この作品は、オーストラリア研究評議会リンケージインフラ機器と設備グラントLEO668421とオーストラリア研究評議会の連携プロジェクト無償LP0669471によってサポートされていました。作者は感謝してKIT -マッコーリー脳研究所設立に金沢工業大学と横河電機の協力を認める。

これらのガイドラインは、脳磁図（MEG）を使用して、未就学児の認知脳機能を測定するための装置と手順を説明します。最初に、私たちは子どもたちの認知機能の実験的なテストを実行する際に考慮しなければならない一般的なガイドラインと問題点を話し合う。第二に、我々は、4歳児の動機づけと行動の容量を持つMEGデータ収集の要件を調整するために開発されたプロトコルについて説明します。 1。 MEG幼い子供を研究するための一般的な考慮事項目を覚まし健康な子供を扱うときMEGの研究者が固有の課題の数に直面している。最初に、荒涼と機能的な実験室環境では、若い子供たちを威嚇または怖がらせる可能性があります。第二に、実験的脳研究の基本的な要件は、動きの制限を含め、積極的に、長時間の作業に多くの大人は、退屈、不快見つけることが条件に参加。子供を勉強するときに、それは子どもの能力や限界に実験手順を適応させることが不可欠となる。このセクションでは、我々は、データ収集のセッションに参加する被験者の準備に関与する最初の手順を説明します。 1.1ゼーション/研修会：就学前児童のための習熟/トレーニングセッションが前に実際のデータ収集のセッションが予定されている。このセッションでは、子どもが自分のペースで環境や研究者に精通することができます。我々は研究者に子と親を紹介し、ラボ環境とルーチンでそれらを理解。入門セッションの終わりまでに親と子は、参加に関わるすべての手順を理解し、彼らはデータの記録時に期待すべきかを理解する。最初の訪問の間に、子供は恥ずかしがり屋や不安が表示される場合、それらをより快適に感じるまで、我々は再生時間の周期を持っている。その後、我々はすべてのステップの導入を進める。 1.2環境を説明：子にMEGの環境を説明するために、我々は周囲のための子供向けの正当化を提供するテーマを採用しています。例えば、MEGシステムは、いくつかのなじみのないイベントが発生する可能性のある宇宙の冒険に子供を取るのを待っている宇宙船です。研究室では、壁のステッカーやおもちゃ（図1）で飾られている、そしてそれは、専用のプレイルームを持っています。子供に話すとき、私たちは子どもたちが容易にそのような代わりに&quot;マーカーコイルキャップ&quot;の代わりに&quot;最小限の動き&quot;の&quot;凍結&quot;、そして&quot;宇宙飛行士のヘルメット&quot;として理解できる言語を使用してください。 1.3子どもへ、両親への手順を説明する：私たちは、子と親の子は磁気シールドルーム（MSR）の内部に入る前に取られるステップを示しています。最初のステップの一つは、被験者の頭の上の5つのマーカーコイルキャップを取付けてであり、これらのコイルは、MEGのヘルメットの中に被写体の頭の位置を示します。子供の助けを借りて、我々デジタル化する人形の頭部の形状、そしてその後、我々は、子供とMSR /宇宙船に人形を伴うために親を招待。一度MSRの内側、我々は映画を鑑賞するためにヘルメットの内部に人形の頭を置く。彼らは人形と一緒に映画を鑑賞する場合に我々は、その子を求める。子供は快適でリラックスであり、彼らの親が私達のプロトコルに満足している場合、我々は彼らが宇宙船の内部に、私たちの研究室を訪問、次回のゲームをプレイするために招待。 1.4ワークブック：私たちは、子どもたちは、テストセッション中に行われる手順を示して宇宙の冒険のテーマに基づいて、ワークブックを持っている。子どもたちは家庭を取ると次の訪問の準備のために両親とそれらを介して動作するようにこれらのworksbooksを与えられている。ワークブックの目的は、テストするには、3つの主要な要件の重要性を強調するためである。a）MSR内の任意の鉄系材料の回避、b）の正確なデータの共同登録とcを確保するためのマーカーのコイルキャップの配置）とデジタル化とデータ収集中にまだ残っていることの重要性。 2。就学前年齢の子どもからMEG信号を測定するためのプロトコル データ収集と実験的なタスクの実行中に使用される2.1機器 マーカーコイルキャップ：主題の構造情報（例えばMRIまたはデジタル化された頭部の形状）、五コイルのセットでMEGのデータを整列するためには、子供がMEGヘルメットの内部に身に着けている水泳キャップに配置されます。これらのコイルは基準点として機能し、その位置は、被験者の頭部は、買収時に移動しているどの程度を測定するタスクの前後に比較されます。 デジタイザ：データ収集に先立ち、五マーカーのコイルと被験者の頭部の形状の位置は、Polhemus社のFを使用してデジタル化されるastrak ®デジタイザ（コルチェスター、バーモント州）。デジタイザは、ペンデジタイザ、3つのレシーバ、トランスミッタ、モーショントラッキングのユニットで構成されています。このプロセスは、コイルの位置を測定します。後でこれらの場所は、取得した磁気データとヘッドの形状を揃えるためにMEGで得られる位置情報と照合されます。 子MEGシステム ：データ収集は全頭子MEGシステム（KIT、金沢、日本）[2]を使用して行われます。データは0.03 Hzから200Hzの間でオンラインのバンドパスフィルタと、1000Hzのサンプリングレートで連続的に取得されます。マーカのコイルの位置は、ヘッドの移動を測定するための録音の前後に取得されます。ヘッドの移動は5mmより大きい場合、データは拒否されます。 MEGシステム周辺機器：実験刺激は、プレゼンテーション®（神経行動学的システム、バニー、CA）を使用してPCで制御されます。視覚的な入力は、MSRの外に座っているとMSRの画面上に反映されているプロジェクターによって配信されます。聴覚刺激は、赤ちゃんサイズの泡のイヤホン（Etymotic Research社、エルクグローブヴィレッジ、イリノイ州）を装着したEtymotic研究モデルER - 30インサートイヤホンによって配信されます。子は常に閉じた回路のカメラによって監視され、実験者がマイクを使って子供と連通している。 2.2データ収集：データのレコーディングセッションでは、次の段階を伴います。 ステージI：前の子供の到着に 早く子供が到着する実験を開始する準備がすべてのコンピュータ、ソフトウェアや機器を持つことが重要です。子と親が研究室にいる間一般的な推奨事項として、2つの経験豊富な研究者が存在する必要があります。研究者、親と子の間に明確なコミュニケーションは、実験の成功にとってきわめて重要である。 ステージII：子どもが到着した後、 <ol><li>同意書に署名し、親が質問がある場合は、実験者はチェックされています。 </li><li>子供が最初の会議中、または小冊子で導入されたすべての手順を記憶し、記憶されているものを再導入した場合実験者がチェックします。デジタル化とデータ収集中に動きを凍結または最小化の重要性が強調されている。これは、人形を使って実証することができます。 </li><li>子供が（グリッターパッチ付き衣類、ジュエリー、ポケット内部に忘れおもちゃ、ヘアピンなど）を、鉄系材料を運んでいるならば、、実験者がチェックします。彼らは鉄系材料を含んでいる衣類の一部を実行する場合、我々は、MEG安全な衣類を提供してください。親が実験中に子どもを同行することを望む場合、我々は、彼らがまた、鉄鋼材料を除去することを確認してください。 </li></ol> ステージIII：MSR内のタスクを実行するために子供が準備 <ol><li>実験者は、5マーカーコイルを保持するキャップに適合します。 </li><li>実験者は、マーカーのコイルの位置と子供の頭部の形状をデジタル化。固定位置で頭を維持するために、我々は3つのレシーバを装着されている首の支柱を使用してください。良好なヘッド形状を得るために定期的に運動や&quot;凍結&quot;を最小限にするという考えを補強することが重要です。子は&quot;凍結&quot;で良い仕事を行うためのステッカーで報われることがあります。 </li><li>子は、MSRを入力するように招待されている。イヤホンが挿入され、子は、仰臥位でMEGデュワーのヘルメットに配置される。 </li><li>子供がMEGヘルメット内部の頭で快適に横になってされると、マーカのコイルは、その電源ユニットに接続され、MSRのドアが閉じている。一つの実験は、全体のセッションの間にお部屋で（必要に応じても、親、）のまま。 </li><li>セットアップ中およびデータ収集のブロック間の娯楽のために、映画や漫画は、MSRの逆投影スクリーン上に投影することができます。 </li></ol> ステージIV：実験の実行 <ol><li> MSRのドアが閉じられると、イカが従事し、データ収集が開始されています。 </li><li>子供が映画を観るように、coregistrationの事前テストは、MEGの信号がデジタル化された頭部の形状にcoregisteredできることを確認するために行われている。このプレテストは、MEGのシステムで示す位置にデジタル化の間に得られたマーカのコイルの位置と一致するで構成されています。 </li><li>いったん最初のテストでは、子供の頭が正しくヘルメットの内側に配置され、研究者が実験を開始できることを示しています：（a）の映画がミュートされ、彼らは&quot;ゲームをプレイ&quot;する準備ができている場合（b）の研究者は、子を要求します。ゲームが開始されると（c）に、実験者は、定期的にフィードバックを持つ子を提供します。例えば、子供は文のシリーズに耳を傾け、そしてできるだけ早く彼らはそれを聞くと、&quot;チーズ&quot;などのターゲットの単語を繰り返すよう求められる場合があります。これらのターゲットの単語がキャッチトリのサブセットに埋め込まれていますALSは、そのプローブは、タスクへの気配りを続けたが、最終的な分析に含まれていません。 </li><li>タスクが完了すると、別のマーカーのコイルの測定は、頭の位置と動きを監視するために実行されます。 </li><li> SQUIDのが外れると、MSRの扉が開かれ、子供は彼らが良い仕事のための賞を選ぶことができるプレイルームに招待されている。 </li></ol> 3。代表的な結果我々の研究室における代表的な研究は、子どもの言語のロジックの開発を研究するためにこのプロトコルを使用していました。 3-4年熟成二十六の子供（4歳と5ヶ月の平均では）少なくとも一つの取得のセッションに参加した。これらの子供たちのうち17秒のテストセッションに返され、5人が第三のテストセッションのために来た。平均的なヘッドの動きがよく5ミリメートルしきい値内未満3.7ミリメートル（SD 3.1ミリメートル）であった。 5mm以上大きな動きは、5.5ミリメートルから15ミリメートル（= 8.6ミリメートル、SD = 2.5 mmの平均）に至るまで変化して、セッションの4.5％に発生しました。 <img src="/files/ftp_upload/1693/1693fig1.jpg" alt="図1" /> 図1：MSRとMEGのヘルメットは、ステッカーやぬいぐるみで飾られています。 <img src="/files/ftp_upload/1693/1693fig2.jpg" alt="図2" /> 図2。成人サイズのMEG（左）の四歳の少女。運動と頭部とヘルメットの間の距離を最小限に抑えるために彼女の頭の上部にあるパディングに注意してください。右の画像は、子のMEGで同じ女の子を示しています。

<ol>
	<li>H&#228;m&#228;l&#228;inen, M., Hari, R., Ilmoniemi, R. J., Knuutila, J., Lounasmaa, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalography-+theory,+instrumentation,+and+applications+to+noninvasive+studies+of+the+working+human+brain.">Magnetoencephalography- theory, instrumentation, and applications to noninvasive studies of the working human brain.</a> Rev. Mod. Physics. 65 (2), 413-497 (1993).</li><li>Johnson, B. W., Crain, S., Thornton, R., Tesan, G., Reid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+brain+function+in+pre-school+children+using+a+custom+sized+whole-head+MEG+sensor+array.">Measurement of brain function in pre-school children using a custom sized whole-head MEG sensor array.</a> Clin. Neurophysiol. , (2009).</li><li>Kado, H., Higuchi, M., Shimogawara, M., Haruta, Y., Adachi, Y., Kawai, J., Ogata, H., Uehara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalogram+system+developed+at+KIT.">Magnetoencephalogram system developed at KIT.</a> IEEE Trans. Appl. Supercond. 9, 4057-4062 (1999).</li><li>Uehara, G., Adachi, Y., Kawai, J., Shimogawara, M., Higuchi, M., Haruta, Y., Ogata, H., Kado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Channel+SQUID+Systems+for+Biomagnetic+Measurement.">Multi-Channel SQUID Systems for Biomagnetic Measurement.</a> IEICE Trans. Electr. E86-C, 43-54 (2003).</li></ol>

このプロトコルは、健康と目を覚まし4歳の子供の認知脳機能の測定を容易にするために設計されています。我々は、就学年齢の子どもの頭に合うように設計されたカスタムサイズのMEGシステムを使用してください。このシステムでは、子供向けの実験室の環境、および機能と子どもたちの動機に適応protocolは、我々は非常に少ないデータが現在存在するする年齢層に認知の脳機能を測定することができます。 KIT -マッコーリーシステムは、就学前年齢の子どもで使用するためのサイズの第一全体のヘッド方式のカスタムです。子どもたちを研究するためのカスタマイズされたヘルメットデュワーの3つの主要な利点があります。最初に、センサーの設定の半径は、子供の頭のneuromagnetic信号の範囲にSQUIDセンサをもたらします。第二に、小さなヘルメットはデュワーに子供の頭を完全に挿入することができます。完全な挿入は、お子様をお連れの肩の距離（図2参照）に小さい王冠の成人デュワーのヘルメットに阻止される。 neuromagnetic信号は距離と共に急速に減衰するため、これら二つの要因は、脳磁図を用いた脳活動を記録するの基本です。第三に、カスタマイズされた子のヘルメットのヘッドの左右対称の位置に補助とは、デュワー内で子供の頭の動きの自由度を制限。これらの機能は大幅にセットアップ、ポジショニング、そして子供からのMEG信号の測定を容易にします。 カスタムサイズの子MEGシステムの出現は、認知神経科学のための重要な前進です。放射性医薬品や強い磁場を用いる代替機能的神経イメージング技術は、小さなお子様連れルーチンの使用に適しているとは限りません。 MEGは、完全に受動的な計測手法であるため、組織を開発するためには、考えられる危険性はありません。従来の大人のMEGシステムは、子どもたちと一緒に使用するのに適していないので、健康な就学前の年齢の子供たちからは非常に少ないMEGのデータは、現在ありません。子MEGシステムは、認知機能の正常と病理学的発展に新たな洞察を提供し、私たちは脳の活動の劇的な構造が発生している年齢でで脳機能を研究することができます。

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Whole head child MEG</td>
<td>&nbsp;</td>
<td>Kanazawa Institute of Technology (Kanazawa) and Yokogawa Electric Corporation (Tokyo)</td>
<td>Model PQ1064R-N2m</td>
<td>&nbsp;</td>
<tr>
<td>Magnetically shielded room</td>
<td>&nbsp;</td>
<td>Fujihara Co. Ltd., (Tokyo)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Digitiser</td>
<td>&nbsp;</td>
<td>Polhemus (Colchester, VT)</td>
<td>Fastrack</td>
<td>&nbsp;</td>
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<td>Experimental control software</td>
<td>&nbsp;</td>
<td>Neurobehavioral Systems (Albany, CA)</td>
<td>Presentation</td>
<td>&nbsp;</td>
<tr>
<td>Earphones</td>
<td>&nbsp;</td>
<td>Etymotic Research Inc. (Elk Grove Village, IL)</td>
<td>ER-30</td>
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measurement of neuromagnetic brain function in pre-school children with custom sized meg

脳磁図は、皮質の活動[1]に関連する磁場を検出する技術です。脳の電気生理学的活性は、電界を生成する - とその付随する磁場 - - 脳波を脳波（EEG）を使用して記録することができるMEGで検出された。 MEGの信号は、量子干渉素子（SQUIDのを）超伝導と呼ばれる特殊なセンサーによって検出されています。超電導センサーは-270で液体ヘリウムで冷却℃を必要とする彼らは、液体で満たされているデュワーと呼ばれるvacumm絶縁ヘルメット、内部に含まれています。 SQUIDのは、ヘリウム冷却材中のヘルメットデュワー内の固定位置に配置され、被験者の頭部は、MEGの測定のためにヘルメットデュワー内に配置されます。ヘルメットデュワーは反対の制約を満たすサイズにする必要があります。明らかに、それは、ほとんどまたはすべての調査される人口の頭のに合わせて十分な大きさでなければなりません。しかし、ヘルメットはまた、測定するためであることが小さな脳分野の範囲内にSQUIDセンサのほとんどを保つために十分に小さくなければならない。従来の全頭MEGシステムは、成人の頭の90％以上を収容するように設計されています。しかし、成人のシステムはよくその頭大人より数cm小さい半径を有する就学前童車での脳機能を測定するには適していません。マッコーリー大学のKIT -マッコーリー脳研究所では、就学前の子どもたちの頭に合わせてサイズMEGシステムのカスタムを使用しています。この子システムが50mmのベースラインを持つ64の一次軸勾配計を持っている[2]と、従来の成人サイズのMEGシステムと一緒に磁気シールドルーム（MSR）の中に含まれている[3,4]。子どもたちを研究するためのカスタマイズされたヘルメットデュワーの3つの主要な利点があります。最初に、センサーの設定の半径は、子どもたちの頭のneuromagnetic信号の範囲にSQUIDセンサをもたらします。第二に、小さなヘルメットはデュワーに子供の頭を完全に挿入することができます。完全な挿入は、お子様をお連れの肩の距離に小さい王冠の成人デュワーのヘルメットに阻止される。 neuromagnetic信号は距離と共に急速に減衰するため、これら二つの要因は、脳磁図を用いた脳活動を記録するの基本です。第三に、カスタマイズされた子のヘルメットのヘッドの左右対称の位置に補助とは、デュワー内で子供の頭の動きの自由度を制限。子どもたちの動機や行動能力とデータ収集の要件を整列プロトコルで使用する場合、これらの機能は大幅にセットアップ、ポジショニング、およびMEG信号の測定を容易にします。

Watch this Scientific Journal Video about Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG at JoVE.com

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

幼児用サイズのMEGシステムの出現は、脳の発達を研究するために重要な新しい機会を開きます。新システムは、一緒に子供たちの能力で実験的な要件を揃えプロトコルと3〜6歳の健康、目を覚まし子どもの認知と言語のプロセスを研究するために使用することができます。

カスタムサイズのMEGと就学前のお子様でNeuromagnetic脳機能の計測

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

measurement-neuromagnetic-brain-function-pre-school-children-with

Nanyang Technological University

Research

JoVE Journal

Biology

16.8K ビュー.  Macquarie University. 幼児用サイズのMEGシステムの出現は、脳の発達を研究するために重要な新しい機会を開きます。新システムは、一緒に子供たちの能力で実験的な要件を揃えプロトコルと3〜6歳の健康、目を覚まし子どもの認知と言語のプロセスを研究するために使用することができます。

ビデオ: Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

広視野顕微鏡を用いて長期的なイメージング哺乳類細胞

生物学

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

近赤外分光法で赤ちゃん、子供と大人の認知機能を探る

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Performing Custom MicroRNA Microarray Experiments

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

カスタムmicroRNAのマイクロアレイの実験を行う

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZrカスタムジンクフィンガーヌクレアーゼによるゲノム編集（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS /タクシーと細胞走化性の測定

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

ゼブラフィッシュ幼生における筋機能を評価するために、収縮時の力測定

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

薬理研究に単離された組織のバース·アプリケーションにおける平滑筋機能の測定

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

サンプル準備および透過電子顕微鏡によるエクソソームのイメージ投射

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...