reduction in light transmission through the cornea.Conclusions:The obtained results indicate changes in physicochemical and morphological modifications in the cornea and insulin modifications after exposed to SPLF.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

Characterization of recrystallized depth and dopant distribution in laser recovery of grinding damage in single-crystal silicon

A nanosecond pulsed Nd:YAG laser was irradiated on a boron-doped single-crystal silicon wafer with a diamond grinding finish to recover the grinding-induced subsurface damage. In order to visualize and measure the depth of the laser melted/recrystallized layer, small-angle beveled polishing was performed in pure water followed by KOH etching. It enabled the direct observation of the recrystallized region using a differential interference microscope and the measurement of its depth using a white light interferometer. Crystallinity analysis of the recrystallized region was carried out by using laser micro-Raman spectroscopy, and the dopant concentration profile was characterized by using radio frequency glow discharge optical emission spectrometry (rf-GD-OES). The results showed that the crystallinity and boron distribution in the recrystallized region changed after laser recovery. The dopant concentration becomes higher at the boundary of the recrystallized region and the bulk. This study demonstrates the possibility of boron concentration control by using suitable laser parameters.

Oocyte-activating capacity of fresh and frozen-thawed spermatids in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) represents a promising nonhuman primate model for the study of human diseases because of its small size, ease of handling, and availability of gene-modified animals. Here, we aimed to devise reproductive technology for marmoset spermatid injection using immature males for a possible rapid generational turnover. Spermatids at each step could be identified easily by their morphology under differential interference microscopy: thus, early round spermatids had a round nucleus with a few nucleolus-like structures and abundant cytoplasm, as in other mammals. The spermatids acquired oocyte-activating capacity at the late round spermatid stage, as confirmed by the resumption of meiosis and Ca2+ oscillations upon injection into mouse oocytes. The spermatids could be cryopreserved efficiently with a simple medium containing glycerol and CELL BANKER®. Late round or elongated spermatids first appeared at 10-12 months of age, 6-8 months before sexual maturation. Marmoset oocytes microinjected with frozen-thawed late round or elongated spermatids retrieved from a 12-month-old male marmoset developed to the 8-cell stage without the need for artificial oocyte activation stimulation. Thus, it might be possible to shorten the intergeneration time by spermatid injection, from 2 years (by natural mating) to 13-15 months including gestation.

Formation of Basal Plane Dislocations Introduced by Collision of Macrosteps on Growth Surface during SiC Solution Growth

The relationship between surface morphology and spatial distribution of basal plane dislocations in 4H-SiC crystal grown by top-seeded solution growth on the C face was investigated by the differential interference microscopy as well as X-ray topography. Basal plane dislocations were generated at the boundaries of the domains with the different macrosteps advance directions. On the other hand, at the position where macrosteps advance to the same direction, BPDs were hardly observed. This results suggest that BPD density can be decreased by the suppression of the collision of macrosteps during the solution growth on the C face controlling the surface morphology.

Effect of Vanillin to Prevent the Dendrite Growth of Zn in Zinc-Based Secondary Batteries

Although considerable research efforts have been taken to minimize the dendrite growth of zinc in zinc-based secondary batteries, still dendrite growth is a severe issue, which needs further research in this field. In this paper, we have investigated that vanillin as an additive material either eliminates, or assist to minimize the formation of dendrite growth during the deposition of Zn. More than 300 cycles of charge-discharge curves are tested and even at relatively large current densities, dendrite-free zinc surfaces are observed with small amount of vanillin (0.5–5 mM). On the other hand, dendrite growth was observed in the vanillin-free solution. In order to observe the dynamic zinc deposition process on ultra-flat Au(111), we have used an advanced optical microscopy with differential interference microscopy which allowed us to follow the time dependent processes of deposition and dissolution of Zn at high speed accusation times (4∼10 frames/sec even in large area of 100 × 100 μm). We also found that the formation of “black” films depends on the applied electrode potentials. It is anticipated that such insoluble films might be a key factor for the formation and growth of zinc dendrites in rechargeable zinc batteries.

In Situ Visualization of Lithium Ion Intercalation into MoS2 Single Crystals using Differential Optical Microscopy with Atomic Layer Resolution.

Atomic-level visualization of the intercalation of layered materials, such as metal chalcogenides, is of paramount importance in the development of high-performance batteries. In situ images of the dynamic intercalation of Li ions into MoS2 single-crystal electrodes were acquired for the first time, under potential control, with the use of a technique combining laser confocal microscopy with differential interference microscopy. Intercalation proceeded via a distinct phase separation of lithiated and delithiated regions. The process started at the atomic steps of the first layer beneath the selvedge and progressed in a layer-by-layer fashion. The intercalated regions consisted of Li-ion channels into which the newly inserted Li ions were pushed atom-by-atom. Interlayer diffusion of Li ions was not observed. Deintercalation was also clearly imaged and was found to transpire in a layer-by-layer mode. The intercalation and deintercalation processes were chemically reversible and can be repeated many times within a few atomic layers. Extensive intercalation of Li ions disrupted the atomically flat surface of MoS2 because of the formation of small lithiated domains that peeled off from the surface of the crystal. The current-potential curves of the intercalation and deintercalation processes were independent of the scan rate, thereby suggesting that the rate-determining step was not governed by Butler-Volmer kinetics.

Comparison of cushioned centrifugation and SpermFilter filtration on longevity and morphology of cooled-stored equine semen

This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and...

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy.

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

Smoothing of GaN Substrate by Ultraviolet Assisted Polishing in KOH Solution

In this study, we investigated the possibility of smoothing a GaN substrate utilizing ultraviolet (UV) assisted polishing method in potassium hydroxide (KOH) solution. In this polishing method, GaN substrate was excited by an UV radiation, and then an oxide layer on the GaN substrate was formed by photochemical reaction. Simultaneously, generated oxide layer was removed by synthetic quartz tool and chemically etched by KOH solution. Finally, smoothed GaN surface could be realized. The surface quality and removal depth were measured and evaluated using a scanning white light interferometer and Normalski type differential interference microscopy. Obtained results show that the surface morphology and the removal rate are strongly dependent on the existence of the UV irradiation. Moreover, the processed surface has revealed that many scratches on the preprocessed GaN surface could be completely removed. The microroughness of the processed GaN surface profile was improved to be 0.18 nm (Rms), 1.06 nm (Rz).

Analysis of the dynamics of a Bacillus subtilis spore germination protein complex during spore germination and outgrowth.

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the "germinosome," and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥ 75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼ 50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼ 2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.