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Could enzymatic activities and their cooperative functions act as cellular temperature-sensing systems? This review introduces recent opto-thermal technologies for microscopic analyses of various types of cellular temperature-sensing system. Optical microheating technologies have been developed for local and rapid temperature manipulations at the cellular level. Advanced luminescent thermometers visualize the dynamics of cellular local temperature in space and time during microheating. An optical heater and thermometer can be combined into one smart nanomaterial that demonstrates hybrid function. These technologies have revealed a variety of cellular responses to spatial and temporal changes in temperature. Spatial temperature gradients cause asymmetric deformations during mitosis and neurite outgrowth. Rapid changes in temperature causes imbalance of intracellular Ca2+ homeostasis and membrane potential. Among those responses, heat-induced muscle contractions are highlighted. It is also demonstrated that the short-term heating hyperactivates molecular motors to exceed their maximal activities at optimal temperatures. We discuss future prospects for opto-thermal manipulation of cellular functions and contributions to obtain a deeper understanding of the mechanisms of cellular temperature-sensing systems.

Drebrin E is a regulatory protein of intracellular force produced by actomyosin complexes, that is, myosin molecular motors interacting with actin filaments. The expression level of drebrin E in nerve cells decreases as the animal grows, suggesting its pivotal but unclarified role in neuronal development. Here, by applying the microscopic heat pulse method to actomyosin motility assay, the regulatory mechanism is examined from the room temperature up to 37 °C without a thermal denaturing of proteins. We show that the inhibition of actomyosin motility by drebrin E is eliminated immediately and reversibly during heating and depends on drebrin E concentration. The direct observation of quantum dot-labeled drebrin E implies its stable binding to actin filaments during the heat-induced sliding. Our results suggest that drebrin E allosterically modifies the actin filament structure to regulate cooperatively the actomyosin activity at the maintained in vivo body temperature., 胎児の神経を形作る仕組みは精密な温度センサー --母体の体温維持が神経の成熟に重要であることを示唆--. 京都大学プレスリリース. 2021-11-10.

Could enzymatic activities and their cooperative functions act as cellular temperature-sensing systems? This review introduces recent opto-thermal technologies for microscopic analyses of various types of cellular temperature-sensing system. Optical microheating technologies have been developed for local and rapid temperature manipulations at the cellular level. Advanced luminescent thermometers visualize the dynamics of cellular local temperature in space and time during microheating. An optical heater and thermometer can be combined into one smart nanomaterial that demonstrates hybrid function. These technologies have revealed a variety of cellular responses to spatial and temporal changes in temperature. Spatial temperature gradients cause asymmetric deformations during mitosis and neurite outgrowth. Rapid changes in temperature causes imbalance of intracellular Ca2+ homeostasis and membrane potential. Among those responses, heat-induced muscle contractions are highlighted. It is also demonstrated that the short-term heating hyperactivates molecular motors to exceed their maximal activities at optimal temperatures. We discuss future prospects for opto-thermal manipulation of cellular functions and contributions to obtain a deeper understanding of the mechanisms of cellular temperature-sensing systems.

Negatively charged nitrogen-vacancy (NV) centers in diamond are promising magnetic field quantum sensors. Laser threshold magnetometry theory predicts improved NV center ensemble sensitivity via increased signal strength and magnetic field contrast. Here, we experimentally demonstrate laser threshold magnetometry. We use a macroscopic high-finesse laser cavity containing a highly NV-doped and low absorbing diamond gain medium that is pumped at 532 nm and resonantly seeded at 710 nm. This enables a 64% signal power amplification by stimulated emission. We test the magnetic field dependency of the amplification and thus demonstrate magnetic field–dependent stimulated emission from an NV center ensemble. This emission shows an ultrahigh contrast of 33% and a maximum output power in the milliwatt regime. The coherent readout of NV centers pave the way for novel cavity and laser applications of quantum defects and diamond NV magnetic field sensors with substantially improved sensitivity for the health, research, and mining sectors.

電子線照射を用いればダイヤモンド中のNV（窒素・空孔）センターを高濃度に形成することが出来ることが知られている。2 MeV電子線照射を室温で実施し、照射を進める過程で熱処理を行い、その都度P1センター（格子中窒素不純物）の消費の様子や形成されたNVセンターの電荷状態、総量と照射量の関係を調べた。その結果、高効率でのNVセンター形成が確認出来た一方でP1センターがNVセンター以外の欠陥に消費されている可能性が示唆された。, 第69回応用物理学会春季学術講演会

The study on NV center formation condition for high Econv and charge-state ratio of NV- and NV0 centers, The 4th International Forum on Quantum Metrology and Sensing

これまで発表者が行ってきた筋肉と温度の関係についての研究の紹介を通して生体内の温度を測ることの面白さ、重要さを導入することで、温度を高感度に計測出来る量子センサー開発の重要性を説明する。量子センサーの一つとしてダイヤモンドNVセンターが挙げられるが、センサーの高感度化にはNVセンターを効率よく高濃度に形成することが求められる。そこで本発表では電子線照射を通したNVセンター形成メカニズムについて我々が行ってきた研究の一つを発表する。電子線照射は室温で実施し、照射を進める過程で熱処理を行い、その都度P1センター（格子中窒素不純物）の消費の様子や形成されたNVセンターの電荷状態、総量と照射量の関係を調べた。その結果、高効率でのNVセンター形成が確認出来た一方でP1センターがNVセンター以外の欠陥に消費されている可能性が示唆された。, The 4th International Forum on Quantum Metrology and Sensing

NV センターの負電荷割合 - 平衡状態方程式による解釈に関する結果を報告する。, 第35回ダイヤモンドシンポジウム

Muscle fiber contraction involves chemical reactions and structural changes that are sensitive to temperature. Ishii et al. show that rapid heating causes cardiac thin filament sliding, and the data could indicate that thin filaments are partially activated during diastole at mammalian body temperature., During the excitation–contraction coupling of the heart, sarcomeres are activated via thin filament structural changes (i.e., from the “off” state to the “on” state) in response to a release of Ca2+ from the sarcoplasmic reticulum. This process involves chemical reactions that are highly dependent on ambient temperature; for example, catalytic activity of the actomyosin ATPase rises with increasing temperature. Here, we investigate the effects of rapid heating by focused infrared (IR) laser irradiation on the sliding of thin filaments reconstituted with human α-tropomyosin and bovine ventricular troponin in an in vitro motility assay. We perform high-precision analyses measuring temperature by the fluorescence intensity of rhodamine-phalloidin–labeled F-actin coupled with a fluorescent thermosensor sheet containing the temperature-sensitive dye Europium (III) thenoyltrifluoroacetonate trihydrate. This approach enables a shift in temperature from 25°C to ∼46°C within 0.2 s. We find that in the absence of Ca2+ and presence of ATP, IR laser irradiation elicits sliding movements of reconstituted thin filaments with a sliding velocity that increases as a function of temperature. The heating-induced acceleration of thin filament sliding likewise occurs in the presence of Ca2+ and ATP; however, the temperature dependence is more than twofold less pronounced. These findings could indicate that in the mammalian heart, the on–off equilibrium of the cardiac thin filament state is partially shifted toward the on state in diastole at physiological body temperature, enabling rapid and efficient myocardial dynamics in systole.

We investigated spin-echo coherence times T2 of negatively charged nitrogen vacancy center (NV−) ensembles in single-crystalline diamond synthesized by either the high-pressure and high-temperature and chemical vapor deposition methods. This study specifically examined the magnetic dipole–dipole interaction (DDI) from the various electronic spin baths, which are the source of T2 decoherence. Diamond samples with NV− center concentration [NV−] comparable to those of neutral substitutional nitrogen concentration [Ns0] were used for DDI estimation. Results show that the T2 of the ensemble NV− center decreased in inverse proportion to the concentration of nitrogen-related paramagnetic defects [NPM], being the sum of [Ns0], [NV−], and [NV0], which is a neutrally charged state NV center. This inversely proportional relation between T2 and [NPM] indicates that the nitrogen-related paramagnetic defects of three kinds are the main decoherence source of the ensemble NV− center in the single-crystalline diamond. We found that the DDI coefficient of NVH− center was significantly smaller than that of Ns0, the NV0 center, or the NV− center. We ascertained the DDI coefficient of the NV− center [Formula: see text] through experimentation using a linear summation of the decoherence rates of each nitrogen-related paramagnetic defect. The obtained value of 89 μs ppm for [Formula: see text] corresponds well to the value estimated from the relation between DDI coefficient and spin multiplicity.

電子線照射を用いればダイヤモンド中のNV（窒素・空孔）センターを高濃度に形成することが出来ることが知られているが、照射中加熱することでより効率の良い形成が可能となることから、2 MeV電子線照射を室温、800℃及び1000℃中にて実施した。P1センター（格子中窒素不純物）の消費の様子や形成されたNVセンターの電荷状態、総量と照射量の関係を調べた。その結果、高温中での照射によりNVセンター形成効率が改善され、特に800℃においてその効果が高いことが分かった。, 量子生命科学会第3回大会

1. Introduction Negatively charged nitrogen-vacancy (NV-) center in diamond is expected as quantum sensor to measure small changes in physical quantities, such as magnetic and electric field, temperature, strain, etc. The quantum sensors with high sensitivity require diamond containing high concentration of NV centers. Electron irradiation is a good method to create high concentration of NV centers. The high energy electrons knock carbon atoms out of the diamond lattice, creating vacancies. Annealing an irradiated diamond causes all the vacancies to become mobile and subsequently trapped by substituted nitrogen (P1 centers), forming NV centers. It is known that the process of NV center creation depends on the initial P1 concentrations, and the irradiation fluence dependence at low initial P1 concentrations below 1 ppm is still uncleared. In this study, we irradiate samples with different initial P1 concentrations, especially low concentration, and estimate the fluence dependency of the charge state and the amount of created NV centers. 2. Experiments The diamonds synthesized by High Pressure and High Temperature (HPHT) and Chemical Vapor Deposition (CVD) were used. The initial concentration of P1 was in the range from 40 ppb to 100 ppm. 2 MeV electrons were irradiated with a fluence up to 2E18 cm-2 at room temperature. Then, the samples were annealed at 1000℃ for 2 hours to create NV center. The concentration of P1 was measured by Electron Spin Resonance (ESR) using JES-X330 (JEOL). The ratio between NV0 and NV- was estimated from photoluminescence (PL) spectrum measurement obtained using LabRAM HR Evolution (HORIBA). All these measurements were performed at room temperature. 3. Results & Discussion In the case of type Ib HPHT diamond containing initial P1 concentration of ~100 ppm, the most of NV centers are thought to be NV-, and PL measurement revealed that only NV- was detected when the fluence of 2E18 cm-2. P1 center acts as electron donor, and provide electron to NV0. In contrast to high P1 concentration, an appreciable fraction of NV center is present as NV0 for samples with low initial P1 concentration (~ 10 ppm). We examined the relationship between the concentration of P1 and the fluence, using HPHT diamonds with initial P1 concentration of ~0.8 ppm. The concentration of P1 decreased to ~0.3 ppm when the fluence of 1.5E17 cm-2. This result suggests that P1 centers of ~0.5 ppm change to either NV center or positively charged substitutional nitrogen. PL spectrum when the fluence of 1.5×1017 cm-2, shows that both NV0 and NVare created. The ratio of NV- to NV0 decreased as irradiation fluence, to be 0.70 after irradiated with 1.0×1017 cm-2 and 0.53 after irradiated with 1.5E17 cm-2. It is noted that NV0 was formed even though P1 center which can act as electron donor to NV0 center remains. Similar results were observed for CVD diamonds with initial P1 concentration of ~40 ppb. The adequate fluence to maximize NV- concentration for diamonds containing low P1 concentration will be discussed by examining the fluence dependency of the charge state and total amount of NV center., New Diamond and Nano Carbons 2020/2021

広帯域の交差コプレーナー導波路を用いてT2*を伸長した結果を報告する。, 第68回 応用物理学会春季学術講演会

ダイヤモンド中のNVセンターのマイナス荷電状態は２つの異なるスピン状態の間で蛍光強度に差を持つ(30％程度)。この特徴を使って、２つのスピン状態を重ねわせた時の蛍光強度によって外部場の検出をすることが可能である。また、その重ね合わせの保持時間(T2)は室温で最長であり、量子センサーへの応用研究が盛んに行われている。上述した蛍光強度の変化(蛍光コントラスト)をあげることは感度向上に大きく貢献する。一方で、コントラストを下げる要因であるNVセンターの中性荷電状態起因のバックラウンド蛍光が挙げられる。本研究では、NV-/NV0の比が材料の観点から何に律速しているかに着目した。結果として、ESR、PL分光を用いて、ダイヤモンド中の置換窒素濃度が荷電状態比の主な律速要因である実験結果を得た。, 第34回 ダイヤモンドシンポジウム

量子センシング・イメージング実現に向けて、ダイヤモンド中NV（窒素−空孔）センターの基礎的物性から高濃度・高輝度発光NVセンターの形成についてナノ粒子とバルク結晶の二つのダイヤモンド材料を用いた研究を進めている。 ナノダイヤモンドにおいて、単一細胞レベルから動物レベルまでの幅広い生体に導入することができ、それらの生命現象により誘起される微小温度変化、細胞活性に伴う磁気・電気的変化の検出やMRI（磁気共鳴画像）法を活用した体内のイメージング材料として期待されている。NVセンターの量子性を利用した量子センシング・イメージングのためには、より効率的にNVセンターを導入することに加え、量子性の優れたナノダイヤモンド中NVセンター形成技術が求められている。今日までにナノダイヤモンドに電子線照射と熱処理により高輝度なNVセンター形成技術の最適化を図ってきており、そのようにして作製した高輝度NVナノダイヤモンドの表面に対して、生体導入に適した化学処理を施す必要があり、それら表面化学修飾についても最適化を進めている。 一方、数mmのバルクダイヤモンドに対して、電子線照射と熱処理を施したのでその結果についても報告する。試料はHPHT（高温高圧）法により合成されたIb型ダイヤモンドを使用した。電子線照射は照射エネルギー2 MeV、照射量を3×1018 cm-2とした。照射後に1000℃（2時間）の熱処理を行い、ダイヤモンド中窒素と原子空孔を結合させることでNVセンターを形成させた。P1(ダイヤモンド中の不純物窒素)濃度はESR（電子スピン共鳴）にて測定し、NVセンター量についてはフォトルミネッセンス（PL）測定法により評価した。結果として照射前のP1濃度は約80 ppmであったが、照射後は約40ppmまで減少した。PLスペクトル測定結果から、NVセンターは負の電荷（NV-）をもつもののみが形成されることが分かった。P1はNVセンターになるだけでなく、NVセンターに電荷を供給するドナーにもなる。そのため減少した40ppmの内、20ppmがNVセンターになり、20ppmが電子を失ったNS+になったものと考えられる。 本研究では、電子線照射量と熱処理条件をさらに突き詰め、それら種々の条件とNVセンター形成への影響を調べた。, 量子生命科学会第2回大会

we consider the total amount of NV center in diamond as an indicator of amount of vacancy assuming all vacancies created in diamond were coupled with nitrogen during the consecutive annealing. In this study, we investigate the dependence of the negative-charge stability of NV centers, [NV-]/[NV0] ratio, on the percentage of vacancies to nitrogen, [NVT]/[P1] ratio. Our result indicates that the negative-charge stability is realized at [NVT]/[P1] < 0.2 except for the case in which [NV-] is compatible to residual [B]., 3rd IFQMS

Diamond growth is a key technology for the developing quantum sensing device using NV centers. Desired concentration of NV centers varies from 1ppb to 10ppm depending on the type of quantum sensing devices. Then, precise control of nitrogen concentration in wide doping range is requested for diamond growth. Carbon isotope control is another important issue of diamond growth to prolong the spin coherent time. Here, research activity of diamond growth in National Institute for Materials Science (NIMS) by chemical vapor deposition (CVD) and high-pressure and high-temperature (HPHT) will be introduced focusing on the NV center formation., 3rd IFQMS

The nitrogen-vacancy (NV) center in diamond is extensively studied for a next-generation high-sensitivity magnetometer. The DC magnetic sensitivity can be improved by extending the inhomogeneous spin dephasing time through suppressing the effects of inhomogeneous fluctuations or by increasing the number of effective NV centers through expanding the sensor volume. It has been demonstrated that strain-induced dephasing can be suppressed by double quantum (DQ) technique with two-tone microwaves (MW), and spin bath-induced dephasing can be suppressed by spin bath drive (SBD) technique with multi-tone radio-frequency (RF) to drive the P1 centers. To combine these T2* -extension techniques with a bulk diamond, it is necessary to apply MW and RF uniformly to the sample. A coplanar waveguide (CPW) circuit has been reported to be useful for uniform microwave irradiation to a bulk sample. In this study, we reports the newly designed and fabricated a cross-aligned two-layer CPW circuit., 3rd IFQMS

Introduction Negatively charged nitrogen-vacancy (NV-) center in diamond is known as quantum sensor, can be used to measure small changes in physical quantities, such as magnetic field and temperature. To enhance the sensitivity of the quantum sensor, increasing NV- and decreasing neutral charge state NV0 in diamonds are needed since NV0 does not act as quantum sensor. The electron irradiation is a good method to create high concentration of NV centers. The high energy electrons create vacancies in diamond. Annealing allows the vacancies to move and to be trapped by substitutional nitrogen (P1 centers), forming NV centers. In this study, we irradiate diamonds containing different initial P1 concentrations with electrons and evaluate the fluence dependence of the charge state and the amount of created NV centers, for the purpose of determining appropriate fluence to form NV centers efficiently. Method The commercial diamonds synthesized by High Pressure and High Temperature (HPHT) were used. 2 MeV electrons were irradiated with fluences up to 3×1018 cm-2 at room temperature. Then, the samples were annealed at 1000℃ for 2 hours to create NV centers. The concentration of P1 (number of electron spins) was measured by Electron Spin Resonance (ESR). The initial concentration of P1 was in the range from 50 ppm to 100 ppm. The ratio of NV- to NV0 was evaluated from photoluminescence (PL) spectrum. All these measurements were performed at room temperature. Results & Discussion The concentration of P1 decreased with increasing fluence. In contrast, total amount of NV centers increased with increasing fluence and only NV- was detected when the fluence of 3×1018 cm-2. The result suggests that remained P1 centers act as electron donor, and provide electron to NV0. Further irradiation with higher fluence will be needed to achieve the adequate one to maximize NV- concentration for diamonds remaining the charge state only in NV-., 3rd IFQMS

The nitrogen-vacancy (NV) center in diamond is a promising quantum system for magnetometry applications exhibiting optical readout of minute energy shifts in its spin sub-levels. Key material requirements for NV ensembles are a high NV- concentration, a long spin coherence time and a stable charge state. However, these are interdependent and can be difficult to optimize during diamond growth and subsequent NV creation. In this work, we systematically investigate the NV center formation and properties in bulk chemical vapor deposition (CVD) diamond. The nitrogen flow during growth is varied by over 4 orders of magnitude, resulting in a broad range of single substitutional nitrogen concentrations of 0.2-20~parts per million. For a fixed nitrogen concentration, we optimize electron-irradiation fluences with two different accelerated electron energies, and we study defect formation via optical characterizations. We discuss a general approach to determine the optimal irradiation conditions, for which an enhanced NV concentration and an optimum of NV charge states can both be satisfied. We achieve spin-spin coherence times T2 ranging from 45.5 to 549 μs for CVD diamonds containing 168 to 1~parts per billion NV- centers, respectively. This study shows a pathway to engineer properties of NV-doped CVD diamonds for improved sensitivity.

電子線照射を用いればダイヤモンド中のNV（窒素・空孔）センターを高濃度に作成することができる。2 MeV電子線によって導入した原子空孔と窒素不純物（P1センター）を熱処理によって結合させてNVセンターを形成し、NVセンターの電荷状態やその総量を調べた。特にP1センター濃度とNVセンターの濃度比から電荷状態について化学平衡式を用いて議論し、その平衡点を決定している。

Actin is the most abundant protein in the cytoplasm of most eukaryotic cells, which plays important roles in such processes as muscle contraction, cell division, morphogenesis, organelle positioning, and vesicle trafficking. In our previous study, we reported the expression method of recombinant beta-actin at high cell density in a shaking Erlenmeyer flask. In the insect cell culture at early stationary phase (1-1.2 x 107 cells per ml), additional nutrients or medium exchange at time of infection restored the expression level of beta-actin per cell equivalent to that in the cell culture at mid-logarithmic phase (1-2 x 106 cells per ml). Therefore, the volumetric yield of recombinant beta-actin, which was determined by the maximal cell density and the yield of recombinant protein per cell, was at least 6.5-fold higher than compared to the manufacturer's protocol. The present study further developed the expression method of recombinant beta-actin at the density of 1.75 x 107 cells per ml, which is ∼1.5-fold higher than the cell density at stationary phase. This method requires medium exchange plus additional nutrients and air ventilation after infection. The volumetric yield of recombinant beta-actin reached 200 mg per liter of insect cell culture. Here we report in detail the effective method for the production of recombinant beta-actin and the biological and the biochemical properties of recombinant beta-actin.

HCM is characterized by thickening of left ventricular wall and interventricular septum, with abnormalities in contractile functions. It has been thought that Tm's flexible association with actin is important for its function. With in vitro motility assays, we investigated α-Tm mutants V95A and D175N (based on human sequence), which are known to cause HCM. Reconstituted thin filaments with rabbit skeletal actin and Tn were placed on HMM-coated glass surface. At the room temperature (24°C), we applied a microscopic heat pulse by focused laser beam, which momentarily generated a concentric temperature gradient with the center reaching 50°C. Because the pulse duration was brief (2 s), this procedure did not denature proteins. This area was viewed in a microscope field, which allowed observations of gliding thin filaments at different temperatures in the same field. We found that the gliding occurred on elevating the temperature even in the absence of Ca (Oyama et al., BBRC, 2012; see also Ishiwata, BBA, 1978). The thermally-activated association of HMM to actin results in a temporary opening of the thin filament, where it is likely that Tm/Tn is partially dissociated from the actin filament, which causes a partial activation of the thin filament. We found that the thin filament reconstituted with V95A was more difficult to become thermally activated at around physiological temperature than that with WT or D175N. This result suggests that the critical temperature for activation is higher for V95A than WT and D175N. These results further suggest that the thermal stability of the association of V95A Tm to the actin filament is higher than that of WT Tm. Our results indicate that extra stability of the actin-Tm association may cause HCM.

It is well established that heart beat is triggered by electrical excitation, followed by an increase in [Ca2+]i and ensuing activation of myofilaments in cardiomyocytes. This property is the basis for the regulation of the heart with electric stimulators or chemical compounds that mediate Ca2+ signaling. Recently, we found that microscopic heat pulses induce contractions in isolated rat cardiomyocytes without a rise in [Ca2+]i (Oyama, K. et al., Biochem. Biophys. Res. Commun., 417, 607-612, 2012). Microscopic heat pulses produced by focusing infrared laser beam increase temperature of the extracellular solution near cardiomyocytes. In our experiments, a microscopic heat pulse (ΔT = 5°C for 0.5 sec) induced contractions at a basal temperature of 36°C. When heat pulses were repeatedly applied at 2.5 Hz, oscillatory contractions were observed. In contrast to the electric field stimulation, Ca2+ transients were not detected during the course of contraction. Moreover, we confirmed that skinned cardiomyocytes are thermally activated in the Ca2+-free solution. To elucidate the molecular mechanism of thermal activation, microscopic heat pulses were applied to an in vitro motility assay system utilizing reconstituted thin filaments (rabbit skeletal actin filaments reconstituted with human cardiac tropomyosin and bovine cardiac troponin). Thin filaments started to move during heating in the absence of Ca2+, and most of filaments immediately stopped after switching the laser off, demonstrating that thin filaments can be thermally activated, presumably due to the weakening of the binding affinity of the troponin-tropomyosin complex with actin (Ishiwata, S., Biochim. Biophys. Acta, 534, 350-357, 1978). These results suggest that heat pulses can regulate cardiac contractions by shifting the “on-off” equilibrium in the thin filament toward the “on” state, without accompanying intracellular Ca2+ transients.