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Microscope Cells Containing Multiple Micromachined Wells

Tech Briefs, May 2003 19 Manufacturing Microscope Cells Containing Multiple Micromachined Wells The cost per cell has been reduced substantially. John H. Glenn Research Center, Cleveland, Ohio An improved design for multiple-well microscope cells and an associated improved method of fabricating them have been devised. [As used here, "well" denotes a cavity that has a volume of about 1 or 2 L and that is used to hold a sample for examination under a microscope. As used here, "cell" denotes a laminate, based on a standard 1- by 3-in. (2.54- by 7.62-cm) microscope slide, that comprises (1) the slide as the lower layer, (2) an intermediate layer that contains holes that serve as the wells, and (3) a top layer that either consists of, or is similar to, a standard microscope-slide cover slip.] The improved design and method of fabrication make it possible to increase (relative to a prior design and method of fabrication) the number of wells per cell while reducing the fabrication loss and reducing the cost per cell to about one-tenth of the prior value. In the prior design and method, the slide, well, and cover-slip layers were made from silicate glass. The fabrication of each cell was a labor-intensive process that included precise cutting and grinding of the glass components, fusing of the glass components, and then more grinding and polishing to obtain desired dimensions. Cells of the prior design were expensive and fragile, the rate of loss in fabrication was high, and the nature of the glass made it difficult to increase the number of cells per well. Efforts to execute alternative prior designs in plastic have not yielded satisfactory results because, for typical applications, plastics are not sufficiently thermally or chemically stable, not sufficiently optically clear, and/or not hard enough to resist scratching. The figure depicts a cell of the present improved type. The slide and cover-slip layers are made of a low-thermal-expansion glass (Pyrex(TradeMark) or equivalent) and the intermediate (well layer) is made of SiO2 - a combination of materials that results in a laminate stronger than one made from layers of silicate glass. Before the layers are assembled into the laminate, the SiO2 layer is micromachined to form the wells plus shallow grooves that, when subsequently covered with the cover slip, become capillary channels that are used to fill the wells with samples. The micromachining is accomplished by use of the same patterning and etching techniques used to fabricate microelectromechanical systems (MEMS).

Turner, Walter↗

Common-Mode Digital Holographic Microscope

A lens-less digital holographic microscope, a reflective digital holographic microscope, and a digital holographic microscope including a plurality of lenses. In one example, the digital holographic microscope includes a single mode fiber collimated light source which provides illumination for both the `science` and `reference` arms, a pair of microscope objectives located side-by side, and illuminated by the common beam, a relay lens whose center is between the two objectives, and a focal plane element where the interference pattern is measured.

Wallace, James K.↗

An interchangeable scanning Hall probe/scanning SQUID microscope

We have constructed a scanning probe microscope for magnetic imaging, which can function as a scanning Hall probe microscope (SHPM) and as a scanning SQUID microscope (SSM). The scanning scheme, applicable to SHPM and SSM, consists of a mechanical positioning (sub) micron-XY stage and a flexible direct contact to the sample without a feedback control system for the Z-axis. With the interchangeable capability of operating two distinct scanning modes, our microscope can incorporate the advantageous functionalities of the SHPM and SSM with large scan range up to millimeter, high spatial resolution (⩽4 μm), and high field sensitivity in a wide range of temperature (4.2 K-300 K) and magnetic field (10{sup −7} T-1 T). To demonstrate the capabilities of the system, we present magnetic images scanned with SHPM and SSM, including a RbFeB magnet and a nickel grid pattern at room temperature, surface magnetic domain structures of a La{sub 2/3}Ca{sub 1/3}MnO{sub 3} thin film at 77 K, and superconducting vortices in a striped niobium film at 4.2 K.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Influence of mechanical noise inside a scanning electron microscope

The scanning electron microscope is becoming a popular tool to perform tasks that require positioning, manipulation, characterization, and assembly of micro-components. However, some of these applications require a higher level of performance with respect to dynamics and precision of positioning. One limiting factor is the presence of unidentified noises and disturbances. This work aims to study the influence of mechanical disturbances generated by the environment and by the microscope, identifying how these can affect elements in the vacuum chamber. To achieve this objective, a dedicated setup, including a high-resolution vibrometer, was built inside the microscope. This work led to the identification and quantification of main disturbances and noise sources acting on a scanning electron microscope. Furthermore, the effects of external acoustic excitations were analysed. Potential applications of these results include noise compensation and real-time control for high accuracy tasks.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Compact scanning transmission x-ray microscope at the photon factory

We report the design and performance of a compact scanning transmission X-ray microscope developed at the Photon Factory. Piezo-driven linear stages are used as coarse stages of the microscope to realize excellent compactness, mobility, and vibrational and thermal stability. An X-ray beam with an intensity of ∼10{sup 7} photons/s was focused to a diameter of ∼40 nm at the sample. At the soft X-ray undulator beamline used with the microscope, a wide range of photon energies (250–1600 eV) is available. The microscope has been used to research energy materials and in environmental sciences.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Set-up of a high-resolution 300 mK atomic force microscope in an ultra-high vacuum compatible {sup 3}He/10 T cryostat

The design of an atomic force microscope with an all-fiber interferometric detection scheme capable of atomic resolution at about 500 mK is presented. The microscope body is connected to a small pumped {sup 3}He reservoir with a base temperature of about 300 mK. The bakeable insert with the cooling stage can be moved from its measurement position inside the bore of a superconducting 10 T magnet into an ultra-high vacuum chamber, where the tip and sample can be exchanged in situ. Moreover, single atoms or molecules can be evaporated onto a cold substrate located inside the microscope. Two side chambers are equipped with standard surface preparation and surface analysis tools. The performance of the microscope at low temperatures is demonstrated by resolving single Co atoms on Mn/W(110) and by showing atomic resolution on NaCl(001).

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Superresolution effect on a microstep phase image in a laser heterodyne microscope

The possibility of achieving superresolution on a microstep phase image in a laser scanning differential heterodyne microscope is studied both heoretically and experimentally. The superresolution is estimated as the width ratio for the amplitude and phase components of the microscope response measured at half the height of the corresponding parts of the response. It is shown theoretically that superresolution greatly exceeding unity can be achieved for an object in the form of a phase microstep introducing a phase shift equal to π. Superresolution of ∼2 is experimentally obtained for certified test micro-objects. A possibility of tuning a test sample into the superresolution regime by shifting a point photodetector in the microscope’s Fourier plane is demonstrated. (laser microscope)

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

TH-CD-201-07: Experimentally Investigating Proton Energy Deposition On the Microscopic Scale Using Fluorescence Nuclear Track Detectors

Purpose: In order to further understand the interplay between proton physics and radiobiology it is necessary to consider proton energy deposition on the microscopic scale. In this work we used Fluorescent Nuclear Track Detectors (FNTDs) to experimentally investigate proton energy deposition, track-by-track. Methods: We irradiated 8×4×0.5mm{sup 3} FNTD chips (Landauer Inc) at seven water depths along a pristine proton Bragg peak with range=12cm. After irradiation, the FNTDs were scanned using a confocal microscope (FV1200, Olympus) with a high-power red laser and an oil-immersion objective lens (UPLSAPO60XO, NA=1.35). 10 slice image stacks were acquired with a slice-thickness of 2µm at multiple positions across each FNTD. Image-based analyses of track radius and track “mass” (integrated signal intensity) were performed using trackpy. For comparison, Monte Carlo simulated data were obtained using TOPAS and TOPAS-nBio. Results: Excellent correlation was observed between median track mass and TOPAS dose-averaged linear energy transfer. The resolution of the imaging system was determined insufficient to detect a relationship between track radius and exposure depth. Histograms of track mass (i) displayed strong repeatability across positions within an FNTD and (ii) varied in peak position and shape as a function of depth. TOPAS-nBio simulations implemented on the nanometer scale using physics lists from GEANT4-DNA yielded energy deposition distributions for individual protons and electrons scored within a virtual FNTD. Good agreement was found between these simulated datasets and the FNTD track mass distributions. Conclusion: Robust experimental measurements of the integral energy deposited by individual proton tracks can be performed using FNTDs. Monte Carlo simulations offer an exceedingly powerful approach to the quantification of proton energy deposition on the microscopic scale, but whilst they have been well validated at the macroscopic level, their microscopic validation is far from complete. Our results demonstrate that FNTD-based study can play an important role in addressing this deficit. Tracy Underwood gratefully acknowledges the support of the European Commission under an FP7 Marie Curie International Outgoing Fellowship for Career Development (#630064).

60 APPLIED LIFE SCIENCES↗

Agentic traffic intelligence: Augmented human-in-the-loop scenario generation for microscopic traffic simulation

Traditional microscopic traffic simulation generation often relies on static datasets and manual design, limiting its ability to simulate complex conditions easily. This paper presents a novel framework, Agentic Traffic Intelligence, which combines human approval large language models (LLMs), the Real-Twin tool, and multi-agent systems to perform realistic microscopic traffic simulation scenario generation. The proposed framework incorporates human-in-the-loop (HIL) control, retrieval-augmented generation (RAG), and multi-agent control mechanisms. HIL mechanisms are used to guide multiple LLMs focused on attributes for microscopic simulation generation and to improve the interpretability and transparency of LLM execution for users. RAG enhances context extraction by dynamically integrating external knowledge sources for traffic scenario generation foundations. A multi-agent architecture with supervisory control coordinates the interaction of simulation components, including traffic simulators, control logic, and calibration tools. This enables the synthesis of simulation-ready scenarios that reflect dynamic demand profiles and behavior controls. Furthermore, the framework fuses multisource traffic data with unstructured context and supports iterative refinement through interactive user feedback. Validated through microscopic simulation using Simulation of Urban Mobility, the generated scenarios demonstrate high-fidelity network generation with inflow and turn movement and behavioral calibration, offering a robust and efficient tool for stress-testing and optimizing urban mobility systems.

Hierarchical multi-agent control↗

Microscopic diffusion processes measured in living planarians

Living planarian flatworms were probed using quasielastic neutron scattering to measure, on the pico-to-nanosecond time scale and nanometer length scale, microscopic diffusion of water and cell constituents in the planarians. Measurable microscopic diffusivities were surprisingly well defined in such a complex system as living animals. The overall variation in the microscopic diffusivity of cell constituents was found to be far lower than the variation in the microscopic diffusivity of water in planarians in a temperature range of 284.5 to 304.1K.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Enhancing traffic safety analysis with digital twin technology: integrating vehicle dynamics and environmental factors into microscopic traffic simulation

Traffic safety is a critical concern in transportation engineering and urban planning. Traditional traffic safety analysis requires trained observers to collect data in the field, which is time-consuming, labor-intensive, and sometimes inaccurate. In recent years, microscopic traffic simulation, which simulates individual vehicles’ movements within a transportation network, have been utilized to study traffic safety. However, microscopic traffic simulation only focuses on traffic-related factors, such as traffic volume, traffic signals, and lane configurations, neglecting vehicle dynamics and environment-related factors like weather and lighting conditions, which can significantly impact traffic safety. In light of this, this paper explores the application of digital twin technology in traffic safety analysis, integrating vehicle simulators, which consider vehicle dynamics and environmental factors, and microscopic traffic simulators, which simulate the operations of traffic flow, for enhanced safety evaluations. Various scenarios, including different weather conditions and visibility levels, are simulated using a digital twin of a road segment in Tuscaloosa, Alabama. The simulations employ Surrogate Safety Measures (SSMs) like Time to Collision (TTC) and Deceleration Rate to Avoid a Crash (DRAC) to assess safety under varying conditions. The results demonstrate that traffic digital twin can identify potential safety issues that traditional microscopic simulation cannot, providing insights for improving traffic control strategies and transportation infrastructure to enhance traffic safety.

Civil engineering↗

Characterization of grain boundary conductivity of spin-sprayed ferrites using scanning microwave microscope

Grain boundary electrical conductivity of ferrite materials has been characterized using scanning microwave microscope. Structural, electrical, and magnetic properties of Fe{sub 3}O{sub 4} spin-sprayed thin films onto glass substrates for different length of growth times were investigated using a scanning microwave microscope, an atomic force microscope, a four-point probe measurement, and a made in house transmission line based magnetic permeameter. The real part of the magnetic permeability shows almost constant between 10 and 300 MHz. As the Fe{sub 3}O{sub 4} film thickness increases, the grain size becomes larger, leading to a higher DC conductivity. However, the loss in the Fe{sub 3}O{sub 4} films at high frequency does not increase correspondingly. By measuring the reflection coefficient s{sub 11} from the scanning microwave microscope, it turns out that the grain boundaries of the Fe{sub 3}O{sub 4} films exhibit higher electric conductivity than the grains, which contributes loss at radio frequencies. This result will provide guidance for further improvement of low loss ferrite materials for high frequency applications.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Microscopic Theory of Nuclear Fission: A Review

This paper reviews how nuclear fission is described within nuclear density functional theory. A distinction should be made between spontaneous fission, where half-lives are the main observables and quantum tunnelling the essential concept, and induced fission, where the focus is on fragment properties and explicitly time-dependent approaches are often invoked. Overall, the cornerstone of the density functional theory approach to fission is the energy density functional formalism. The basic tenets of this method, including some well-known tools such as the Hartree–Fock–Bogoliubov (HFB) theory, effective two-body nuclear potentials such as the Skyrme and Gogny force, finite-temperature extensions and beyond mean-field corrections, are presented succinctly. The energy density functional approach is often combined with the hypothesis that the time-scale of the large amplitude collective motion driving the system to fission is slow compared to typical time-scales of nucleons inside the nucleus. In practice, this hypothesis of adiabaticity is implemented by introducing (a few) collective variables and mapping out the many-body Schrödinger equation into a collective Schrödinger-like equation for the nuclear wave-packet. The region of the collective space where the system transitions from one nucleus to two (or more) fragments defines what are called the scission configurations. The inertia tensor that enters the kinetic energy term of the collective Schrödinger-like equation is one of the most essential ingredients of the theory, since it includes the response of the system to small changes in the collective variables. For this reason, the two main approximations used to compute this inertia tensor, the adiabatic time-dependent HFB and the generator coordinate method, are presented in detail, both in their general formulation and in their most common approximations. The collective inertia tensor enters also the Wentzel–Kramers–Brillouin (WKB) formula used to extract spontaneous fission half-lives from multi-dimensional quantum tunnelling probabilities (For the sake of completeness, other approaches to tunnelling based on functional integrals are also briefly discussed, although there are very few applications.) It is also an important component of some of the time-dependent methods that have been used in fission studies. Concerning the latter, both the semi-classical approaches to time-dependent nuclear dynamics and more microscopic theories involving explicit quantum-many-body methods are presented. One of the hallmarks of the microscopic theory of fission is the tremendous amount of computing needed for practical applications. In particular, the successful implementation of the theories presented in this article requires a very precise numerical resolution of the HFB equations for large values of the collective variables. This aspect is often overlooked, and several sections are devoted to discussing the resolution of the HFB equations, especially in the context of very deformed nuclear shapes. In particular, the numerical precision and iterative methods employed to obtain the HFB solution are documented in detail. Finally, a selection of the most recent and representative results obtained for both spontaneous and induced fission is presented, with the goal of emphasizing the coherence of the microscopic approaches employed. In conclusion, although impressive progress has been achieved over the last two decades to understand fission microscopically, much work remains to be done. Several possible lines of research are outlined in the conclusion.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Microscopic origins of hydrodynamic transport in the type-II Weyl semimetal WP 2

A microscopic understanding of hydrodynamic transport in Dirac and Weyl semimetals has remained elusive in theoretical descriptions and experimental measurements. We investigate the structure and microscopic properties of transport in WP 2 , a type-II Weyl semimetal exhibiting hydrodynamic transport. Here, we characterize the nature of the microscopic properties of WP 2 as a function of temperature through ab initio calculations of the relevant scattering processes, including electron-phonon and electron-electron lifetimes, and provide equivalent calculations for copper as a point of reference. Additionally, we present an approach to calculate phonon drag, a mechanism invoked in recent experiments, through predictions of a phonon-mediated electron-electron lifetime. We show that the resistivity is very well described by the electron-phonon interaction alone, indicating WP 2 exhibits conventional metallic electrical behavior in which strong electron-electron correlations do not play a significant role. After establishing the zone-averaged behavior of the calculated lifetimes, we further investigate specific features of the spatial distribution of the electron-phonon lifetime across the Fermi surfaces of WP 2 to study possible scattering channels involved in hydrodynamic transport and quantify the degree of lifetime anisotropy in electron and hole pockets. This description of microscopic dynamics in WP 2 prompts additional investigation of specific scattering channels and indicates the importance of phonon interactions in understanding connections between transport in hydrodynamic materials and strongly correlated systems including unconventional metals.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

Absence of hindrance in a microscopic 12 C + 12 C fusion study

Studies of low-energy fusion of light nuclei are important in astrophysical modeling, with small variations in reaction rates having a large impact on nucleosynthesis yields. Due to the lack of experimental data at astrophysical energies, extrapolation and microscopic methods are needed to model fusion probabilities. To investigate deep sub-barrier 12 C+ 12 C fusion cross sections and establish trends for the S factor. Microscopic methods based on static Hartree-Fock and time-dependent Hartree-Fock (TDHF) mean-field theory are used to obtain 12 C+ 12 C ion-ion fusion potentials. Fusion cross sections and astrophysical S factors are then calculated using the incoming wave boundary condition method. Both density-constrained frozen Hartree-Fock (DCFHF) and density-constrained TDHF (DC-TDHF) predict a rising S factor at low energies, with DC-TDHF predicting a slight damping in the deep sub-barrier region (≈1 MeV). Comparison between DC-TDHF calculations and maximum experimental cross sections in the resonance peaks are good. However, the discrepancy in experimental low-energy results inhibits interpretation of the trend. Using the fully microscopic DCFHF and DC-TDHF methods, no S factor maximum is observed in the 12 C+ 12 C fusion reaction. In addition, no extreme sub-barrier hindrance is predicted at low energies. Furthermore, the development of a microscopic theory of fusion including resonance effects, as well as further experiments at lower energies must be done before the deep sub-barrier behavior of the reaction can be established.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Microscopic silicon-based lateral high-aspect-ratio structures for thin film conformality analysis

Film conformality is one of the major drivers for the interest in atomic layer deposition (ALD) processes. This work presents new silicon-based microscopic lateral high-aspect-ratio (LHAR) test structures for the analysis of the conformality of thin films deposited by ALD and by other chemical vapor deposition means. The microscopic LHAR structures consist of a lateral cavity inside silicon with a roof supported by pillars. The cavity length (e.g., 20–5000 μm) and cavity height (e.g., 200–1000 nm) can be varied, giving aspect ratios of, e.g., 20:1 to 25 000:1. Film conformality can be analyzed with the microscopic LHAR by several means, as demonstrated for the ALD Al{sub 2}O{sub 3} and TiO{sub 2} processes from Me{sub 3}Al/H{sub 2}O and TiCl{sub 4}/H{sub 2}O. The microscopic LHAR test structures introduced in this work expose a new parameter space for thin film conformality investigations expected to prove useful in the development, tuning and modeling of ALD and other chemical vapor deposition processes.

36 MATERIALS SCIENCE↗

Smart align -- A new tool for robust non-rigid registration of scanning microscope data

Many microscopic investigations of materials may benefit from the recording of multiple successive images. This can include techniques common to several types of microscopy such as frame averaging to improve signal-to-noise ratios (SNR) or time series to study dynamic processes or more specific applications. In the scanning transmission electron microscope, this might include focal series for optical sectioning or aberration measurement, beam damage studies or camera-length series to study the effects of strain; whilst in the scanning tunnelling microscope, this might include bias voltage series to probe local electronic structure. Whatever the application, such investigations must begin with the careful alignment of these data stacks, an operation that is not always trivial. In addition, the presence of low-frequency scanning distortions can introduce intra-image shifts to the data. Here, we describe an improved automated method of performing non-rigid registration customised for the challenges unique to scanned microscope data specifically addressing the issues of low-SNR data, images containing a large proportion of crystalline material and/or local features of interest such as dislocations or edges. Careful attention has been paid to artefact testing of the non-rigid registration method used, and the importance of this registration for the quantitative interpretation of feature intensities and positions is evaluated.

36 MATERIALS SCIENCE↗

How microscopic epistasis and clonal interference shape the fitness trajectory in a spin glass model of microbial long-term evolution

The adaptive dynamics of evolving microbial populations takes place on a complex fitness landscape generated by epistatic interactions. The population generically consists of multiple competing strains, a phenomenon known as clonal interference. Microscopic epistasis and clonal interference are central aspects of evolution in microbes, but their combined effects on the functional form of the population’s mean fitness are poorly understood. Here, we develop a computational method that resolves the full microscopic complexity of a simulated evolving population subject to a standard serial dilution protocol. Through extensive numerical experimentation, we find that stronger microscopic epistasis gives rise to fitness trajectories with slower growth independent of the number of competing strains, which we quantify with power-law fits and understand mechanistically via a random walk model that neglects dynamical correlations between genes. We show that increasing the level of clonal interference leads to fitness trajectories with faster growth (in functional form) without microscopic epistasis, but leaves the rate of growth invariant when epistasis is sufficiently strong, indicating that the role of clonal interference depends intimately on the underlying fitness landscape. The simulation package for this work may be found at https://github.com/nmboffi/spin_glass_evodyn .

59 BASIC BIOLOGICAL SCIENCES↗