This work has been financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (No. 25248027 to M.S.).

1.試料の調製<ol><li> 温度応答性モノマーの精製 <ol><li>トルエン100mL中のN -isopropylacrylamide 20gの（NIPA）を溶解します。 </li><li>ほこりを除去するために吸引ソリューションをフィルタリングします。 </li><li>石油エーテル500mLでろ液を混ぜます。 </li><li>氷水浴中で反応容器を置きます。 </li><li>モノマーは、（典型的には30分）を沈殿させるまで溶液をかき混ぜます。 </li><li>沈殿したモノマーを得るために、吸引下でソリューションをフィルタリングします。 </li><li>一晩、減圧（100 Pa）で下にモノマーを乾燥させます。 </li></ol></li><li> 温度応答性高分子溶液の調製 <ol><li>ドガダイヤフラムポンプを用いて1.0分間、脱イオン水20mL。 </li><li>脱気し、脱イオン水9.5 mLの精製されたNIPAの780.8 mgの溶かします。 </li><li>氷水浴中で反応容器を置きます。 </li><li>からの反応を保護アルミ箔で装置を覆うことにより、光。 </li><li>優しくチューブとガスシリンダーに取り付けられたピペットチップを経由してArガスの適度な流れを導入しながら10分間、溶液を穏やかに攪拌します。 </li><li>マイクロピペットを介して溶液に、Nの11.9μL、N、N &#39;、N&#39; -tetramethylethylenediamineを追加します。 </li><li>ステップ1.2.5で述べたように、Arガスを導入しながら1.0分間溶液を撹拌しました。 </li><li>試料を撹拌しながら、脱気および脱イオン水0.5 mLの過硫酸アンモニウム4.0ミリグラムを溶解します。 </li><li> （ステップ1.2.7）から試料溶液と（ステップ1.2.8から）、過硫酸アンモニウム溶液を混合します。 </li><li>ステップ1.2.5で述べたように、Arガスを導入しながら30秒間、溶液を撹拌しました。 </li><li>アルミホイルでソリューションをカバーし、一晩冷蔵庫（4℃）に保管してください。 </li></ol></li><li> サンプルマウントの準備 <ol><li>から（試料溶液の60μLを配置キャビティスライドステップ1.2.11）。 </li><li>円形のカバーガラスで溶液をカバーしています。トラップ気泡に注意しないこと。 </li><li>マイクロピペットおよび実験ワイプを使用して余分な溶液を除去します。 </li><li>接着剤でサンプルを密封します。室温（典型的には6時間）での接着剤の乾燥をしてみましょう。 </li><li>手順1.3.1-1.3.4に従うことによって、0.1重量％のポリスチレンラテックス（100-nmの粒子径）懸濁液で満たされた別のスライドを準備します。このスライドは、標準として使用されます。 </li></ol></li></ol>動的光散乱顕微鏡による2粒径測定<ol><li> 楽器の最適化 <ol><li>倒立顕微鏡のステージ上で（ステップ1.3.5から）ポリスチレンラテックス懸濁液のスライドを置きます。カバーガラス側が下を向く必要があります。 </li><li>検出器の前のビームダンパー（アバランシェフォトダイオードと自己相関器）を配置します。 </li><li>連続的な、レーザービーム（固体レーザ、λ= 488 nmの30 mWのを適用します対物レンズを通して試料に波）（10×）。反射された光の一部は、顕微鏡の打ち上げミラーを通過し、CCDカメラで観察し、顕微鏡（ 図1）のサイドポートに取り付けられています。 </li><li>ローからハイへの位置から対物レンズの高さをシフトさせることにより試料懸濁液に焦点を設定するために、対物レンズの高さを調整します。カバーガラスと試料との界面に、カバーガラスの表面に、サンプルと正孔スライドガラスとの界面でこの手順の間に、反射像を三回集光されます。第二と第三の点の間の焦点を設定します。 </li><li>レーザパワーを変化させることにより、散乱光強度を減衰させます。 </li><li>検出器の前にビームダンパーを除去することにより、検出器に散乱光を導入します。このユニットは、光強度の時間相関を測定します。 </li><li> betwピンホール（φ= 50μm）を設定します共焦点効果を達成するために、顕微鏡、検出器をEEN。検出器での光強度を最大化するために、ピンホールの位置を調整します。 </li><li>コンピュータを介して相関器の動作を開始して30秒間散乱光強度の時間相関関数を測定します。測定された相関関数は、多くの場合、G（2）（t）と表現される- 1、tは相関時間4と<img alt="式（1）" src="/files/ftp_upload/54885/54885eq1.jpg" /> 。ここで、I（t）は時刻tにおける散乱光強度であり、（•••）Tは、時間平均です。減衰時間は約0.1ミリ秒となります。 </li><li>時間相関関数の初期振幅のための広い範囲を得るために焦点を調整（G（2）、（T = 0） - 1）。 注：初期振幅を強く反射された光の量によって影響されます。焦点トウを移動させることによりカバーガラス及びサンプル反射光の量が増加するとの間のインタフェースをARD。ポリスチレンラテックスのような強い光散乱体については、初期振幅が0から1に変更することができ、反射光の強度よりもはるかに高いので、より一般的なポリマー溶液のための1に近い初期振幅を設定することは困難です散乱光のこと。 </li><li>サイズ分布関数を取得するために取得した時間相関関数に（拘束正則プログラムCONTIN 13,14を使用）の逆ラプラス変換を適用します。初期振幅が0.2未満に設定されている例では、流体力学的半径の分布関数は（詳細については、議論を参照）を2回、実際の半径である100nmで、周りに鋭いピークが表示されます。 </li></ol></li><li> サンプル測定 <ol><li> 25°Cまでのステージ温度を設定します。 </li><li>ポリNIPA（PNIPA）solutを用いて調製したスライドを配置イオン顕微鏡のステージ上で（ステップ1.3.4）。 </li><li>手順2.1.4-2.1.8に従うことによって、散乱光強度の時間相関関数を測定します。初期振幅が0.2より大きい場合、ステップ2.1.9に従うことによって、0.2未満の時間相関関数の初期振幅を作るために焦点を合わせます。小さな初期振幅は、分析を簡素化します。 </li><li> 35°Cまでのステージ温度を設定し、溶液が濁るまで待ちます。 PNIPA溶液の下限臨界溶液温度（LCST）は、32°C 15です。 </li><li>以下の手順により、時間相関関数を測定2.1.4-2.1.8。可能であれば、時間相関関数が0.2未満の初期振幅を作るために焦点位置を調整します。濁った溶液は、その初期振幅は、反射光のそれは一定のままで散乱光が増加する強度は、増加する傾向にあります。 </li><li>逆ラプラス変換transformatiを適用します得られた時間相関関数上にサイズの分布関数を得ることができます。実際のサイズは、初期振幅が0.2未満である場合に半求めた値であることに注意してください。 </li></ol></li></ol>

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The authors have nothing to disclose.

図2（A）に示すように、時間相関関数の初期振幅は重く、焦点に依存します。これは、一見溶液8（界面における薄層を除いて）均一であるという事実と矛盾します。初期振幅のこの変化は、反射光の量の変化に起因します。部分的ヘテロダイン理論16は、初期振幅、A、散乱光の強度は、I S、および反射光強度、I Rは 、以下の式1を満たすことを予測します<img alt="式（2）" src="/files/ftp_upload/54885/54885eq2.jpg" />この式は、 私はrを大きくはなることを示し、小さいAになります 。従って、Aは、インタフェースに近い焦点位置を設定することによって低減されます。見かけの拡散定数D A CAnは、単分散液の場合には、時間相関関数をフィットさせることによって得られます。 <img alt="式3" src="/files/ftp_upload/54885/54885eq3.jpg" />どこ<img alt="式4" src="/files/ftp_upload/54885/54885eq4.jpg" /> 。ここで、nは溶媒（水、1.33）の屈折率であり、θは散乱角（180°）であり、λは光（514.5 nm）での波長です。我々は、後方散乱ジオメトリを適用するので、qの値が固定されています。しかし、この点は、異なる波長の光を使用することによって解決されます。連続波レーザ光源のいずれかの種類がDLS顕微鏡を構築するために利用可能であることに注意してください。小さい照射量のおかげで、コヒーレンスファクター17を超え150であると推定され、無視できる程度です。多分散溶液については、D Aの分布関数は、逆ラプラス変換することによって得られます。部分的なヘテロダイン番目eoryはまた、D Aは実際の拡散定数Dと同じではないと予測します。これら二つの拡散定数は、次式を満たします。 <img alt="式（5）" src="/files/ftp_upload/54885/54885eq5.jpg" />拡散定数Dは、アインシュタイン-ストークス方程式4を用いて、流体力学的半径R Hに変換されます。 A = 1の場合には、この関係は、D A = Dとなります。この場合、データ変換処理は、一般的な動的光散乱用のものと同じです。 図2（B）に示す赤線がこの場合に相当します。対照的に、この関係は、によって示されるように、Aは 、（0.2よりも実質的に少ない）が小さいときにA→0の極限におけるD A = 0.5 Dは、従って、サイズは実際のサイズの2倍であると推定されてなります図2（b）は青線</strong&gt;。我々は、Aが著しく小さいことがわかっている場合は、 図2（c）に示すように、横軸は、シフトさせることができます。原則的に、我々はAの任意の値のためにDにD Aに変換することができます。単純な近似DのA〜D 0.5 が成立するので、実際には、しかしながら、0.2未満の初期振幅を設定した方がよいです。 動的光散乱顕微鏡法の顕著な特徴は、PNIPA溶液を用いて実証されました。 LCST以下と上記PNIPAの立体構造が広く15,18を小角散乱中性子を用いて研究されてきました。対照的に、動的光散乱は、その濁度19のLCST上記PNIPAの分析に利用されていません。 図3（a）及び（b）に示すように、この問題は、動的光散乱顕微鏡によって解決されます。これらの凝集体の大きさは、いくつかあります＆＃181; M、小角X線/中性子散乱または従来の光散乱法のいずれかによって得ることができません。このシステムを用いた時間分解測定は、温度変化の間に凝集プロセスについての情報を与えます。 動的光散乱顕微鏡の欠点はまた、図3に示されています。 LCST以下の結果は、時間相関関数が強くダスト本非常に少量の（ 図3の黒線）によって影響されます。例えば、時間相関関数があっても1.0秒程度の相関時間で、完全に減衰しません。この装置（約1.0ミクロン）を照射量は、通常の動的光散乱装置（約100μm）とその照射よりも著しく小さいためです。散乱光の強度が弱い場合には、信号は、Sによって引き起こされるような、ノイズによって隠されています溶液中の塵のモールの量。サイズの一般的な順序は意味があるが、そのため、 図3（b）に示す三つのピークは、定量的な重要性を持っていないかもしれません。そのような弱い散乱体は、従来の動的光散乱装置によって測定することができることに注意してください。 我々は、動的光散乱顕微鏡は、同じ設定を持つ透明で濁ったサンプルの両方を測定することを可能にすることを実証しました。試料中の光路長が短いため、この技術は、カーボンナノチューブ懸濁液20のように、強い光吸収サンプルに適用することができます。さらに、その高い空間分解能のために、この技術は、生物学的細胞に適用することができます。生物学への応用のために、この方法はまた、蛍光およびラマンイメージングのような他の画像化技術と組み合わせることができます。したがって、我々は、動的光散乱顕微鏡は、研究分野の広い範囲のための強力なツールであると考えています。

Particle size is one of the most fundamental properties of colloidal and polymer solutions. Numerous techniques are used to measure the particle size. Particle sizes of 1.0 &#181;m or larger can be measured directly using an optical microscope. For smaller particles, alternative techniques, such as laser diffraction, electron microscopy, or atomic force microscopy, are used2,3. Dynamic light scattering is a commonly-used technique for the measurement of particle size distributions in solutions4. The results obtained using this technique are not derived from images of the particles but from the characteristic time of the fluctuations in scattered light intensity. These fluctuations originate from Brownian motion, which is characterized by a diffusion constant. The size distribution is obtained from the distribution of diffusion constants using the Einstein-Stokes equation. Due to its simplicity, dynamic light scattering is widely used for the routine assessment of solutions, such as paints and food colloids.
Pretreatment is required for most of the techniques used for the particle size measurement of solution samples. In the case of electron microscopy and atomic force microscopy, the sample must be analyzed under vacuum conditions. Therefore, it is difficult to observe the samples in their native forms. Furthermore, for laser diffraction and dynamic light scattering, only diluted samples that are free from multiple scattering and light absorption can be measured. To overcome this difficulty, several new techniques have been proposed for the measurement of dynamic light scattering from undiluted, concentrated solutions, such as cross-correlation spectroscopy5,6, low-coherence dynamic light scattering7,8, diffusing-wave spectroscopy9,10, and differential dynamic microscopy11,12.
We have developed a new apparatus called a dynamic light scattering microscope1. This apparatus enables us to measure turbid samples without dilution by means of a confocal optical system in which multiple scattering is eliminated using a pinhole. However, the measurement procedure and data analysis are slightly more complicated than those of commercially-available instruments. This video explains the measurement procedure and data analysis in detail using the analysis of the temperature-responsive polymer, poly(N-isopropylacrylamide), as an example.

<table border="0" cellpadding="0" cellspacing="0" width="591">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">N-isopropylacrylamide, 98%</td>
			<td style="width:89px;">Tokyo Chemical Industry Co., Ltd.</td>
			<td style="width:119px;">I0401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">toluene, 99%</td>
			<td style="width:89px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:119px;">201-01876</td>
			<td></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">petroleum ether, distillation temperature 30 ~ 60 &#176;C</td>
			<td style="width:89px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:119px;">169-22565</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">N,N,N&#39;,N&#39;-tetramethylethylenediamine, 99%</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:119px;">T9281</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">ammonium persulfate, 98%</td>
			<td style="width:89px;">Sigma</td>
			<td style="width:119px;">248614</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">polystyrene latex suspension, 1 wt%</td>
			<td style="width:89px;">Duke Scientific Corporation</td>
			<td style="width:119px;">3500A</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">argon</td>
			<td style="width:89px;">Koike Sanso Kogyo Co., Ltd.</td>
			<td style="width:119px;"></td>
			<td>purity &#62; 99.999 vol.%</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;"></td>
			<td style="width:89px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">cavity slide</td>
			<td style="width:89px;">Matsunami Glass Ind.,Ltd.</td>
			<td style="width:119px;">83-0336</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">inverted microscope</td>
			<td style="width:89px;">Nikon Instech Co., Ltd.</td>
			<td style="width:119px;">ECLIPSE Ti-U</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Thermo Plate</td>
			<td style="width:89px;">Tokai Hit CO.,Ltd</td>
			<td style="width:119px;">TP-108R-C</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Ar-Kr ion laser</td>
			<td style="width:89px;">Spectra-Physics</td>
			<td style="width:119px;">Stabilite 2018</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">avalanche photodiode</td>
			<td style="width:89px;">ALV-GmbH</td>
			<td style="width:119px;">ALV-High Q.E. Avalanche Photo Diode</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">correlator</td>
			<td style="width:89px;">ALV-GmbH</td>
			<td style="width:119px;">ALV-5000/EPP</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of particle size distribution in turbid solutions by dynamic light scattering microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

ポリスチレンラテックス懸濁液（粒子径：50 nm）のための散乱光強度の時間相関関数は、（a）は、図2に示すように、異なる焦点で測定しました。この相関関数は、逆ラプラス変換による流体力学的半径の分布関数に変換した（ 図を参照して、図2（b）及び（c））。同じ手順を使用して、PNIPA溶液の流体力学的半径の時間相関関数と分布関数は、それぞれ、25℃及び35℃で得ました。 図3（a）および（b）散乱光強度の時間相関関数、（25°C）以下PNIPA溶液の対応するサイズ分布関数を示し、（35°C）上記LCST。粒度分布関数は、続いて、逆ラプラス変換によって得られました部分的なヘテロダインの補正。 LCST未満の平均流体力学的半径は、ポリマー溶液のために典型的である数十nmです。これとは対照的に、LCST以上の流体力学的半径は約1.0μmです。この結果は、溶液がLCST上記混濁であるという事実と一致しています。解決策は、白濁した後、図3の赤と青の線はそれぞれ、PNIPAの直後に得られた溶液を、20分の大きさの分布を表しています。 図3（b）は、明らかに凝集の成長を示しています。 <img alt="図1" src="/files/ftp_upload/54885/54885fig1.jpg" /> 図動的光散乱顕微鏡の1回路図。ピンホール（PH）、ビームスプリッタ（BS）、偏光子（ポル）、アバランシェフォトダイオード（APD）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54885/54885fig1large.jpg" target="_blank">ご覧になるにはこちらをクリックしてください。</a>この図の拡大版。 <img alt="図2" src="/files/ftp_upload/54885/54885fig2.jpg" /> ポリスチレンラテックス懸濁液2.代表的な結果を図。 （a）は、ポリスチレンラテックス懸濁液のための散乱光強度の時間相関関数。公称半径は50nmであり、濃度は0.1重量％です。 2つのデータセットは、異なる散乱点から得ました。 （B）、（C）に対応するサイズ分布関数（A）の逆ラプラス変換により得られたポリスチレンラテックス懸濁液のため。赤い線は、その初期振幅は約1.0である時間相関関数に対応し、青い線は約0.2である初期振幅とに対応しています。横軸は、部分的なヘテロダイン（PHD）の効果を考慮して（B）を含まない、および（c）を用いて計算した場合&lt;&lt;<html> ; 1. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54885/54885fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54885/54885fig3.jpg" /> 図PNIPAソリューション3.代表的な結果。 （A）PNIPA液の散乱光強度の時間相関関数。 （b）は （A）の逆ラプラス変換により得られたPNIPA溶液のための対応するサイズ分布関数。横軸は、各データセットのための部分的なヘテロダインの影響を考慮して計算しました。黒い線は、25°Cで得られたデータを表します。赤い線は、溶液が濁った（35℃）になった直後に得られたデータを表します。青い線は赤い線の20分間の測定後に得られたデータを表します。/54885/54885fig3large.jpg &quot;ターゲット=&quot; _空白 &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

Watch this Scientific Journal Video about Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy at JoVE.com

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

動的光散乱顕微鏡を用いて濃縮された溶液中で粒度分布の直接測定のためのプロトコルが提供されます。

動的光散乱顕微鏡による濁りソリューションにおける粒度分布の測定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

measurement-particle-size-distribution-turbid-solutions-dynamic-light

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14.1K ビュー.  University of Tokyo. 動的光散乱顕微鏡を用いて濃縮された溶液中で粒度分布の直接測定のためのプロトコルが提供されます。 

ビデオ: Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

コロイドプローブナノスを使用して、粒子間相互作用の定量的および定性的調査

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

化学

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

塩化物イオンの動的電気化学測定

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

NMRによるリアルタイム酵素反応速度測定のための溶解動的核分極計装

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

光温度計の作製と試験

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

バインドの動的・電気泳動光散乱による解析 DNA 磁性粒子

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

シリアル結晶の結晶 x 線回折その場その場での動的光散乱のためのマイクロ流体チップ

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

軟 x 線吸収分光と共鳴非弾性 x 線散乱による電池の化学元素高感度検出

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

F− 310 Nm 剥離によって示されている陰イオンの光電子イメージング

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

動的光散乱粒子径解析における電圧の応用

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam&#160;rapidly induces radiation damage of photosensitive&#160;cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (&#62; 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein&#160;crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps.