Review of laser scanning methods for microelectronic semiconductor structures investigation

The development and widespread of high-tech microelectronic products impose increased requirements on the quality and reliability of microcircuits. The most effective methods for reliability improvement of electronic systems include diagnostic non-destructive testing (NDT) methods and selective destructive testing in special cases. Studies using visual inspection and electrical testing, consisting of functional and parametric testing, do not provide enough information to detect latent defects (for example, macro-defects in SiO 2 layers in CMOS chips) and to detect fakes and counterfeits. A fake integrated circuit (IC) may contain an undeclared malicious modification of the circuit, called hardware bugs. The common ICs studying tools are systems based on microfocus X-ray sources, scanning acoustic microscopes, optical and scanning electron microscopes, and X-ray fluorescence spectroscopes. Products destruction avoidance is a fundamental point, for example, for the technological process control in crystal manufacturing. Investigation of ICs using a light microscope is one of the most accessible and widespread method of microchip NDT. Semiconductor ICs structure scanning from the side of the device layer is limited by the shielding effect of metallization, since the metal is opaque for light. This limitation can be overcome by an alternative approach to microchip scanning based on irradiating the IC from the side of the substrate with laser sources in the near-IR range. This paper provides a brief overview of the major methods used in laser scanning microscopy to analyze the structures, responses, and features of the operating modes of semiconductor circuits. The main advantages and limitations in the use of optical methods are described, as well as what information about the product can be obtained as a result of laser scanning

Formation of Iron Oxide Nanoparticles in the Internal Cavity of Ferritin-Like Dps Protein: Studies by Anomalous X-Ray Scattering

DNA-binding protein from starved cells (Dps) takes a special place among dodecamer mini-ferritins. Its most important function is protection of bacterial genome from various types of destructive external factors via in cellulo Dps–DNA co-crystallization. This protective response results in the emergence of bacterial resistance to antibiotics and other drugs. The protective properties of Dps have attracted a significant attention of researchers. However, Dps has another equally important functional role. Being a ferritin-like protein, Dps acts as an iron depot and protects bacterial cells from the oxidative damage initiated by the excess of iron. Here we investigated formation of iron oxide nanoparticles in the internal cavity of the Dps dodecamer. We used anomalous small-angle X-ray scattering as the main research technique, which allows to examine the structure of metal-containing biological macromolecules and to analyze the size distribution of metal nanoparticles formed in them. The contributions of protein and metal components to total scattering were distinguished by varying the energy of the incident X-ray radiation near the edge of the metal atom absorption band (the K-band for iron). We examined Dps specimens containing 50, 500, and 2000 iron atoms per protein dodecamer. Analysis of the particle size distribution showed that, depending on the iron content in the solution, the size of the nanoparticles formed inside the protein molecule was 2 to 4 nm and the growth of metal nanoparticles was limited by the size of the protein inner cavity. We also found some amount of iron ions in the Dps surface layer. This layer is very important for the protein to perform its protective functions, since the surface-located N-terminal domains determine the nature of interactions between Dps and DNA. In general, the results obtained in this work can be useful for the next step in studying the Dps phenomenon, as well as in creating biocompatible and solution-stabilized metal nanoparticles

Insulin receptor-related receptor in the pancreas and in a β-cell line

Insulin receptor-related receptor (IRR) belongs to the family of the insulin receptor (IR) along with the IR itself and the insulin-like growth factor receptor. Whereas the ligands of the latter receptors are known, identification of an IRR ligand has eluded investigations so that IRR has been considered an orphan receptor. A recent breakthrough in the understanding of IRR functional role came from the finding that IRR can be activated by mildly alkali media in absence of any protein agonist [1]. IRR shows highly specific tissue distribution, with highest concentration in kidney intercalated cells. However, significant amounts of the receptor are also found in the stomach and in α- and β-cells of the islets of Langerhans. Recent reports indicate that the pancreatic duct system is frequently associated with islet cells. Here, we show that those islet cells that are in contact with the excretory ducts are also IRR-expressing cells. Thus, when the exocrine pancreas is in an active state of secretion duct-associated islet cell behavior is potentially influenced by an IRR-mediated alkaline-induced signalling pathway. To explore this issue, we analyzed the effects of alkaline media on the pancreatic β-cell line MIN6. Activation of endogenous IRR was detected and could be inhibited with linsitinib, a synthetic inhibitor of the IR family of receptors. IRR autophosphorylation correlated with pH-dependent linsitinib-sensitive activation of IR substrate 1 (IRS-1). In contrast to insulin stimulation, no protein kinase B (Akt/PKB) phosphorylation was detected as a result of the alkali treatment. The alkaline medium but not insulin also triggered actin cytoskeleton remodeling in MIN6 cells that was blocked by pre-incubation with linsitinib. We propose that the activation of IRR by alkali is a component of a local loop of signaling between the exocrine and endocrine parts of the pancreas

ECG recordings of cardiac pacing in mice carriyng TRPV1

The data are ECG recordings acquired from mice whose cardiac muscle tissue carries the human TRPV1 channel. ECG was recorded with standard procedure on the limbs with disposable electrodes, with one or two electrodes. Myocardial stimulation was applied with an IR laser. Each archive contains the result of an ECG recording of one animal. Initial recordings are cut so that the unsuccessful recordings are excluded. File name Recording date Vector type Vector Amount No1 2022 06 22 AAV488 6x10-11 No2 2022 06 16 AAV479 6x10-12 No3 2022 06 22 AAV488 6x10-11 No4 2021 10 1

Spatial organization of Dps and DNA–Dps complexes

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA–Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA–Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA–Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA–Dps complexes. Angle analysis has demonstrated that in DNA–Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet

The dimeric ectodomain of the alkali-sensing insulin receptor–related receptor (ectoIRR) has a droplike shape

Insulin receptor–related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid–base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a “droplike” shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment

FLIM-Based Intracellular and Extracellular pH Measurements Using Genetically Encoded pH Sensor

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems