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><tbody><tr><td>&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide, 98%</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>I0401</td><td></td></tr><tr><td>toluene, 99%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>201-01876</td><td></td></tr><tr><td>petroleum ether, distillation temperature 30 ~ 60 &amp;deg;C</td><td>Wako Pure Chemical Industries, Ltd.</td><td>169-22565</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetramethylethylenediamine, 99%</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>ammonium persulfate, 98%</td><td>Sigma</td><td>248614</td><td></td></tr><tr><td>polystyrene latex suspension, 1 wt%</td><td>Duke Scientific Corporation</td><td>3500A</td><td></td></tr><tr><td>argon</td><td>Koike Sanso Kogyo Co., Ltd.</td><td></td><td>purity &gt; 99.999 vol.%</td></tr><tr><td>cavity slide</td><td>Matsunami Glass Ind.,Ltd.</td><td>83-0336</td><td></td></tr><tr><td>inverted microscope</td><td>Nikon Instech Co., Ltd.</td><td>ECLIPSE Ti-U</td><td></td></tr><tr><td>Thermo Plate</td><td>Tokai Hit CO.,Ltd</td><td>TP-108R-C</td><td></td></tr><tr><td>Solid-state laser</td><td>Coherent</td><td>OBIS 488LX</td><td></td></tr><tr><td>avalanche photodiode</td><td>ALV-GmbH</td><td>ALV-High Q.E. Avalanche Photo Diode</td><td></td></tr><tr><td>correlator</td><td>ALV-GmbH</td><td>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="http://ecsource.jove.com/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="http://ecsource.jove.com/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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The Laplace Transform

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14.2K 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

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

ナノサイズの脂質小胞の調製のための一定の圧力制御押出法

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

生物工学

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

水溶液中におけるナノ材料の分散: プロトコルの最適化に向けて

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

環境学

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

制御された合成および均一性の高いポリの蛍光トラッキング（<em&gt; N</em&gt; -isopropylacrylamide）ミクロゲル

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

化学

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 ...

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. ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

高分子電解質複合体ミセルの組み立てと特徴付け

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板輸血で微粒子のスクリーニング法のルーチン

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

医学

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

Differential dynamic microscopy (DDM) combines features of dynamic light scattering and microscopy. Here, the process of using DDM to characterize reconstituted cytoskeleton networks by quantifying the subdiffusive and caged dynamics of particles in vimentin networks and the ballistic motion of active myosin-driven actin-microtubule composites is presented.

微分動的顕微鏡を用いた細胞骨格ダイナミクスの定量化

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

エキソソームの定量化とサイズの測定のための革新的方法

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

生物学

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose.

鉄-炭水化物複合体の粒径測定のための動的光散乱分析

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally ...