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Abstract Genetically encoded Ca2+ indicators (GECIs) are versatile for live imaging of cellular activities. Besides the brightness and dynamic range of signal change of GECIs, Ca2+ affinity is another critical parameter for successful Ca2+ imaging, as the concentration range of Ca2+ dynamics differs from low nanomolar to sub-millimolar depending on the celltype and organism. However, ultrahigh-affinity GECIs, particularly the single fluorescent protein (1FP)-type, are lacking. Here, we report a simple strategy that increases Ca2+ affinity through the linker length optimization in topology mutants of existing 1FP-type GECIs. The resulting ultrahigh-affinity GECIs, CaMPARI-nano, BGECO-nano, and RCaMP-nano (K d = 17–25 nM), enable unique biological applications, including the detection of low nanomolar Ca2+ dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity.

Abstract Bacterial phytochromes are attractive molecular templates for engineering fluorescent proteins (FPs) because their near-infrared (NIR) emission significantly extends the spectral coverage of GFP-like FPs. Existing phytochrome-based FPs covalently bind heme-derived tetrapyrrole chromophores and exhibit constitutive fluorescence. Here we introduce Rep-miRFP, an NIR imaging probe derived from bacterial phytochrome, which interacts non-covalently and reversibly with biliverdin chromophore. In Rep-miRFP, the photobleached non-covalent adduct can be replenished with fresh biliverdin, restoring fluorescence. By exploiting this chromophore renewal capability, we demonstrate NIR PAINT nanoscopy in mammalian cells using Rep-miRFP.

Summary: cIPAD is a fluorescent indicator that allows the visualization of trans-interactions of clustered protocadherin (Pcdh), a cell adhesion molecule that mediates neuronal self-recognition. We describe steps for using HEK293T cells to visualize Pcdh trans-interactions across cells as a preliminary experiment before using dissociated mouse neurons. We then detail procedures for visualizing Pcdh trans-interactions between processes originating from the same neurons, which are considered as Pcdh-mediated neuronal self-recognition.For complete details on the use and execution of this protocol, please refer to Kanadome et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Background/Aims Endoscopic submucosal dissection (ESD) for superficial non-ampullary duodenal tumors (SNADETs) is associated with a high rate of en bloc resection. However, the technique for ESD remains challenging. Recent studies have demonstrated the effectiveness of S-O clips in colonic and gastric ESD. We evaluated the efficacy and safety of duodenal ESD using an S-O clip for SNADETs. Methods Consecutive patients who underwent ESD for SNADETs between January 2011 and December 2021 were retrospectively enrolled. Propensity score matching analysis was used to compare patients who underwent duodenal ESD with the S-O clip (S-O group) and those who underwent conventional ESD (control group). Intraoperative perforation rate was the primary outcome, while procedure time and R0 resection rate were the secondary outcomes. Results After propensity score matching, 16 pairs were created: 43 and 17 in the S-O and control groups, respectively. The intraoperative perforation rate in the S-O group was significantly lower than that in the control group (p=0.033). A significant difference was observed in the procedure time between the S-O and control groups (39±9 vs. 82±30 minutes, respectively; p=0.003). Conclusions The S-O clip reduced the intraoperative perforation rate and procedure time, which may be useful and effective in duodenal ESD.

Abstract Over the past century, understanding the nature of shock compression of condensed matter has been a major topic. About 20 years ago, a femtosecond laser emerged as a new shock-driver. Unlike conventional shock waves, a femtosecond laser-driven shock wave creates unique microstructures in materials. Therefore, the properties of this shock wave may be different from those of conventional shock waves. However, the lattice behaviour under femtosecond laser-driven shock compression has never been elucidated. Here we report the ultrafast lattice behaviour in iron shocked by direct irradiation of a femtosecond laser pulse, diagnosed using X-ray free electron laser diffraction. We found that the initial compression state caused by the femtosecond laser-driven shock wave is the same as that caused by conventional shock waves. We also found, for the first time experimentally, the temporal deviation of peaks of stress and strain waves predicted theoretically. Furthermore, the existence of a plastic wave peak between the stress and strain wave peaks is a new finding that has not been predicted even theoretically. Our findings will open up new avenues for designing novel materials that combine strength and toughness in a trade-off relationship.

We demonstrate multiscale mechanical testing using miniature and microscale tensile tests to elucidate the crack initiation and propagation behaviors corresponding to interfacial nanostructures in dissimilar joints of 6061 aluminum alloy and 304 stainless steel. The initial nucleation and subsequent growth of intermetallic compounds (IMCs) can be controlled by heat treatment after friction stir lap welding. The miniature tensile tests exhibited the relationship between the joint strength and IMC layer thickness with changes in the fracture location. Microscale tensile testing using a single edge-notch tension geometry initiates the crack and subsequent propagations near the interface, which helps identify the crack initiation site and understand the dependence of the local strength on the type of IMCs. Crack deviation behavior toward the Al side, attributed to the difference of local strength, is confirmed by in situ observations, which indicated that continuous failure can easily occur within the grown IMCs, particularly in macroscale joints. It was concluded that the crack propagation is controlled by the local crack deviation toward metal matrix, which tends to occur for the dissimilar interface with thin IMC layer. This miniature/microscale tensile testing approach is expected to clarify the essential relationship between the interfacial microstructure and the fracture behavior.

Intensiometric N-cadherin (NCad) interaction indicators, named INCIDERs, visualize reversible NCad-mediated cell-cell interactions.

Summary: Clustered protocadherin (Pcdh), a cell adhesion protein, is involved in the self-recognition and non-self-discrimination of neurons by conferring diversity on the cell surface. Although the roles of Pcdh in neurons have been elucidated, it has been challenging to visualize its adhesion activity in neurons, which is a molecular function of Pcdh. Here, we present fluorescent indicators, named IPADs, which visualize the interaction of protocadherin-α4 isoform (α4). IPADs successfully visualize not only homophilic α4 trans-interactions, but also combinatorial homophilic interactions between cells. The reversible nature of IPADs overcomes a drawback of the split-GFP technique and allows for monitoring the dissociation of α4 trans-interactions. Specially designed IPADs for self-recognition are able to monitor the formation and disruption of α4 trans-interactions between processes originating from the same neurons. We expect that IPADs will be useful tools for obtaining spatiotemporal information on Pcdh interactions in neuronal self-recognition and non-self-discrimination processes.

Abstract Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

Background and study aims Esophagogastroduodenoscopy (EGD) is an effective and important diagnostic tool to detect gastric cancer (GC). Although previous studies show that examiner, patient, and instrumental factors influence the detection of GC, we analyzed whether assigning a different examiner to surveillance EGD would improve the detection of GC compared to assigning the same examiner as in the previous endoscopy. Patients and methods We retrospectively reviewed patients who underwent two or more consecutive surveillance EGDs at a single center between 2017 and 2019. We identified factors associated with GC detection using multivariable regression analysis and propensity-score matching. Results Among 7794 patients, 99 GC lesions in 93 patients were detected by surveillance EGD (detection rate; 1.2 %), with a mean surveillance interval of 11.2 months. Among the detected 99 lesions, 87 (87.9 %) were curatively treated with endoscopy. There were no differences in the clinicopathologic characteristics of GC detected by the same or different endoscopists. GC detection in the group examined by different endoscopists was more statistically significant than in the group examined by the same endoscopist, even after propensity-score matching (1.6 % and 0.7 %; P

This paper demonstrates the in situ sinter bonding of Ag microparticle pastes to a Si substrate via the temporal formation and decomposition of Ag carboxylate on the surface of Ag microparticles. This was proposed via the investigation of Ag sinter bonding using the redox reaction between Ag2O and ethylene glycol, which achieved a bonding strength above 30 MPa even for the bonding temperature at 220 °C. Thermal analysis was used to identify the product of the redox reaction between Ag2O and ethylene glycol and determine the bonding temperature because the final reaction facilitates the interfacial sinter bonding with the substrate. Fourier-transform infrared spectroscopy and nuclear magnetic resonance results indicated the in situ formation of Ag salts of carboxylic acids, such as Ag oxalate on the surface of Ag microparticles. Therefore, the sinter bonding process enabled by the in situ formation and subsequent decomposition of these Ag salts was investigated using Ag microparticles and oxalic acid. Observations of the surface and interfacial morphology of the Ag particles after heating revealed the formation of Ag nanoparticles on the surfaces of the microparticles and the formation of sintering necks between the particles. The bonding experiments demonstrated a significant increase in strength with the addition of oxalic acid to the Ag paste due to the enhanced interfacial sinter bonding with the substrate. The in situ formation and decomposition of Ag salts are promising strategies for improving sintered bonds in electronic devices because they can provide enhanced localized sinter bonding using stable insert materials.

Background and study aims Although the Japan Esophageal Society’s magnifying endoscopic classification for Barrett’s epithelium (JES-BE) offers high diagnostic accuracy, some cases are challenging to diagnose as dysplastic or non-dysplastic in daily clinical practice. Therefore, we investigated the diagnostic accuracy of this classification and the clinicopathological features of Barrett’s esophagus cases that are difficult to diagnose correctly. Patients and methods Five endoscopists with experience with fewer than 10 cases of magnifying observation for superficial Barrett’s esophageal carcinoma reviewed 132 images of Barrett’s mucosa or carcinoma (75 dysplastic and 57 non-dysplastic cases) obtained using high-definition magnification endoscopy with narrow-band imaging (ME-NBI). They diagnosed each image as dysplastic or non-dysplastic according to the JES-BE classification, and the diagnostic accuracy was calculated. To identify risk factors for misdiagnosed images, images with a correct rate of less than 40 % were defined as difficult-to-diagnose, and those with 60 % or more were defined as easy-to-diagnose. Logistic regression analysis was performed to identify risk factors for difficult-to-diagnose images. Results The sensitivity, specificity and overall accuracy were 67 %, 80 % and 73 %, respectively. Of the 132 ME-NBI images, 34 (26 %) were difficult-to-diagnose and 99 (74 %) were easy-to-diagnose. Logistic regression analysis showed low-grade dysplasia (LGD) and high-power magnification images were each significant risk factors for difficult-to-diagnose images (OR: 6.80, P = 0.0017 and OR: 3.31, P = 0.0125, respectively). Conclusions This image assessment study suggested feasibility of the JES-BE classification for diagnosis of Barrett’s esophagus by non-expert endoscopists and risk factors for difficult diagnosis as high-power magnification and LGD histology. For non-experts, high-power magnification images are better evaluated in combination with low-power magnification images.

Potassium-sensing oligonucleotide, PSO, a conjugate of a quadruplex structure-forming oligonucleotide with a peptide incorporating a Förster Resonance Energy Transfer (FRET) chromophore pair, has been developed for fluorescent detection of potassium ion (K+) in aqueous medium. PSO 1 could be introduced into cells for real-time imaging of cytoplasmic K+ concentrations. To perform fluorescent imaging of K+ on the cell surface, we synthesized twelve PSO derivatives with different types of peptide types and lengths, and oligonucleotide sequences including thrombin-binding aptamer (TBA) sequences with FAM and TAMRA as a FRET chromophore pair, and evaluated their performance. 1 was shown to respond selectively to K+, not to most ions present in vivo, and to show reciprocal fluorescence changes in response to K+ concentration. For the peptide chains and oligonucleotide sequences examined in this study, the PSO derivatives had Kd values for K+ in the range of 5–30 mM. All PSO derivatives showed high K+ selectivity even in the presence of excess Na+. The PSO derivatives were successfully localized to the cell surface by biotinylated concanavalin A (ConA) or sulfo-NHS-biotin via streptavidin (StAv). Fluorescence imaging of extracellular K+ upon addition of apoptosis inducers was successfully achieved by 1 localized to the cell surface.

Dissimilar material joining via friction stir spot welding (FSSW) has been implemented in some automobiles. However, the effectiveness of this method is easily affected by the surface condition of the lower plate. To solve this problem, a method that combines tool contact to the lower plate and material refilling via refill FSSW (RFSSW) was developed. This method was named scrubbing RFSSW (Sc-RFSSW). In Sc-RFSSW, the plunging depth of the shoulder is larger than that in conventional RFSSW. In this study, the relationship between the process parameters and maximum joint load of an Sc-RFSSW joint composed of an aluminum alloy and non-coated mild steel was determined using design of experiments (DoE). The results of DoE showed that the maximum joint load was improved by reduction of the tool plunging depth into the lower plate, such that it was only scrubbing the lower plate interface. In the optimized Sc-RFSSW joining condition, a nugget pull out was stably obtained in both TSS and CTS. In addition, it was demonstrated that, in joining an aluminum alloy and non-coated mild steel, Sc-RFSSW provides a higher maximum joint load than that of conventional FSSW. The unloading test results confirmed that the maximum joint load of Sc-RFSSW was within the welded area, which was equivalent to the tool outer diameter. Moreover, an extremely thin amorphous layer with a thickness of 4–6 nm was observed at the interface of the Sc-RFSSW joint.

The macroscale mechanical properties of dissimilar joints are generally influenced by the fracture behavior of joint interface. However, little is known about the dominant factor for the joint properties related with both macroscale fracture behavior and microscale interfacial properties. Herein, microscale tensile testing of the joint interface was coupled with the macroscale fracture evaluation to elucidate the dominant factor of strength in dissimilar joints between 6061 aluminum alloy and high tensile strength galvannealed steel via friction stir spot welding (FSSW). Microstructural analyses revealed the characteristic formation of interfacial microstructure accompanied by friction and Zn discharge during the FSSW process. Microscale tensile testing was performed on the specimen on which the pre-notch was introduced to induce local fracture at the intended position on the joint interface. Results showed the presence of the weakest interface attributed to initial joining defects formed by Zn concentration at the outer area, which were consistent with the outside of the crack arresting area confirmed by macroscale evaluation. These results indicated that the joint strength of dissimilar joints between 6061 aluminum alloy and galvannealed steel is dominated by the local strength of the joining area without defects and suggested a process design for improving dissimilar joints.

In this study, the synergistic effects of a hybrid surface treatment involving hydrochloric acid (HCl) immersion and silanization on friction stir spot welding (FSSW) of aluminum and carbon fiber reinforced thermoplastic (CFRTP) were investigated. Maximum tensile shear strength of 10.2 kN and maximum cross-tension strength of 1.92 kN were achieved. An unloading test and a miniature tensile test revealed that the fracture behavior changed from interfacial to CFRTP fracture due to increased interfacial strength with the hybrid treatment and utilization of the CFRTP strength enhanced the joint strength. The nanostructures at the interface with HCl immersion treatment indicated that the interfacial properties were improved mechanically in addition to the chemical enhancement by silanization. Owing to the hybrid treatment, aluminum and CFRTP were efficiently joined by the adhesion behavior of spreading melted plastic resin during the FSSW process.

Herein, we investigated the fracture behavior of thermally aged Ag–Cu composite sinter joints through nano-X-ray computed tomography (nano-CT) combined with microscale tensile testing. Nano-CT results showed that the joint comprised Ag/oxidized Cu flake sintered layer and Cu oxide, which was formed on the Cu substrate; further, pores were present at the interface between the substrate and Cu oxide layer. The tensile test showed that the stress increased with plastic deformation up to a maximum of 308 MPa, before a subsequent steep decrease due to crack initiation inside the sintered layer. The nano-CT and in situ observation results suggest that the microscale joint fractured through the interface between the oxidized Cu flake and Ag. Results obtained herein suggest that microscale tensile testing coupled with nano-CT is an effective approach to describe the intrinsic fracture behavior of joints.

Thin metallic Cu and Ni meshes were successfully embedded within SAC305 solder joints. Defects (residues) within the joint were removed by a vacuum step in the bonding sequence. The metallic mesh inlets were metallurgically bonded by the formation of scallop-like Cu6Sn5 and faceted Ni3Sn4 intermetallic compounds (IMCs) at the Cu mesh-SAC305 solder (CMS) and Ni mesh-SAC305 solder (NMS) interfaces, respectively. After process optimization, the averaged shear strengths were found to be 44.1 (reference), 44.7 (+1%), and 51.4 MPa (+16%) for SAC305, NMS, and CMS composite joints, respectively. The solder-substrate-mesh interaction resulted in a characteristic fracture pattern, with the cracking path partly within the solder and partly at the solder-mesh interface. Finite element (FE) simulations suggested a mesh-induced stiffening effect of the solder joint by 5%. The additional reinforcement (11%) introduced by the Cu mesh inlets was attributed to a locally enhanced fracture resistance. Keywords: Solder-mesh composite joints, Microstructure, Shear strength, Finite element modeling, Fracture

Abstract Objective Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening. Results GmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

Abstract Background Photosensitizing fluorescent proteins, which generate reactive oxygen species (ROS) upon light irradiation, are useful for spatiotemporal protein inactivation and cell ablation. They give us clues about protein function, intracellular signaling pathways and intercellular interactions. Since ROS generation of a photosensitizer is specifically controlled by certain excitation wavelengths, utilizing colour variants of photosensitizing protein would allow multi-spatiotemporal control of inactivation. To expand the colour palette of photosensitizing protein, here we developed SuperNova Green from its red predecessor, SuperNova. Results SuperNova Green is able to produce ROS spatiotemporally upon blue light irradiation. Based on protein characterization, SuperNova Green produces insignificant amounts of singlet oxygen and predominantly produces superoxide and its derivatives. We utilized SuperNova Green to specifically inactivate the pleckstrin homology domain of phospholipase C-δ1 and to ablate cancer cells in vitro. As a proof of concept for multi-spatiotemporal control of inactivation, we demonstrate that SuperNova Green can be used with its red variant, SuperNova, to perform independent protein inactivation or cell ablation studies in a spatiotemporal manner by selective light irradiation. Conclusion Development of SuperNova Green has expanded the photosensitizing protein toolbox to optogenetically control protein inactivation and cell ablation.

Current smartphones equipped with high-sensitivity and high-resolution sensors in the camera can respond to the needs of low-light imaging, streaming acquisition, targets of various scales, etc. Therefore, a smartphone has great potential as an imaging device even in the scientific field and has already been introduced into biomolecular imaging using fluorescence tags. However, owing to the necessity of an excitation light source, fluorescence methods impair its mobility. Bioluminescence does not require illumination; therefore, imaging with a smartphone camera is compact and requires minimal devices, thus making it suitable for personal and portable imaging devices. Here, we report smartphone-based methods to observe biological targets in various scales using bioluminescence. In particular, we demonstrate, for the first time, that bioluminescence can be observed in an organelle in a single living cell using a smartphone camera by attaching a detachable objective lens. Through capturing color changes with the camera, changes in the amount of target molecules was detected using bioluminescent indicators. The combination of bioluminescence and a mobile phone makes possible a compact imaging system without an external light source and expands the potential of portable devices.

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.

Sensory processing is regulated by the coordinated excitation and inhibition of neurons in neuronal circuits. The analysis of neuronal activities has greatly benefited from the recent development of genetically encoded Ca2+ indicators (GECIs). These molecules change their fluorescence intensities or colours in response to changing levels of Ca2+ and can, therefore, be used to sensitively monitor intracellular Ca2+ concentration, which enables the detection of neuronal excitation, including action potentials. These GECIs were developed to monitor increases in Ca2+ concentration; therefore, neuronal inhibition cannot be sensitively detected by these GECIs. To overcome this difficulty, we hypothesised that an inverse-type of GECI, whose fluorescence intensity increases as Ca2+ levels decrease, could sensitively monitor reducing intracellular Ca2+ concentrations. We, therefore, developed a Ca2+ indicator named inverse-pericam 2.0 (IP2.0) whose fluorescent intensity decreases 25-fold upon Ca2+ binding in vitro. Using IP2.0, we successfully detected putative neuronal inhibition by monitoring the decrease in intracellular Ca2+ concentration in AWCON and ASEL neurons in Caenorhabditis elegans. Therefore, IP2.0 is a useful tool for studying neuronal inhibition and for the detailed analysis of neuronal activities in vivo.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

We have proposed a low-temperature bonding technology utilizing the sintering of Cu particles with transient liquid-phase of Sn-based solder, called transient liquid-phase sintering (TLPS), as a die-attach solution for high-temperature power modules. A copper-intermetallic compound-resin (Cu-IMC-resin) microstructure, which consists of Cu particles connected with Cu−Sn intermetallic compounds (IMCs) partially filled with polyimide resin, is obtained by the pressureless TLPS process at 250 °C for 1 min using a novel Cu-solder-resin composite as the bonding material in a nitrogen atmosphere. Macro- and micro-deformation properties of the unique microstructure of the TLPS Cu-IMC-resin are evaluated by finite element analysis using a three-dimensional image reconstruction model. The macroscopic computational uniaxial tensile tests of the Cu-IMC-resin model reveal that the utilization of the IMCs and the addition of the easily-deformable resin facilitates the temperature-stability and low-stiffness of the mechanical properties. The microstructure exhibits a significantly low homogenized Young’s modulus (11 GPa). Microscopic investigations show that the local stresses are broadly distributed on the IMC regions under uniaxial macroscopic tensile displacement, indicating highly reliable performance of the joint within a specific macroscopic strain condition. Numerical and experimental investigations demonstrate the excellent thermal cyclic reliability of die-attached joints between silicon carbide chips and directly bonded copper substrate.

Thermal fatigue generally occurs in ultrasonically bonded copper joints in electronic devices as the bonding substrate is composed of plural materials, leading to differences in the coefficient of thermal expansion. In this study, we found that the thermal fatigue resistance of the ultrasonically bonded copper joints was influenced by the grain size and hardness of the bonding substrate through the evaluation of the thermal fatigue properties. Copper alloys C1020 and C1940 were used as substrate materials to investigate the influence of the initial properties of the bonding material on the thermal fatigue resistance. We evaluated the crack propagation due to thermal fatigue via thermal cycle tests. Microstructural observations of the region fractured because of thermal fatigue revealed that cracks resulting from thermal fatigue did not progress in the fine grain region formed at the bonded interface. It was inferred that grain boundaries were an obstacle to crack propagation. C1940 has higher hardness and finer grains than C1020, and showed a lower decreasing rate of the peel strength and bonding area after the thermal cycling test than C1020 joints. Thus, a hard copper material with fine grains is effective in suppressing thermal fatigue fracture of ultrasonically bonded copper joints.

The evolution of the transient liquid-phase sintered (TLPS) Cu–Sn skeleton microstructure during thermal aging was evaluated to clarify the thermal reliability for die-attach applications. The Cu–Sn skeleton microstructure, which consists of Cu particles connected with Cu–Sn intermetallic compounds partially filled with polyimide resin, was obtained by the pressure-less TLP sintering process at 250 °C for 1 min using a novel Cu-solder-resin composite as a bonding material in a nitrogen atmosphere. Experimental results indicate that the TLPS joints were mainly composed of Cu, Cu6Sn5, and Cu3Sn in the as-bonded state, where submicron voids were observed at the interface between Cu3Sn and Cu particles. After thermal aging at 150, 175, and 200 °C for 1000 h, the Cu6Sn5 phase fully transformed into Cu3Sn except at the chip-side interface, where the number of the submicron voids appeared to increase. The averaged shear strengths were found to be 22.1 (reference), 22.8 (+3%), 24.0 (+9%), and 19.0 MPa (−14%) for the as-bonded state and specimens aged at 150, 175, and 200 °C for 1000 h, respectively. The TLPS joints maintained a shear strength over 19 MPa after thermal aging at 200 °C for 1000 h because of both the positive and negative impacts of the thermal aging, which include the transformation of Cu6Sn5 into Cu3Sn and the formation of submicron voids at the interface, respectively. These results indicate an excellent thermal reliability of the TLPS Cu–Sn skeleton microstructure.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported Pcdh-B2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we -B2 designed-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism. [ABSTRACT FROM AUTHOR]