Intra- and Interspecies Variability of Single-Cell Innate Fluorescence Signature of Microbial Cell

Here we analyzed the innate fluorescence signature of the single microbial cell, within both clonal and mixed populations of microorganisms. We found that even very similarly shaped cells differ noticeably in their autofluorescence features and that the innate fluorescence signatures change dynamically with growth phases. We demonstrated that machine learning models can be trained with a data set of single-cell innate fluorescence signatures to annotate cells according to their phenotypes and physiological status, for example, distinguishing a wild-type Aspergillus nidulans cell from its nitrogen metabolism mutant counterpart and log-phase cells from stationary-phase cells of Pseudomonas putida We developed a minimally invasive method (confocal reflection microscopy-assisted single-cell innate fluorescence [CRIF] analysis) to optically extract and catalog the innate cellular fluorescence signatures of each of the individual live microbial cells in a three-dimensional space. This technique represents a step forward from traditional techniques which analyze the innate fluorescence signatures at the population level and necessitate a clonal culture. Since the fluorescence signature is an innate property of a cell, our technique allows the prediction of the types or physiological status of intact and tag-free single cells, within a cell population distributed in a three-dimensional space. Our study presents a blueprint for a streamlined cell analysis where one can directly assess the potential phenotype of each single cell in a heterogenous population by its autofluorescence signature under a microscope, without cell tagging.IMPORTANCE A cell\u27s innate fluorescence signature is an assemblage of fluorescence signals emitted by diverse biomolecules within a cell. It is known that the innate fluoresce signature reflects various cellular properties and physiological statuses; thus, they can serve as a rich source of information in cell characterization as well as cell identification. However, conventional techniques focus on the analysis of the innate fluorescence signatures at the population level but not at the single-cell level and thus necessitate a clonal culture. In the present study, we developed a technique to analyze the innate fluorescence signature of a single microbial cell. Using this novel method, we found that even very similarly shaped cells differ noticeably in their autofluorescence features, and the innate fluorescence signature changes dynamically with growth phases. We also demonstrated that the different cell types can be classified accurately within a mixed population under a microscope at the resolution of a single cell, depending solely on the innate fluorescence signature information. We suggest that single-cell autofluoresce signature analysis is a promising tool to directly assess the taxonomic or physiological heterogeneity within a microbial population, without cell tagging

Transmission electron microscopy/electron energy loss spectroscopy measurements and ab initio calculation of local magnetic moments at nickel grain boundaries

We have determined local magnetic moments at nickel grain boundaries using a transmission electron microscopy/electron energy loss spectroscopy method assuming that the magnetic moment of Ni atoms is a linear function of the L3/L2 (white-line ratio) in the energy loss spectrum. The average magnetic moment measured in the grain interior was 0.55 μB, which agrees well with the calculated magnetic moment of pure nickel (0.62 μB). The local magnetic moments at the grain boundaries increased up to approximately 1.0 μB as the mis-orientation angle increased, and showed a maximum around 50°. The respective enhancement of local magnetic moments at the Σ5 (0.63 μB) and random (0.90 μB) grain boundaries in pure nickel was approximately 14 and 64% of the grain interior. In contrast, the average local magnetic moment at the (111) Σ3 grain boundary was found to be 0.55 μB and almost the same as that of the grain interior. These results are in good agreement with available ab initio calculations

Multimodal assessment of mechanically induced transformation in metastable multi‐phase steel using X‐ray nano‐tomography and pencil‐beam diffraction tomography

【研究成果】次世代自動車用鋼板の外力による内部組織の変化を直接観察 --複合X線CT解析技術の開発--. 京都大学プレスリリース. 2022-05-16.A combination of X-ray nano-tomography and pencil-beam diffraction tomography was utilized for multimodal assessment of the mechanically induced transformation of individual retained austenite grains during tensile deformation in a 0.1C-5Mn-1Si multi-phase steel. In the present study, a newly developed high energy (20 - 30 keV) and high resolution (spatial resolution of 0.16 µm in this study) X-ray nano-tomography technique was applied for the first time to the in-situ observation of a steel under external loading. The gradual transformation, plastic deformation, and rotation behaviour of the individual austenite grains were clearly observed in 3D. It was revealed that the early stage of the transformation was dominated by the stress-assisted transformation that can be associated with measured mechanical driving force, whilst the overall transformation was dominated by the strain-induced transformation that is interrelated with measured dislocation multiplication. The transformation behaviour of individual grains was classified according to their initial crystallographic orientation and size. Noteworthy was the high stability of coarse austenite grains (i.e., 2.5 μm or larger in diameter), contrary to past reports in the literature. Characteristic rotation behaviour and wide data dispersion were also observed in the case of the individual austenite grains. It was conclusively demonstrated that such characteristic behaviour partly originated from interactions with surrounding soft and hard phases. The origins of these characteristics are discussed by combining the image-based and diffraction-based information

High-energy x-ray nanotomography introducing an apodization Fresnel zone plate objective lens

In this study, high-energy x-ray nanotomography (nano-computed tomography, nano-CT) based on full-field x-ray microscopy was developed. Fine two-dimensional and three-dimensional (3D) structures with linewidths of 75 nm-100 nm were successfully resolved in the x-ray energy range of 15 keV-37.7 keV. The effective field of view was similar to 60 mu m, and the typical measurement time for one tomographic scan was 30 min-60 min. The optical system was established at the 250-m-long beamline 20XU of SPring-8 to realize greater than 100x magnification images. An apodization Fresnel zone plate (A-FZP), specifically developed for high-energy x-ray imaging, was used as the objective lens. The design of the A-FZP for high-energy imaging is discussed, and its diffraction efficiency distribution is evaluated. The spatial resolutions of this system at energies of 15 keV, 20 keV, 30 keV, and 37.7 keV were examined using a test object, and the measured values are shown to be in good agreement with theoretical values. High-energy x-ray nano-CT in combination with x-ray micro-CT is applied for 3D multiscale imaging. The entire bodies of bulky samples, similar to 1 mm in diameter, were measured with the micro-CT, and the nano-CT was used for nondestructive observation of regions of interest. Examples of multiscale CT measurements involving carbon steel, mouse bones, and a meteorite are discussed

Assessment of 3D Short Crack Closure in Ti-6Al-4V Alloy Utilizing Synchrotron X-ray Microtomography

Synchrotron X-ray microtomography was utilized to observe the complex 3D crack morphology and the closure behavior of a short crack in Ti-6Al-4V alloy. The aim of the study was to investigate the effect of the crack path evolution on the 3D short crack closure behavior. In situ fatigue tests at R = 0.1 were carried out using microtomography with a spatial resolution of 1 μm. The 3D crack morphology was observed in detail consisting of non-facets (zigzag), branching, and facets with deflection angles indicating the presence of mode II and mode III displacements. The crack grows with facet-like paths mainly in α grains as compared to the non-facet paths in the α+β grains. The change in the crack path from facet-like paths to non-facet-like paths in the larger crack front induces an increase in the fractional area of closed patches

Assessment of 3D Short Crack Closure in Ti-6Al-4V Alloy Utilizing Synchrotron X-ray Microtomography

Synchrotron X-ray microtomography was utilized to observe the complex 3D crack morphology and the closure behavior of a short crack in Ti-6Al-4V alloy. The aim of the study was to investigate the effect of the crack path evolution on the 3D short crack closure behavior. In situ fatigue tests at R = 0.1 were carried out using microtomography with a spatial resolution of 1 μm. The 3D crack morphology was observed in detail consisting of non-facets (zigzag), branching, and facets with deflection angles indicating the presence of mode II and mode III displacements. The crack grows with facet-like paths mainly in α grains as compared to the non-facet paths in the α+β grains. The change in the crack path from facet-like paths to non-facet-like paths in the larger crack front induces an increase in the fractional area of closed patches

A Surrogate Approach to Reveal Microstructural Mechanisms Controlling the 3D Short Crack Growth in a Ti-6Al-4V Alloy

Microstructural features and short crack growth behavior were characterized and linked in a Ti-6Al-4V by employing X-ray micro-tomography combined with EBSD serial sectioning. Statistical analysis was used to rank the contributing features to the crack behavior. Afterwards, by creating surrogate models, the microstructural mechanism controlling the short crack behavior were revealed. Short crack preferably grows inside the predominant α phase above the average microstructural fraction. A high number of grains in contact with cracked α grains elongated in the loading direction may impose a constraint on the crack opening resulting in low crack growth rates. As the crack front becomes larger, the increase in the shear stress field away from the cracked grain leads to crack bifurcations, resulting in a decrease in crack driving forces with low crack growth rates. This leads to a preferable growth in α+β phase and along the interface above the average microstructural fractions

A Surrogate Approach to Reveal Microstructural Mechanisms Controlling the 3D Short Crack Growth in a Ti-6Al-4V Alloy

Microstructural features and short crack growth behavior were characterized and linked in a Ti-6Al-4V by employing X-ray micro-tomography combined with EBSD serial sectioning. Statistical analysis was used to rank the contributing features to the crack behavior. Afterwards, by creating surrogate models, the microstructural mechanism controlling the short crack behavior were revealed. Short crack preferably grows inside the predominant α phase above the average microstructural fraction. A high number of grains in contact with cracked α grains elongated in the loading direction may impose a constraint on the crack opening resulting in low crack growth rates. As the crack front becomes larger, the increase in the shear stress field away from the cracked grain leads to crack bifurcations, resulting in a decrease in crack driving forces with low crack growth rates. This leads to a preferable growth in α+β phase and along the interface above the average microstructural fractions