シュレヤ・シャーの校正とアドバイスに感謝します。

1. ドップラーファントム準備のセットアップ (図2A)
<ol><li>蠕動ポンプ、血液模擬流体貯蔵所、パルスダンパー、ドップラーフローファントムをプラスチックチューブに接続します。</li>
	<li>直径4mmの運河を選ぶ。</li>
	<li>ポンプを0.3 sの流体720 mL/minに排出し、0.7 sで50 mL/minを排出して収縮期と拡張期心相をそれぞれ模倣するようにプログラムする</li>
	<li>ポンプを実行し、静かに潜在的な気泡を排出するためにパイプを振ります。 注:オペレータは別の運河の直径および異なったポンプ速度を選びることができるが、超音波の順序が最も速い流れの速度を得るのに十分な速さであることを保障しなければならない。Eq. 3 は後で提示され、シーケンスを設計するのに役立ちます。</li>
</ol>2. 超高速超音波スキャナーのセットアップ (図 2A)
<ol><li>超高速対応のリサーチ スキャナを、PCI Express リンクを使用してホスト コンピュータに接続します。</li>
	<li>超音波スキャナーのトランスデューサアダプタをプローブコネクタに合わせて交換し、プローブを接続します。</li>
	<li>Matlabを実行し、超音波スキャナのライセンスをアクティブにします。 注:このセクションと以下は、Verasonics Vantageシステムの使用を暗黙のうちに想定しています。</li>
</ol>3. 超音波シーケンスプログラミング
<ol><li>例のスクリプトを使用して、プローブの位置決めに使用される従来の焦点を合わせられる「Bモード」(すなわち、echography)シーケンスを設計する。<ol>
<li>撮像深さを50mmに設定します。</li>
		<li>焦点深さを35mmに設定します。</li>
	</ol>
</li>
	<li>スクリプトの例を使用して、超高速超音波シーケンスを設計します。<ol>
<li>撮像深さを50mmに設定します。</li>
		<li>プログラム3は[-3,0,3]度で平面波を傾けた。</li>
		<li>パルス繰り返し周波数(PRF)を12 kHzに設定します。</li>
		<li>使用するプローブに応じて、中心周波数で、超音波波形に4半サイクルを使用してください。中心周波数は 5.2 MHz と想定されます。</li>
		<li>合計期間を 1 s に設定します。</li>
	</ol>
</li>
</ol>4. プローブの位置決めとデータ取得
<ol><li>プローブのレンズに超音波ゲルを塗布します。</li>
	<li>プローブをファントムに置き、Bモード超音波シーケンスを起動します。</li>
	<li>興味のある運河を見つけます。流体は周囲の組織よりも暗く見えます。プローブを縦方向のビューに配置します。</li>
	<li>プローブを対象の位置に手動で維持します。</li>
	<li>Bモードシーケンスを終了し、超高速シーケンス取得スクリプトを起動します。</li>
</ol>5. 画像再構成 (図2B)
<ol><li>シーケンスが終了したら、生データ(無線周波数データ「RF」とも呼ばれる)を保存します。</li>
	<li>超音波システムのデフォルトソフトウェアを使用して画像再構築スクリプトを起動します。プロセスの最後に、IQ データマトリックスを作成する必要があります。 注:超音波エコーは、プローブの各要素と各放出/受信に記録され、RFデータマトリックスに保存されます。画像再構成は、各チャンネルに適切な遅延則を適用し、いわゆる「IQ」(位相/直交)行列を生み出した。複雑なIQマトリックスは、スペース(画像の深さと幅)と時間用の3次元を持っています</li>
</ol>6. クラッターフィルタリング (図 2C)
注: 手順 6 ~ 7 については、「 補足資料」に記載されている Matlab スクリプトを参照してください。
<ol><li>3D (空間 x 空間 x 時間) の IQ 行列を 2D (空間 x 時間) の Casorati 行列に変更します。</li>
	<li>IQrの単数分解15(Eq. 1)を計算する。 <img alt="Equation 1" src="/files/ftp_upload/61838/61838equ01v3.jpg"> Eq. 1</li>
	<li>バランゲルら (II,D)で記述された空間的特異ベクトルU(II,D)を用いて空間類似行列Cを計算し、血液部分空間境界Nを特定する。</li>
	<li>このカットオフNを使用して、Demeneら(II,C)に記載されているIQデータをフィルタリングする。</li>
</ol>7. 流れの視覚化と速度の測定 (図 2C)
<ol><li>時間ディメンションに沿ってフィルターされたデータ IQt のエンベロープを統合することによって、パワードップラーマップPDを計算します(Eq. 2)。3D 座標 z、x、t はそれぞれ深さ、幅、時間寸法 、nt であり、取得したフレーム数です。 <img alt="Equation 2" src="/files/ftp_upload/61838/61838equ02v5.jpg"> Eq. 2</li>
	<li>PD マップを対数スケールで表示します。ダイナミック レンジを設定するには、運河の外側の領域で平均 PD を計算し、この値を dB でダイナミック レンジの下限として使用します。一般的なダイナミック レンジは [-30, 0] dB です。</li>
	<li>1 ~ 30 ピクセルを含む、画像上の対象となる円形領域 (ROI) を定義します。</li>
	<li>そのROIのピクセル上のIQf信号を平均して <img alt="Equation 3" src="/files/ftp_upload/61838/61838equ03v2.jpg"> 、nt時間ポイントのベクトルを得る。</li>
	<li><img alt="Equation 4" src="/files/ftp_upload/61838/61838equ04v2.jpg">短時間フーリエ変換 (STFT) の平方マグニチュードを使用して、ドップラースペクトログラムを計算して表示します。<ol>
<li>STFT ウィンドウを 60 サンプルの「Hann」ウィンドウに設定します。</li>
		<li>STFTのオーバーラップをウィンドウ長の90%に設定します。</li>
	</ol>
</li>
	<li>スペクトログラムの各時点で中心周波数をオーバーレイします。</li>
	<li>ドップラー式(Eq. 3)を使用して、周波数f値を血液軸速度vzに変換します。c0は、送信された超音波波形の中心周波数(ここでは5.2MHz)の媒体とfTWの音速です。 <img alt="Equation 5" src="/files/ftp_upload/61838/61838equ05v4.jpg"> Eq. 3</li>
</ol>

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超音波画像診断は、臨床現場および研究活動において最も一般的に使用される画像化技術の1つである。生物組織における超音波放出の組み合わせと後方散乱エコーの記録により、解剖学的画像、いわゆる「Bモード」の再構築が可能となる。この方法は、通常、≈1540 m/sの伝播速度で、通常、数センチメートルにわたって超音波の浸透を可能にする生体組織などの軟部組織イメージングに完全に適合しています。超音波プローブの中心周波数に応じて、30μmから1mmまでの解像度の画像が得られます。さらに、音響源の動きが、関連する波の物理的特性に影響を及ぼすことはよく知られている。特に、波の周波数シフトとソースの速度に対する周波数シフト間のリンクは、ドップラー効果1と呼ばれ、その最も単純な症状は、移動する救急車のサイレンのピッチの変化である。超音波画像は長い間、この物理的効果を使用して移動する赤血球2を観察し、一般的に「ドップラーイメージング」とラベル付けされる様々なイメージングモードを提案している。これらのモードは、脳、心臓、腎臓または末梢動脈などの非常に異なる用途および器官における血液の流れの評価を可能にする。
驚くべきことに、現在利用可能な超音波システムのほとんどは、従来の超音波と呼ばれる同じ技術に依存しています。基礎となる原理は次のとおりです:音響ビームは視野を内在化し、超音波トランスデューサ開口部に沿って掃引されます。ビームの各位置について、エコーが記録され、最終的な画像のラインに変換されます。ビームをトランスデューサに沿って徐々に移動させることで、全視野をライン毎に画像化することができます(図1、左パネル)。この戦略は、21世紀初めまで普及していた電気的制約と計算能力にうまく適応していました。それにもかかわらず、それはいくつかの欠点を持っています。これらの中で、最終的なフレームレートは、ビームスキャンプロセスによって毎秒数百枚の画像に制限されています。血流の面では、この比較的低いフレームレートは、シャノン・ナイキスト3のサンプリング基準によって決定される検出可能な最大流速に影響を与える。さらに、従来のドップラーは複雑なトレードオフに対処する必要があります。特定の関心領域(ROI)における血流速度を評価するためには、そのROIから来るいくつかのエコーを連続的に記録する必要があります。これは、超音波ビームが一定の位置に一時的に維持されることを意味します。エコーアンサンブルが長ければ長いほど、そのROIに対する速度推定が良くなります。しかし、視野の完全な画像を生成するには、ビームは媒体をスキャンする必要があります。したがって、1 つの線に沿って速度を正確に評価するためにビームを保持するか、ビームを移動してイメージを生成するかという 2 つの拘束の間の競合を感知できます。異なる従来のドップラーモード(すなわち、カラードップラーまたはパルス波ドップラー)は、このトレードオフを直接反映しています。通常、カラードップラーは、容器4を局所化するために使用される低忠実度のフローマップを生成し、パルス波ドップラーは、以前に同定された容器5内の流れを正確に定量するために使用される。
これら 2 つの制限 (低フレームレートとローカリゼーション/定量のトレードオフ) は、非常に高フレームレートの新しい手法で克服されます。これらの中でも、合成開口アプローチ6またはマルチライン送信技術は7を挙げられる。本研究では、いわゆる超高速超音波法に焦点を当てる。20年前に導入された8、9、10、この方法はまた、超音波の放出/受信に依存していますが、根本的に異なるパターンを持っています。確かに、走査に焦点を当てたビームを使用する代わりに、超高速イメージングは平面波または発散波を使用し、単一の放出で視野を内振することができます。その単一の放出に続いて、関連する電子工学はまた全体の視野から発生する多数のエコーを受け取り、処理することができる。最後に、画像は単一の放出/受信パターン11(図1、右パネル)から再構築することができる。これらの非焦点放出は音響エネルギーの普及による低い信号対雑音比(SNR)を有することができる。これは、いくつかのタイトルの平面波(または異なるソースで波を発散する)を放出し、結果の画像を追加することによって取り組むことができます。この方法は「コヒーレント・コンパウンディング」12と名付けられています。2つの大きな結果が生じます。まず、フレームレートは飛行の超音波時間にのみ依存し、1〜10 kHzの典型的な値に達することができます。第 2 に、空間ディメンションと時間ディメンションの両方でデータの連続性が保証されます。したがって、従来のローカリゼーション/定量化のトレードオフは破られます。高いフレームレートと時空的な一貫性のこの組み合わせは、超音波で血液の流れを検出する能力に大きな影響を与えます。従来の超音波と比較して、超高速超音波は、血流の完全な特性を提供します 3.実際には、ユーザーは、取得の全期間(通常は≈1秒)、フレームレート(通常は、時間分解能200μsの場合は5kHzのフレームレート)で、画像のすべてのピクセルの速度時間コースにアクセスできます。この高いフレームレートは、冠状マイクロ灌流14を有する心臓室13や心筋のような移動器官における速い流れのような幅広い適用に適した方法を作る。さらに、その時空間的な一貫性が、バックグラウンド移動組織から遅い血流を分離する能力を強く向上させ、したがって微小血管流れ15に対する感受性を増加させることが示されている。この容量は、動物16と人間17の両方の脳の微小血管系へのアクセスを提供します。
したがって、超高速超音波は、様々な状況での血流を画像に適しています。それは軟らかい生物学的組織に限定され、骨、または肺のようなガス腔のような硬いインターフェースの存在によって強く影響を受ける。超音波シーケンスの物理的なパラメータのチューニングは、遅い(1 mm / s11、16まで)と高速流れ(数m / sまで)の両方の研究を可能にします。空間解像度と浸透深度の間にはトレードオフがあります。通常、50 μmの分解能は、5 mm程度の浸透を犠牲にして達成できます。逆に、1mmの分解能を犠牲にして、浸透を15〜20cmまで拡張することができます。この記事で使用されているような超高速スキャナの多くは、2D画像のみを提供していることは注目に値します。
ここでは、生体組織に埋め込まれた血管(動脈または静脈)を模した超音波スキャナーとドップラーファントムをプログラム可能な研究を用いて、超高速ドップラーイメージングの概念を導入する簡単なプロトコルを提案する。

<table>
	<tbody>
		<tr>
			<td>Blood-mimicking fluid</td>
			<td>CIRS Inc, Norfolk, Virginia, USA</td>
			<td>069DTF</td>
			<td></td>
		</tr>
		<tr>
			<td>Doppler flow phantom</td>
			<td>CIRS Inc, Norfolk, Virginia, USA</td>
			<td>ATS523A</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks, Natick, Massachusetts, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump / Doppler flow pump</td>
			<td>CIRS Inc, Norfolk, Virginia, USA</td>
			<td>769</td>
			<td>Include tubings and pulse dampener</td>
		</tr>
		<tr>
			<td>Transducer adpter</td>
			<td>Verasonics, Kirkland, Washington, USA</td>
			<td>UTA 408-GE</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafast ultrasound research scanner</td>
			<td>Verasonics, Kirkland, Washington, USA</td>
			<td>Vantage 256</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound probe/transducer</td>
			<td>GE Healthcare</td>
			<td>GE 9L-D</td>
			<td></td>
		</tr>
	</tbody>
</table>

blood flow imaging with ultrafast doppler

パルスドップラー効果は、血流を評価するために臨床エクログラフィーで使用される主な技術です。従来の焦点を当てた超音波ドップラーモードで適用され、それはいくつかの制限を有する。まず、血液の流れと周囲の動く組織を区別するために、細かく調整された信号フィルタリング操作が必要です。第二に、オペレータは血液の流れを局所化するか、それらを定量化するかを選択する必要があります。過去20年間で、超音波画像は、焦点を合っていない波を使用して超高速超音波の出現とパラダイムシフトを受けています。この新しい技術は、フレームレートの100倍の増加(最大10000Hz)に加えて、従来の定量化/局在化トレードオフを破り、視野の完全な血流マッピングと、単一ピクセルレベル(50μmまで)での細かい速度測定への同時アクセスを提供します。空間的および時間的な次元の両方のこのデータの連続性は、組織/血液のフィルタリングプロセスを強力に改善し、小さな血流速度(1 mm/sまで)に対する感受性を高める結果となる。本論文では、超高速ドップラーの概念と主なパラメータを紹介することを目指しています。まず、非焦点波イメージングの物理的原理を要約する。次に、ドップラー信号処理の主なステップを提示します。特に、重要な組織/血流分離アルゴリズムの実用的な実装と、これらのフィルタリングされたデータからの速度の抽出について説明する。この理論的な記述は、インビトロの経験によって補完されます。流れる血液模倣液を含む運河を埋め込む組織ファントムは、研究プログラム可能な超音波システムで画像化される。血流画像が得られ、運河の数画素に流動特性が表示されます。最後に、生体内応用の検討が提案され、頸動脈、腎臓、甲状腺、脳および心臓などのいくつかの器官における例を示す。

取得と後処理の品質は、まず目視検査によって評価されます。運河の形状は、パワードップラー画像ではっきりと見える必要があり、組織領域は暗く見える必要があります。パワードップラー信号が運河に限定されない場合、クラッタフィルターステップが間違っていた(SVDの閾値が低すぎる)か、またはプローブが取得中に強い動きを経験したことを意味することができます。
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Watch this Scientific Journal Video about Blood Flow Imaging with Ultrafast Doppler at JoVE.com

Blood Flow Imaging with Ultrafast Doppler

このプロトコルは、血液の流れを定量化するために超高速超音波ドップラーイメージングを適用する方法を示しています。1秒の長い取得の後、実験者は各ピクセルの軸速度値を持つ全視野のムービーにアクセスできます ≈0.3 ms(飛行の超音波時間に応じて)。

超高速ドップラーによる血流イメージング

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

blood-flow-imaging-with-ultrafast-doppler

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Unit 1

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Blood Vessels and Circulation

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7.3K ビュー.  The Hospital for Sick Children, PGCRL Research Institute. このプロトコルは、血液の流れを定量化するために超高速超音波ドップラーイメージングを適用する方法を示しています。1秒の長い取得の後、実験者は各ピクセルの軸速度値を持つ全視野のムービーにアクセスできます ≈0.3 ms(飛行の超音波時間に応じて)。

ビデオ: Blood Flow Imaging with Ultrafast Doppler

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

生物工学

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

人間の細胞に基づく、血液脳関門の接触モデルの作製のための改善された方法

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRETフローサイトメトリーでProteopathic播種活動の高感度検出

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

血管細胞リモデリングの研究への応用生理学的および病理学的血流のインビトロモデル

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

マイクロ流体チャンバーモデル止血や血小板輸血に再構成された血液を使用しました<em&gt;インビトロ</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

血小板輸血や止血のためのマイクロ流体フローチャンバーモデルは、リアルタイムでの血小板沈着およびフィブリン形成を測定します

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

脳血流ベースの安静状態機能性コネクティビティ(光学拡散相関分光法)

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

蛍光顕微鏡によるせん断流下の単分子の立体構造変化の特徴

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

解剖されたインプラント組織の免疫機能解析のための高次元フローサイトメトリー

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

カーブレット変換ベースのツールを使用したフィブリラーコラーゲンの定量化

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...