Hemicyanine-based High Resolution Ratiometric near-Infrared Fluorescent Probe for Monitoring pH Changes in Vivo
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Intracellular pH is an important parameter associated with cellular behaviors and pathological conditions. Quantitative sensing pH and monitoring its changes by near-infrared (NIR) fluorescence imaging with high resolution in living systems are essential but challenging due to the lack of effective probes. To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable NIR hemicyanine skeleton bearing different substituents, are rationally designed and synthesized, enabling detection through noninvasive optical imaging of organisms. Based on the protonation/deprotonation of the hydroxy group, a marked NIR emission shift provides a ratio signal in response to pH. Meanwhile, two states exhibit good photostability, sensitivity and reversibility, conducive to longtime monitoring of persistent pH changes without disturbance of other biological active species. Among the series, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole exhibited the largest emission shift of about 76 nm from 672 to 748 nm with the pH environment changing from acidic to basic, which could be considered as a good candidate for high resolution pH imaging in live cells, tissues and organisms. Moreover, NIR-Ratio-BTZ has an ideal pKa value (pKa ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. The ratiometric fluorescence measurement is beneficial to ensure the accuracy of quantitative measuring pH changes, as well as the real-time monitoring pH-related physiological effects both in living cells and living mice. The results demonstrate that NIR-Ratio-BTZ is a highly sensitive ratiometric pH probe in vivo, giving it potential for biological applications.Five 2-(2-hydroxyphenyl)benzimidazole fluorescent compounds were synthesized,their structures and fluorescent properties were measured.The paper discussed the relationship of fluorescent characteristics of the fluorescent intensity,fluorescent wave,Stoke′s shift,quantum yield of all the fluorescent compounds synthesized between the substituting groups.
Benzimidazole
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本研究においては,マウス精巣細胞へのクロラムフェニコールアセチルトランスフェラーゼ遺伝子のトランスフェクション効率をin vivoおよびin vitroで行ったリポフェクション法,エクレクトロポーレーション法ならびにパーティクルガン法にっいて比較した.併せて,in vivoエレクトロポーレーションによってマウスの生体精巣における大腸菌βガラクトシダーゼ遺伝子の三次元空間的な発現の獲得を試みた.4週齢のICR系統雄マウスを用いて全部で3回実験を行った.その結果,用いた3種類のトランスフェクション法のうちではin vivoエレクトロポーレーション法とin vivoパーティクルガン法が最も効率が良いことが判明した.in vivoエレクトロポーレーションを用いて大腸菌βガラクトシダーゼ遺伝子を導入したところ,マウス精巣において三次元空間的な明白な遺伝子発現がX-gal染色によって確認され,X-gal染色された細胞の中には精母細胞もしくは精子細胞に類似したものも含まれていた.最大導入可能DNA量,組織の損傷およびトランスフェクション効率を考慮に入れると, in vivoエレクトトポーレーションはマウスの生体精巣細胞への外来遺伝子導入に対して優れている方法であると結論された.
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The lack of a precise definition for "ultra-high resolution chromatography and the common confusion of resolution with plate number is discussed. Examples are given in which high resolution separations are achieved through a combination of chemical and physical insights. Cases in which plate number, relative retention and control of capacity are the dominat factors in producing high, practical resolution are discussed.
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皮膚血管は冷却刺激に応答して収縮し，体表面からの熱放散を制限する．この反応は，交感神経の興奮を介する全身性メカニズムと，局所的に皮膚血管の収縮反応性が増大する局所性メカニズムとが相乗的に機能して引き起こされる．局所性メカニズムの存在は，摘出血管を用いたin vitro実験によって証明されてきた．我々は，ラットやマウスを使ってin vivoで皮膚血流調節を解析する実験方法を確立した．皮膚循環は，様々な因子に起因する神経性の影響を強く受けるため，in vivoで皮膚血流を定量的に測定することは難しい．そこで，電位依存性Na+チャネル阻害薬であるテトロドトキシン（TTX）処置により神経伝導を遮断した条件下で皮膚血流を測定することを試みた．この条件下においても，ラットおよびマウスの後肢を局所冷却することにより，足底部の皮膚血流量が減少することを証明し，局所性メカニズムによる反応をin vivoで定量的に評価できることを示した．興味深いことに，マウスとラットでは，この反応の主要なメカニズムが異なっていた．すなわち，ラットでは，冷却刺激により血管あるいは周囲の細胞からATPが遊離され，これが交感神経終末のP2受容体に作用することでノルアドレナリン遊離を誘発し，平滑筋細胞の主にα1受容体の活性化を介して皮膚血管が収縮するのに対し，マウスでは，冷却刺激はRhoキナーゼの活性化を介して血管平滑筋のα2C受容体を介した収縮反応性を増大させることで皮膚血管を収縮させることが示唆された．In vivoでの解析は，レイノー病はもちろん，糖尿病やホルモン異常など末梢循環障害を来たす病態と皮膚循環調節との関係を解析する際に不可欠であり，この解析方法はこうした病態の治療薬や予防薬の開発にも役立つであろう．
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Under the experimental conditions, the fluorescence reflectance of fabrics improved with the increase of the concentration of fluorescent dyes, but it showed downtrend over a certain concentration; the fluorescence reflectance was the highest at about pH = 5; The fluorescence reflectance of dyed fabrics was improved by fluorescence brightening agent (FBA), when the concentration of FBA in Fluorescent Yellow 2GL was increased from 0.1% to 5%, the fluorescence reflectance was improved from 8.43% to 18.59%, FBA only improved the brightness of non-fluorescent dyes, and no fluorescence was observed, the fluorescence reflectance of fabrics dyed with fluorescent dyes was lowered with the adding of non-fluorescent dyes.
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