The measurement of the volume of blood cells is important for clinical diagnosis and patient management. While digital holography microscopy has been used to obtain such information, previous off-axis setups usually involve a separated reference beam and are thus not very easy to implement. Here, we use the simple in-line Gabor setup without separation of a reference beam to measure the shape and volume of cells mounted on glass slides. Inherent to the in-line holograms, the reconstructed phase of the object is affected by the virtual image noise, producing errors in the cell volume measurement. We optimized our approach to use a single hologram without phase retrieval, increasing distance between cell and hologram plane to reduce the measurement error of cell volume to less than 6% in some instances. Therefore, the in-line Gabor setup can be a useful and simple tool to obtain volumetric and morphologic cellular information.
Alveolar bone repair after tooth extraction is essential after oral surgeries. Various grafting materials are used to promote the regeneration of lost alveolar bone. This study analysed the morphological features of the tissue regeneration process using deproteinized bovine bone mineral (DBBM). DBBM was used to densely fill the extraction sockets in beagle dogs. Following resin casting of the vasculature, stereomicroscopy and scanning electron microscopy were used to observe blood vessels and hard tissues in haematoxylin and eosin-stained sections on postoperative days 14, 30 and 90 in conjunction with vascular endothelial growth factor (VEGF) immunostaining to evaluate alveolar bone vascularization. On day 14 post-operation, the DBBM granules tightly filled the extraction sockets, maintained alveolar margin height and formed a scaffold for aiding angiogenesis and new bone formation. On day 30, new bone formation was observed around the DBBM granules. By day 90, bone tissue regeneration progressed in both groups but was more pronounced in the DBBM group. Alveolar margin height was maintained in the DBBM group throughout the study. Furthermore, VEGF expression in the DBBM group was detected around newly formed bone. We conclude that DBBM acts as a suitable scaffold for new bone generation, as well as angiogenesis around healing alveolar bone, and that it has the potential to play a key role in vascularization and bone formation.
The microstructure, chemical composition and mechanical strength of heterogeneous materials of mantis shrimp (Oratosquilla oratoria) saddle were studied. As the key component of the striking system, the saddle comprised two distinct layers including outer layer and inner layer. The outer layer contained blocky microtubules and exhibited compact appearance. The inner layer presented a typical periodic lamellar structure. Due to the change of the thickness of the mineralized outer layer, the organic multilamellar structure became the foundation and enhanced the connection strength (4.55 MPa) at the connect regions between the saddle and merus exoskeleton and membrane, respectively. In the process of fracture, the lamellar structure dispersed the stress effectively by the change of the crack deflection direction and the microfibrils ordered arrangement. The exploration of mantis shrimp saddle region is beneficial to understand the striking system and provided the possibility for the stable connection of heterogeneous materials in engineering fields. The microstructure, heterogeneous material connection characteristics and high mechanical strength of saddle provide bionic models for the preparation of fiber-reinforced resin composites and soft composites.
Abstract The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized by both small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind weakly to specific modifications, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformational changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms.
Here we describe the development and application of miniature integrated microscopes (miniscopes) paired with microendoscopes that allow for the visualization and manipulation of neural circuits in superficial and subcortical brain regions in freely behaving animals. Over the past decade the miniscope platform has expanded to include simultaneous optogenetic capabilities, electrically-tunable lenses that enable multi-plane imaging, color-corrected optics, and an integrated data acquisition platform that streamlines multimodal experiments. Miniscopes have given researchers an unprecedented ability to monitor hundreds to thousands of genetically-defined neurons from weeks to months in both healthy and diseased animal brains. Sophisticated algorithms that take advantage of constrained matrix factorization allow for background estimation and reliable cell identification, greatly improving the reliability and scalability of source extraction for large imaging datasets. Data generated from miniscopes have empowered researchers to investigate the neural circuit underpinnings of a wide array of behaviors that cannot be studied under head-fixed conditions, such as sleep, reward seeking, learning and memory, social behaviors, and feeding. Importantly, the miniscope has broadened our understanding of how neural circuits can go awry in animal models of progressive neurological disorders, such as Parkinson's disease. Continued miniscope development, including the ability to record from multiple populations of cells simultaneously, along with continued multimodal integration of techniques such as electrophysiology, will allow for deeper understanding into the neural circuits that underlie complex and naturalistic behavior.
Until now, studies on nail diseases have been performed through microscopic diagnosis and microscopic computed tomography (micro-CT). However, these kinds of conventional methods have some limitations. Firstly, the microscopic method is considered the gold standard for medical diagnosis. However, due to the use of fluorescent materials, the sample is damaged and it takes a long time to get results. Secondly, while micro-CT is a noninvasive method to get inner structure images of the sample with high resolution, the penetration and spatial resolution are insufficient for studying the microstructures of the sample, such as the sponge bone and the muscle fibers. In contrast, synchrotron radiation (SR) X-ray imaging technology has the advantage of very vividly demonstrating the anatomic structure of the sample with high penetration, sensitivity and resolution. In this study, we compared the optical microscopic method using hematoxylin and eosin staining and SR imaging to analyze the nail tissue in a mouse model. The results showed that SR could depict the inner structures of a mouse nail without any physical damage. Additionally, we could divide the important anatomical structures of the nail unit into three parts with three-dimensional (3D) images: the nail bed, nail matrix and hyponychium. The images showed that SR could be used for analyzing nails by visualizing the relatively clear and medically semantic structures in a 3D section. We expect that the results of this study will be applied to study nail diseases and conduct pharmaceutical research on their treatment.
The stability of beta-precipitates in the Zr-1Nb alloy has been studied under Ne ion irradiation of energy 250 keV by in situ transmission electron microscope as a function of irradiation dose. The irradiation was carried out up to similar to 136 dpa at 573 K. Microstructural investigations have shown that up to similar to 38 dpa, precipitates showed an increase in size, and for irradiation doses >38 dpa, the size of the precipitates was noticed to reduce. Post-irradiation energy-dispersive spectrometry of the specimens revealed the Nb concentration throughout the matrix to be similar to 0.8-1.5%. Three-dimensional atom probe tomography was also carried out for irradiated specimens to look for the presence of any nanoclusters. However, Nb clustering was not observed in the specimens. It is proposed that the dissolution of the precipitates may be facilitated by an increase in the solubility limit of Nb in Zr caused by irradiation. The solubility limit may increase by the introduction of defects generated by irradiation and by the destabilization of the beta-phase. This may result in back-diffusion of Nb atoms to the matrix by radiation-enhanced diffusion to lower the strain produced by the defects, resulting in the dissolution of the precipitates.
This is the first report on analyzing the chemical state of Li-ion battery electrodes at different states of charge by using a wavelength-dispersive spectrometer, which has a two-order improved energy resolution in the soft X-ray energy region compared with that of a conventional energy-dispersive X-ray analyzer. Electrodes containing LiMn1.5Ni0.5O4 were charged to prepare Li0.5Mn1.5Ni0.5O4 and λ-Mn0.75Ni0.25O2. The soft X-ray emission spectra obtained from those materials show that the O-K emission signal was drastically decreased throughout the charging process. This suggests that O-2p electron contributed to the electrochemical oxidation. The density of states and Bader charge evaluated from ab initio calculation support this result.
An image identification method was developed with the aid of a deep convolutional neural network (CNN) and applied to the analysis of inorganic particles using electron holography. Despite significant variation in the shapes of α-Fe2O3 particles that were observed by transmission electron microscopy, this CNN-based method could be used to identify isolated, spindle-shaped particles that were distinct from other particles that had undergone pairing and/or agglomeration. The averaging of images of these isolated particles provided a significant improvement in the phase analysis precision of the electron holography observations. This method is expected to be helpful in the analysis of weak electromagnetic fields generated by nanoparticles showing only small phase shifts.
Operando observation using spin-polarized scanning electron microscopy (spin SEM) has been demonstrated by detecting changes in the magnetization in the writing head of a hard disk drive (HDD) during operation. A current-applying system developed for use in the sample stage of a spin SEM enables imaging of the magnetization changes in the writing head of an HDD while the writing head is activated. Focused ion beam (FIB) technology is used to fabricate electric contacts between the head terminals and the sample holder electrodes. Tungsten film is deposited by FIB technology on the insulator around the writing head to prevent electrostatic charge buildup in the insulators during SEM measurement. This system is well suited for studying the characteristics of writing heads in HDDs in an activated state.
Scanning ion conductance microscopy (SICM) is useful for imaging soft and fragile biological samples in liquids because it probes the samples' surface topography by detecting ion currents under non-contact and force-free conditions. SICM acquires the surface topographical height by detecting the ion current reduction that occurs when an electrolyte-filled glass nanopipette approaches the sample surface. However, most biological materials have electrically charged surfaces in liquid environments, which sometimes affect the behavior of the ion currents detected by SICM and, especially, make topography measurements difficult. For measuring such charged samples, we propose a novel imaging method that uses a double-barrel nanopipette as an SICM probe. The ion current between the two apertures of the nanopipette desensitizes the surface charge effect on imaging. In this study, metaphase chromosomes of Indian muntjac were imaged by this technique because, owing to their strongly negatively charged surfaces in phosphate-buffered saline, it is difficult to obtain the topography of the chromosomes by the conventional SICM with a single-aperture nanopipette. Using the proposed method with a double-barrel nanopipette, the surfaces of the chromosomes were successfully measured, without any surface charge confounder. Since the detailed imaging of sample topography can be performed in physiological liquid conditions regardless of the sample charge, it is expected to be used for analyzing the high-order structure of chromosomes in relation to their dynamic changes in the cell division.
In transmission electron microscope (TEM), both the amplitude and the phase of electron beam change when electrons traverse a specimen. The amplitude is easily obtained by the square root of the intensity of a TEM image, while the phase affects defocused images. In order to obtain the phase map and verify the theoretical model of the interaction between electron beam and specimen, a lot of simulations have to be performed by researchers. In this work, we have simulated defocus images of a SiC nanowire in TEM with the method of electron optics. Mean inner potential and charge distribution on the nanowire have been considered in the simulation. Besides, due to electron scattering, coherence loss of the electron beam has been introduced. A dynamic process with Bayesian optimization was used in the simulation. With the infocus image as input and by adjusting fitting parameters, the defocus image is determined with a reasonable charge distribution. The calculated defocus images are in a good agreement with the experimental ones. Here, we present a complete solution and verification method for solving nanoscale charge distribution in TEM.
Methodology for quantitative evaluation of electron radiation damage and calculation of tolerable electron dose was developed to achieve damage-less scanning electron microscope (SEM) observation of beam-sensitive polymer film. The radiation damage is typically evaluated with visual impressions of SEM images; however, this method may be unreliable because observer's subjectivity may affect the results. Evaluation with quantitative value is crucial to improve reliability. In this study, the radiation damage was evaluated by using normalized correlative coefficient (RNCC) between an initial frame and latter frames of the multiple SEM images that were taken consecutively. Tolerable dose was obtained by defining a threshold point of RNCC where rapid reduction of RNCC started. A SEM image with less damage and acceptable signal-to-noise ratio was obtained by integrating the images from the initial frame to the tolerable frame.
Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called 'Durcupan™'. Specimens were embedded in Durcupan™ or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan™ was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.
Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.
This paper presents a quick and straightforward method to evaluate image distortion in scanning electron microscopy using a certified reference material (CRM) as a test specimen. The CRM has a square dot-array structure, whose dot-pitch has an accredited value. By calculating the distance between each dot of the CRM via image analysis, we can detect the distortion in the image as variations of dot interval. Furthermore, by considering the uncertainty of the certified value, it is possible to quantitatively evaluate the significance of the distortion in the image. This method enables us to easily estimate the uncertainty from image distortion, which can improve the reliability of measurement by scanning electron microscopy.
Degradation of the crystalline quality of transmission electron microscopy specimens in silicon prepared with different conditions has been examined using convergent-beam electron diffraction (CBED). The specimens are prepared using focused ion beam (FIB) with different accelerating voltages, Ar-ion milling and crushing method. Symmetry breaking of CBED patterns was quantitatively evaluated by symmetry breaking index S, which has been previously reported. The degradation and inhomogeneity of the FIB specimen were suppressed by decreasing the accelerating voltages of the FIB fabrication in the final process.
Nickel nanocrystals have received much attention for their ferromagnetic properties. The crystal properties are strongly dependent on their facets and therefore detailed study of their morphology, facets and orientation is critical for magnetic applications. In this work, equilibrium crystal shapes of self-assembled nickel nanocrystals on the (111) termination of strontium titanate (SrTiO3) at room temperature and under ultra-high vacuum (UHV) conditions have been investigated using scanning tunneling microscope (STM). SrTiO3 (111) substrate was sputtered (0.5 keV, 2.5 µA, 10 min) and annealed (900 °C, 1 h) under UHV conditions. Three different periodicities were observed: (2.21 ± 0.01) nm corresponding to (4 × 4) reconstruction, (3.31 ± 0.02) nm corresponding to (6 × 6) reconstruction, and (2.85 ± 0.05) nm, rotated at 30° with respect to (4 × 4) reconstruction, corresponding to (3√3 × 3√3)R30° reconstruction. Nickel (~1 ML) was deposited using an e-beam evaporator on the substrate preheated to 320 °C and the sample was post-annealed multiple times. Nickel took platonic shapes of supported icosahedron comprising of (111) facets and truncated octahedron comprising of (001) and (111) facets. Based on surface energy ratios of truncated octahedrons at equilibrium the work of adhesion was calculated to be (3.889 ± 0.167) J/m2.
Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 mu m x 40 mu m) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.
We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.
