3D Microscopy Research Articles

Micro-scale graded mechanical metamaterials exhibiting versatile Poisson’s ratio

The ability to control Poisson’s ratio of functional materials has been one of the main objectives of researchers attempting to develop structures efficient from the perspective of protective, biomedical and soundproofing devices. This task becomes even more challenging at small scales, such as the microscale, where the possibility to control mechanical properties of functional materials is very significant, like in the case of flexible electronics. In this work, we propose novel microscopic 2D and 3D functionally-graded mechanical metamaterials capable of exhibiting a broad range of Poisson’s ratio depending on their composition. More specifically, we show that upon adjusting the number of structural elements corresponding to one type of the substructure at the expense of another, it is possible to change the resultant Poisson’s ratio of the entire system from highly positive to highly negative values as well as to achieve arbitrary intermediate values. Finally, in addition to static properties, we also analyze the dynamic properties of these structures. Namely, we show how the variation in the composition of the considered mechanical metamaterials affects the velocity of a wave propagating through the system. This, in turn, could be essential in the case of applications utilizing localized wave attenuation or sensors.

Cleaning testing of nineteenth-century plaster surface models with thin polyacrylamide-based gel layers attached to flexible polyethylene films

This paper explores the cleaning efficacy of polyethylene-supported 15-min photo-crosslinked poly (acrylamide-co-benzophenone) surface-attached gels (SAGs) on gypsum plaster surface models inspired by nineteenth-century casts. Cleaning tests were performed on plaster surface models with and without organic and inorganic coatings, which had been exposed to accelerated ageing by heat, humidity, and light after artificial soiling. The specific types of SAG systems were selected based on their water loading and dehydration capacities. The SAGs were loaded with customized solutions and applied on the plaster models for one minute. Cleaning efficacy was evaluated with visible reflectance, UV fluorescence photography, scanning electron microscopy, colorimetry, UV/Vis spectrophotometry, glossimetry, high resolution 3D microscopy, attenuated total reflection Fourier transform infrared spectroscopy and 2D Fourier transform infrared imaging. The SAGs provided fast and minimal wetting of the substrates, prevented excessive liquid spreading and allowed the effective liquid contact with the soiled gypsum plaster surface. A striking removal of soils from the gypsum plaster surface models was observed, which suggests convenient application of the SAG systems for the cleaning of historical plaster objects.

Investigating the buckling deformation of thin metal sheets during the blanking process

This article describes the effect of the generated stresses due to the cutting process on the possibility of the occurrence of buckling deformation in a thin sheet, which could affect the performance of the cut part, especially for some applications such as the cutting of the thin sheet laminations of motor parts. To achieve the research objectives, an experimental approach was followed. Two different thicknesses and two-grain orientations of silicon steel material were applied to investigate the influence of thickness and grain orientation upon the induced buckling deformation. Blanking dies were designed to cut thin sheets of steel at different cutting speeds. Buckling height was measured optically using a 3D microscope with regard to the cutting parameters. The results showed the appearance of buckling deformation only at low sheet thickness. The maximum buckling height was about 164 µm (82% of the material thickness) in the transverse grain direction at the lower cutting speed. Meanwhile, the results also demonstrated the presence of an elongation in the material of approximately 90 µm (1.8%) on the cutting sides, which played a role in the application of pressure to all sides of the cut plate, resulting in the formation of a plastic buckling deformation prior to the completion of the cutting process, and thus the occurrence of the buckling phenomenon.

In vivo ocular microvasculature imaging in rabbits with 3D ultrasound localization microscopy

Morphological and hemodynamic changes in the ocular vasculature are important signs of various ocular diseases. The evaluation of the ocular microvasculature with high resolution is valuable in comprehensive diagnoses. However, it is difficult for current optical imaging techniques to visualize the posterior segment and retrobulbar microvasculature due to the limited penetration depth of light, particularly when the refractive medium is opaque. Thus, we have developed a 3D ultrasound localization microscopy (ULM) imaging method to visualize the ocular microvasculature in rabbits with micron-scale resolution. We used a 32 × 32 matrix array transducer (center frequency: 8 MHz) with a compounding plane wave sequence and microbubbles. Block-wise singular value decomposition spatiotemporal clutter filtering and block-matching 3D denoising were implemented to extract the flowing microbubble signals at different imaging depths with high signal-to-noise ratios. The center points of microbubbles were localized and tracked in 3D space to achieve the micro-angiography. The in vivo results demonstrate the ability of 3D ULM to visualize the microvasculature of the eye in rabbits, where vessels down to 54 μm were successfully revealed. Moreover, the microvascular maps indicated the morphological abnormalities in the eye with retinal detachment. This efficient modality shows potential for use in the diagnosis of ocular diseases.

Design and Study of a Reflector-Separated Light Dispersion-Compensated 3D Microscopy System.

The secondary-phase grating-based tomographic microscopy system, which is widely used in the biological and life sciences, can observe all the sample multilayer image information simultaneously because it has multifocal points. However, chromatic aberration exists in the grating diffraction, which seriously affects the observation of the image. To correct the chromatic aberration of the tomographic microscope system, this paper proposes a system that adopts blazed gratings and angle-variable reflectors as chromatic aberration correction devices according to the principle of dispersion compensation and Fourier phase-shift theory. A reflector-separated light dispersion-compensated 3D microscopy system is presented to achieve chromatic aberration correction while solving the problem of multilayer image overlap. The theoretical verification and optical design of the system were completed using ZEMAX software. The results show that the proposed system reduced the chromatic aberration of ordinary tomographic microscopy systems by more than 90%, retaining more wavelengths of light information. In addition, the system had a relatively wide range in the color difference compensation element installation position, reducing the difficulty of dispersion compensation element installation. Overall, the results indicate that the proposed system is effective in reducing chromatic aberration in grating diffraction.

The effect of incidence angle on the interaction of a pulse laser with ultra-high-performance concrete in the scabbling process

After prolonged service, concrete surfaces can develop a range of issues such as cracking, spalling, scaling, delamination, and contamination. Meanwhile, laser scabbling can be used as an advanced method for removing concrete surface defects. Besides, the downward direction is still mainly used in the scabbling process and variation in inclination angles of the laser beam has not been investigated in the laser scabbling process. In this study, nanosecond laser scabbling on Ultra-High Performance Concrete (UHPC) with different angles of the sample was carried out. The change in sample angle leads to changes in laser beam shape and the laser intensity distribution in the processing zone. The surface morphology and depth of the glassy layer were revealed by using a 3D Confocal Microscope corresponding to different incident angles of the laser beam. Moreover, a Scanning Electron Microscope (SEM) was also used to examine the pore size distribution in the glassy layer and heat-affected zone as compared to the non-processed zone. The molten material moved easily during the laser scabbling process as the inclination of the sample increased, causing the pores in the molten zone to collide and form a larger size.

Thermal model for nanosecond laser ablation Al2O3 considering temperature dependence

This paper presented a thermal model based on heat-conduction and vaporization to predict the ablation profile by nanosecond pulsed laser in Al2O3. The temperature distribution is estimated by heat transfer in the laser irradiated zone, considering temperature dependence of material properties. The ablation profile was analyzed and predicted, which has considered the laser spatial and time-distribution and the effective number of laser pulses within the irradiated area. To validate the accuracy of thermal model proposed, micro-channel experiment has been performed. The morphology and profile of micro-channel was measured by Scanning electron microscope (SEM) and an 3D microscope. The difference in microstructure of SEM can be attributed to the laser-material interaction associated with the temperature distribution. Good agreement between calculated depth and width of microchannel and that of experimentally measured and its dependence on incident laser parameters validated theoretical model. Whereas, the degree of error marginally increases at high laser power fluences due to the model proposed ignored the optical properties. The errors of side-wall of micro-channel can be attributed to the effect of multi-reflection and plasma on the channel side-wall.

A practical method for assigning uncertainty and improving the accuracy of alpha-ejection corrections and eU concentrations in apatite (U–Th) ∕ He chronology

Abstract. Apatite (U–Th) / He (AHe) dating generally assumes that grains can be accurately and precisely modeled as geometrically perfect hexagonal prisms or ellipsoids in order to compute the apatite volume (V), alpha-ejection corrections (FT), equivalent spherical radius (RFT), effective uranium concentration (eU), and corrected (U–Th) / He date. It is well-known that this assumption is not true. In this work, we present a set of corrections and uncertainties for V, FT, and RFT aimed (1) at “undoing” the systematic deviation from the idealized geometry and (2) at quantifying the contribution of geometric uncertainty to the total uncertainty budget for eU and AHe dates. These corrections and uncertainties can be easily integrated into existing laboratory workflows at no added cost, can be routinely applied to all dated apatite, and can even be retroactively applied to published data. To quantify the degree to which real apatite deviates from geometric models, we selected 264 grains that span the full spectrum of commonly analyzed morphologies, measured their dimensions using standard 2D microscopy methods, and then acquired 3D scans of the same grains using high-resolution computed tomography (CT). We then compared our apatite 2D length, maximum width, and minimum width measurements with those determined by CT, as well as the V, FT, and RFT values calculated from 2D microscopy measurements with those from the “real” 3D measurements. While our 2D length and maximum width measurements match the 3D values well, the 2D minimum width values systematically underestimate the 3D values and have high scatter. We therefore use only the 2D length and maximum width measurements to compute V, FT, and RFT. With this approach, apatite V, FT, and RFT values are all consistently overestimated by the 2D microscopy method, requiring correction factors of 0.74–0.83 (or 17 %–26 %), 0.91–0.99 (or 1 %–9 %), and 0.85–0.93 (or 7 %–15 %), respectively. The 1σ uncertainties in V, FT, and RFT are 20 %–23 %, 1 %–6 %, and 6 %–10 %, respectively. The primary control on the magnitude of the corrections and uncertainties is grain geometry, with grain size exerting additional control on FT uncertainty. Application of these corrections and uncertainties to a real dataset (N=24 AHe analyses) yields 1σ analytical and geometric uncertainties of 15 %–16 % in eU and 3 %–7 % in the corrected date. These geometric corrections and uncertainties are substantial and should not be ignored when reporting, plotting, and interpreting AHe datasets. The Geometric Correction Method (GCM) presented here provides a simple and practical tool for deriving more accurate FT and eU values and for incorporating this oft neglected geometric uncertainty into AHe dates.

Postnatal Inhibition of TrkB Kinase Reduces the Number of Phrenic Motor Neurons

During embryonic development, the number of phrenic motor neurons (PhMNs) declines precipitously during the final trimester and continues into the early postnatal period. When BDNF/TrkB signaling is absent in utero, animals do not survive after birth, most likely due to excessive loss of vital motor neurons (e.g., PhMNs). Far less is known about the early postnatal effect of BDNF/TrkB signaling. We have shown that BDNF/TrkB signaling is important in promoting PhMN neuroplasticity in mature rodents. Taken together, we hypothesized that inhibition of BDNF/TrkB signaling during early postnatal development results in fewer PhMNs. Chemo-genetic TrkBF616A mutant mice were used to induce inhibition of TrkB kinase activity in response to oral administration (via drinking water) of a phosphoprotein phosphatase 1 inhibitor derivative (1NMPP1). 1NMPP1-induced TrkB kinase inhibition was initiated at P0 and continued until P21 and compared to untreated control mice. At P28, PhMNs were retrogradely labeled via rhodamine phrenic nerve dip. One day later, mice were euthanized, perfused with 4% paraformaldehyde, and the cervical spinal cord was excised and processed for longitudinal cryosectioning (70 μm). Labeled PhMNs were imaged by 3D high resolution confocal microscopy. The number of PhMNs were counted. PhMN somal surface area and number of primary dendrites were also determined. 1NMPP1-induced inhibition of TrkB kinase resulted in 24% fewer PhMNs (P=0.01) and 21% more primary dendrites (P=0.0002) than control mice. The distribution of PhMN somal surface areas was not affected. We conclude that BDNF/TrkB signaling is essential in promoting survival of PhMNs in the early postnatal period. BDNF/TrkB signaling during postnatal development also plays a role in paring primary dendrites. While inhibition of TrkB kinase did not significantly alter the distribution of PhMN somal surface areas at P28, it is possible that further growth of PhMNs into maturity might have been altered as larger PhMNs emerge as a result of disproportionate growth beyond this age in mice. Supported by National Institutes of Health grants R01-AG044615 (GCS), R01-HL96750 (GCS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we have developed a cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 110 nm enough for the subsequent cryo-lamella fabrication. We have successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

Erosion evolution and mass loss of polyimide in AO environment using 3D Monte Carlo method

Polyimide films coated on the surface of satellites are exposed to atomic oxygen (AO) environment during service. This will suffer variation of microscopic structure and morphology, leading to corrosion and mass loss in macroscopic. In this work, a 3D undercutting model based on Monte Carlo method (MC) and Ray Tracing method was constructed, with which the AO related erosion feature of polyimide film was evaluated. Then, we predicted the evolution of erosion cross-section of the coated material with defect of different sizes and shapes exposed in higher AO fluence. The macroscopic mass loss was derived by the eroded microscopic volume and material density, during which the actual roughness was considered in our proposed undercutting model. This work provides insights into the study of microscopic 3D erosion morphology and connects with macroscopic mass loss in experiment at higher fluences.

Training procedure for scanning electron microscope 3D surface reconstruction using unsupervised domain adaptation with simulated data

Accurate metrology techniques for semiconductor devices are indispensable for controlling the manufacturing process. For instance, the dimensions of a transistor’s current channel (fin) are an important indicator of the device’s performance regarding switching voltages and parasitic capacities. We expand upon traditional 2D analysis by utilizing computer vision techniques for full-surface reconstruction. We propose a data-driven approach that predicts the dimensions, height and width (CD) values, of fin-like structures. During operation, the method solely requires experimental images from a scanning electron microscope of the patterns concerned. We introduce an unsupervised domain adaptation step to overcome the domain gap between experimental and simulated data. Our model is further fine-tuned with a height measurement from a second scatterometry sensor and optimized through a tailored training scheme for optimal performance. The proposed method results in accurate depth predictions, namely 100% accurate interwafer classification with an root-mean-squared error of 0.67 nm. The R2 of the intrawafer performance on height is between 0.59 and 0.70. Qualitative results also indicate that detailed surface features, such as corners, are accurately predicted. Our study shows that accurate z-metrology techniques can be viable for high-volume manufacturing.

3D microscope image acquisition method based on zoom objective.

Microscopy is being pursued to obtain richer and more accurate information, and there are many challenges in imaging depth and display dimension. In this paper, we propose a three-dimensional (3D) microscope acquisition method based on a zoom objective. It enables 3D imaging of thick microscopic specimens with continuous adjustable optical magnification. The zoom objective based on liquid lenses can quickly adjust the focal length, to expand the imaging depth and change the magnification by adjusting the voltage. Based on the zoom objective, an arc shooting mount is designed to accurately rotate the objective to obtain the parallax information of the specimen and generate parallax synthesis images for 3D display. A 3D display screen is used to verify the acquisition results. The experimental results show that the obtained parallax synthesis images can accurately and efficiently restore the 3D characteristics of the specimen. The proposed method has promising applications in industrial detection, microbial observation, medical surgery, and so on.

Large depth-of-field microscopic structured-light 3D imaging with focus stacking

State-of-the-art microscopic structured-light (SL) three-dimensional (3D) imaging systems typically use conventional lenses with a fixed focal length achieving a limited depth of field (DOF). This paper proposes to drastically increase the DOF of the microscopic 3D imaging by leveraging the focus stacking technique and developing a novel computational framework. We first capture fringe images with various camera focal lengths using an electrically tunable lens (ETL) and align the recovered phase maps using phase constraints. Then, we extract the focused pixel phase using fringe contrast and stitch them into an all-in-focus phase map via energy minimization. Finally, we reconstruct the 3D shape using the all-in-focus phase map. Experimental results demonstrate that our proposed method can achieve a large DOF of approximately 2 mm and field of view (FOV) of approximately 4 mm × 3 mm with a pixel size at the object space of approximately 2.6 μm. The achieved DOF is approximately 10 × the DOF of the system without the proposed method.

The Influence of the Metalworking Fluid on Tool Wear in Milling Inconel 718

The understanding of tool wear mechanisms is fundamental to developing more productive machining processes. Therefore, the influence of different metalworking fluids on tool wear is studied when milling Inconel 718 using a rotating dynamometer, a stationary dynamometer, and a 3D microscope. A newly developed force model allows the interpretation of more than the maximal cutting force and explains the relation between tribological conditions, tool wear and process forces. In a novel setup, the coolant type was changed in the middle of the tool life to differentiate the direct influence of the coolant on the process forces and the indirect effect via tool wear. By fitting the model to the data, it is found that the metalworking fluid and the process parameters not only influence the wear rate globally, but also lead to different wear mechanisms. The 3D data of worn cutting edges confirms the existence of different wear mechanisms.

Experimental Investigation on Self-Healing Efficiency of Mortar with Bacillus subtilis and Bacillus cereus

With the development of cracks on their surfaces, mortar’s service life dramatically shortens. Self-healing concrete by Microbiologically Induced Calcite Precipitation (MICP) is one of the high-tech concretes being used to address these issues. This type of mortar can start biological processes to repair itself and deal with its cracks. The self-healing effectiveness of two different bacteria, in this paper, Bacillus subtilis and Bacillus cereus, added to the mortar is examined experimentally. In order to conduct this investigation, artificial cracks were made in the mortar. A 3D optical microscope was used to take repeated pictures of the cracked mortar. The mechanical and durability tests conducted on the bacterial mortar were used to gauge the efficacy of self-healing. Mortar samples were left for 7, 14, and 28 days to cure. Compressive strength, flexural strength, water absorption, and sorptivity were measured during various times of the curing process. The test results showed that the mortar with bacteria had an increase in strength and durability compared to the control mix. In the sample of mortar containing bacteria, Bacillus subtilis and Bacillus cereus a maximum increase of 17.29% and 11.31% in flexural strength, 17.77% and 12.84% in compressive strength were observed and a 34.48% and 26.43% decrease in water absorption in the mortar sample containing bacteria, Bacillus subtilis and Bacillus cereus at 28 days, respectively. The results of the mortar absorption test showed that the addition of bacteria to the mortar matrix significantly reduced the primary and secondary absorption rates of bacterial mortars B-M-1 and B-M-2. Using a 3D light microscope, the cracks in the bacterial mortar showed that larger amounts of white crystal precipitates were generated that nearly filled the surface of the crack. Overall, Bacillus subtilis appeared to be superior to Bacillus cereus based on the results of mechanical and mortar durability tests because calcium carbonate precipitates more rapidly.

Femtosecond Laser Direct‐Writing Ablation of Transparent Fluoropolymer Toward Super‐Resolution Imaging of Cell Movements

AbstractLive imaging of living cells in 3D micro/nano‐environment is important for advanced biological studies. An issue for super‐resolution imaging is refractive index mismatch between the materials used for imaging platform and medium containing cells (water). Fluoropolymer CYTOP is the ideal platform material due to its refractive index close to that of water. Herein, femtosecond laser (220 fs) is exploited to ablate CYTOP at three laser wavelengths of 257, 515, and 1030 nm. Linear power dependences of the ablation rate obtained for all wavelengths indicate that ablation mechanisms of CYTOP are attributed to the production of fragmented CYTOPs (frag‐CYTOPs) inside the film to induce efficient light absorption for subsequent laser pulses. The frag‐CYTOPs can effectively absorb UV–Vis light, whereas those left behind generate green fluorescence. Importantly, the shortest wavelength of 257 nm offers the best ablation quality in terms of surface smoothness and low fluorescence with the highest ablation rate. We elucidate that the refractive‐index matching of CYTOP/water is effective for clear imaging of shapes/fluorescence of cells on 3D structures, e.g., HeLa cells on V‐shaped trenches. The fabrication of a microscopic 3D fluorescence code tag embedded in the CYTOP films is further demonstrated.

Focused and coherent X-ray beams for advanced microscopies

The use of focused X-ray beams in the micron and sub-micron range has fostered the progress of a wide range of scanning microscopy approaches that exploit the numerous interactions of X-rays with matter. These methods are becoming crucial for an increasing number of research fields, such as energy materials, electronic devices, biology, to cite a few. Nanobeams produced at 4th generation synchrotrons have coherence properties that make them ideal for a 3D diffraction-based microscopy with unique sensitivity to strain and defects in crystalline materials and allow delving into materials heterogeneities. We provide here a short overview on the use of nanobeams for diffraction experiments, and perspectives for their use in spectroscopy studies.

Programmable Aperture Light‐Field Microscopy

AbstractA computational three‐dimensional (3D) microscopy technique, termed programmable aperture light‐field microscopy (PALFM), for motion‐free, high‐resolution volumetric imaging of fluorescent or self‐luminous samples is proposed. The well‐known Fourier slice theorem is extended to incoherent tomographic imaging, which states that a detected image under an ideal aperture corresponds to a central slice in the 3D object spectrum, so the spectrum coverage can be accomplished based on the motion‐free aperture modulation. When further considering frequency extension and coverage, a hybrid aperture modulation scheme is designed consisting of non‐centrosymmetric circular and annular apertures for high‐efficiency, non‐ambiguous depth discrimination. A PALFM system with an easy‐to‐build programmable aperture module attached to an off‐the‐shelf inverted fluorescence microscope is constructed, where annular apertures can manipulate Bessel‐like beams for spectrum modulation. Experimental results on near‐diffraction‐limited imaging of a resolution target across a large depth range and high‐resolution, multi‐color 3D imaging of a mouse kidney section verify the validity and effectiveness of PALFM. High‐speed, long‐term time‐lapse volumetric imaging of HeLa cells in vitro further demonstrates that PALFM is a promising tomographic imaging tool for studying dynamic cellular processes and events without requiring complicated sample rotation or beam scanning.

Visual acuity of Empoasca onukii (Hemiptera, Cicadellidae)

Empoasca onukii is a common tea plant pest with a preference for the color yellow. Past work has shown that host leaf color is a key cue for habitat location for E. onukii. Before studying the effect of foliage shape, size, or texture on habitat localization, it is necessary to determine the visual acuity and effective viewing distance of E. onukii. In this study, a combination of 3D microscopy and X-ray microtomography showed that visual acuity did not significantly differ between females and males, but there were significant differences in the visual acuity and optical sensitivity among five regions of E. onukii's compound eyes. The dorsal ommatidia had the highest visual acuity at 0.28 cycles per degree (cpd) but the lowest optical sensitivity (0.02 μm2sr), which indicated a trade-off between visual resolution and optical sensitivity for E. onukii. The visual acuity determined from the behavioral experiment was 0.14 cpd; E. onukii exhibited low-resolution vision and could only distinguish the units in a yellow/red pattern within 30 cm. Therefore, visual acuity contributes to the limited ability of E. onukii to distinguish the visual details of a distant target, which might be perceived as a lump of blurred color of intermediate brightness.