This work was supported by the National Institutes of Health (R01-EB014496). The authors would like to thank Iason Apostolakis for his contribution to the experiments.

このプロトコルはコロンビア大学の施設内動物管理使用委員会によって承認されました。すべてのデータ収集及び処理は、MATLAB環境を用いて行きました。 1.実験のセットアップ<ol><li> 90分の間にex vivoでの犬の肝臓試料を脱気。脱気したリン酸緩衝液（ 図1）を充填したタンク内に肝臓試料を置きます。肝臓の先端に針を持つ音響吸収の肝臓試料を固定します。 </li><li> 4.5 MHzの中​​心周波数、70ミリメートル焦点深度とで（治療）93要素半球状配列HIFUトランスデューサの中心部に位置する円形の穴を通って、64要素0.32 mmピッチ、2.5 MHzの中​​心周波数フェーズドアレイ（画像）を挿入1.7ミリメートルX 0.4ミリメートル焦点サイズ11。同軸の両方の変換器の位置を合わせ、調整ネジで治療用トランスデューサに撮像トランスデューサを修正。 <ol><li> AVとHIFUトランスデューサをカバーそれを冷却するために脱気流水で満たされたポリウレタン膜をolume制御。コンピュータ制御の3次元ポジショナーにトランスデューサアセンブリをマウントします。 </li></ol></li><li> 500 mVの最大振幅の25 Hzの振幅変調された正弦波形を送信する関数発生器にHIFUトランスデューサを接続します。ソフトウェアMATLABを使用して、完全にプログラム可能な超音波システムに撮像トランスデューサを接続します。 注：ソフトウェアの超音波システムに関連し、MATLAB環境を使用してシステムに接続されたコンピュータにインストールする必要があります。 50デシベルRF増幅器及び整合ネットワークはそれぞれの電力を増幅し、インピーダンスを整合させるHIFUトランスデューサと関数発生器との間に配置されるべきです。 </li><li> 9.625ミクロンの128行で方位角方向に90°の空間ステップとアレイの表面から50ミリメートル径方向に深い40ミリメートルを開始するには、MATLABを使用して、極格子を作成した起源はFOであります発散波のCUS。アレイの表面の背後に発散波10.24ミリメートル（開口部の半分のサイズ）のソースを定義し、横方向の中央に配置。 <ol><li>広い平面波の場合は64行で横方向に9.625ミクロンと20ミリメートルの空間ステップで軸方向に深いアレイの表面から50ミリメートルと40ミリメートルを開始するには、MATLABを使用して、デカルトグリッドを作成します。アレイの表面上の平面波のソースを定義します。各グリッドについて、バック配列の各要素に、グリッドの各点にソースから時間を計算し、。 </li></ol></li><li> MATLABコマンドウィンドウで平面波イメージングのため波イメージングまたは「ReconMat_PW」を発散するための「ReconMat_DW」と入力し、各グリッドのための標準的な遅延和アルゴリズムに関連した再構築マトリックスを作成するために、「入力」を押します。標準基底の各ベクトルに遅延和アルゴリズムを適用し、非ゼロelemenを取得結果の行列11のTS。対応する位置でのスパース行列に結果の行列から得られた非ゼロ要素を割り当てます。コンピュータのハードドライブ上の復元行列を保存します。 注意：発散と平面波法は、2つの別個の復興行列を使用しています。 <ol><li> GPU行列に復元行列をキャストします。フェーズドアレイに関連するスクリプトを使用して超音波チャンネルデータ取得のためのセットアップファイルを作成するには、「入力」、MATLABコマンドウィンドウとプレスで平面波イメージングのため波イメージングまたは「SetUpP4_2Flash_4B_streaming_PW」を発散するための「SetUpP4_2Flash_4B_streaming_DW」と入力し、製造業者によって提供超音波システム。波イメージングと平面波イメージングのための「P4-2Flash_PlaneWave.mat」を発散するための設定ファイル「P4-2Flash_DivergingWave.matを」という名前を付けます。 注：市販のソフトウェアパッケージは、コンピュータtに設置されなければなりませんO GPU行列に再建疎行列をキャスト。 </li></ol></li><li>肝臓の高フレームレート超音波データ取得は、HIFUと同時に起動するように外部トリガを使用して、関数発生器と超音波システムを同期します。 </li><li> MATLABを開きます。 Bモード画像を使用するには、超音波システムの製造元から提供されたセットアップスクリプト」SetUpP4_2Flash_4B.m」を実行します。 「P4-2Flash_4B_Bmode.mat」：作成したセットアップファイルに名前を付けます。 「プロセスに.MATファイル名： &quot;&quot; VSX」コマンドと時を使用促され、セットアップファイル「P4-2Flash_4B_Bmode.mat」の名前を入力します。両方のトランスデューサを移動し、切除する肝​​臓の標的領域でそれらを配置するためにコンピュータの画面上に現れたBモード表示を使用しています。吸収による高い超音波減衰を避けるために、肝臓の表面下約1cm地域を対象とします。コンピュータ上の肝臓の従来のBモード画像を保存します。 注意：ここでは、それぞれの切除のための3次元位置決め装置で変換器を移動することにより、2つの肝臓標本における11の異なる場所でのHIFU切除を行いました。 </li></ol> 2.超音波データ集録<ol><li> MATLABを開きます。 「VSX」コマンドを使用して、「プロセスへの.MATファイル名： &quot;ときにプロンプ​​トが表示され、平面波のための波イメージングまたは「P4-2Flash_PlaneWave.mat」を発散するための設定ファイル&quot; P4-2Flash_DivergingWave.mat」の名前を入力します。イメージング。 HIFUを起動し、標的領域へ​​の2分の間にそれを適用します。 </li><li>発散波を使用して2分の間​​に第2の1,000フレームでのRFチャネルのデータを取得します。代替的に、平面波を使用して2分の間​​に第2の1,000フレームでRFチャネルデータを取得します。 </li><li> PCI Expressのケーブルを介してすべての200フレームホストコンピュータにデータを転送します。あるいは、リアルタイムストリーミングでは、平面波とTRANSFを用いて2分間にわたって毎秒167フレームでRFチャネルデータを取得しますホストコンピュータにデータ毎に2フレームER。 注：200フレームのセットでイメージング方法は、各セット内で高い時間分解能を提供しますが、各セット間のギャップを作成し、オフライン処理に適しています。 167 fpsで撮影方法は、低時間分解能を持っていますが、全体の切除時間全体で任意のギャップを作成しません、リアルタイムストリーミングに適しています。 </li><li> MATLABに単精度GPUマトリックスにRFチャネルデータ行列をキャストします。再構成されたRF データ 11 を取得するために再構成マトリクスによってRFチャネルデータ行列を乗算します。 </li></ol> 3.変位イメージング<ol><li> MatlabののDSP System Toolboxのを使用して、4 MHzのカットオフ周波数で第 6次バタワースローパスフィルタを作成します。 4.5 MHzのHIFUコンポーネントをフィルタリングするために再構成されたRFデータには、このローパスフィルタを適用します。 </li><li> 1-D、正規化相互相関を用いて、連続するフレームの間の軸方向の変位を推定します3.1ミリメートルウィンドウの長さと90％のオーバーラップを有します。 </li><li> MatlabののDSP System Toolboxのを使用して100 Hzのカットオフ周波数で第 6次バタワースローパスフィルタを作成します。このローパスフィルタは50Hzで、発振周波数成分を取得するためにMATLABを使用して、一時的な変位データに適用します。 </li><li>で-6dBの（水中1.7×0.4 mm）の焦点領域として関心領域（ROI）を定義し、離れて、変換器表面から70ミリメートルに位置。このROI内の変位データを抽出します。平均変位とROIの変位の標準偏差の比として切除の2分後に、焦点領域における変位信号対雑音比（SNR さd）を推定します 。 </li><li>変位行列データから焦点に50 Hzの一時的な変位信号を抽出します。 MATLABを使用して可聴音に焦点に時間的な変位信号に変換します。 </li></ol>

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	<li>Al-Bataineh, O., Jenne, J., Huber, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+future+applications+of+high+intensity+focused+ultrasound+in+cancer.">Clinical and future applications of high intensity focused ultrasound in cancer.</a> Cancer Treat Rev. 38, 346-353 (2012).</li><li>Dewhirst, M. W., Viglianti, B. L., Lora-Michiels, M., Hanson, M., Hoopes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+principles+of+thermal+dosimetry+and+thermal+thresholds+for+tissue+damage+from+hyperthermia.">Basic principles of thermal dosimetry and thermal thresholds for tissue damage from hyperthermia.</a> Int J Hyperthermia. 19, 267-294 (2003).</li><li>Napoli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR-guided+high-intensity+focused+ultrasound:+current+status+of+an+emerging+technology.">MR-guided high-intensity focused ultrasound: current status of an emerging technology.</a> Cardiovasc Intervent Radiol. 36, 1190-1203 (2013).</li><li>Gudur, M. S., Kumon, R. E., Zhou, Y., Deng, C. 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The authors declare that they have no competing financial interests.

HIFU病変のリアルタイムモニタリングは、適切かつ効率的な病変の配信を保証することが重要です。病変の形態としては、組織が硬直し、励起下での運動の振幅は減少します。組織変位を誘発する音響放射力で組織の結果の領域にHIFUを適用します。変位の相対的な変化は、組織の剛性の相対的な変化の代理です。この技術は、他の超音波ベースの方法とは対照的に治療を停止することなく、H...

High Intensity Focused Ultrasound (HIFU) is a technique that generates temperature elevation at the focal region and can be used to ablate cancerous tissue 1. Temperature elevation at the focus causes thermal lesions in the tissue 2. In order to avoid overtreating a region and to reduce treatment duration, it is imperative to reliably monitor the ablation. Magnetic resonance-guided focused ultrasound (MRgFUS) is the main technique used in clinic to guide and monitor HIFU treatment 3. MRI provides high spatial resolution images of the treated region with tissue displacement or thermal dose but has a frame rate of 0.1-1 Hz and is costly. Several ultrasound-based techniques such as B-mode imaging 4, passive acoustic mapping 5, shear wave imaging 6 and acoustic radiation force impulse 7 have been developed to guide and monitor thermal ablation. However, B-mode imaging and passive acoustic mapping do not provide imaging of mechanical properties of the ablated region which is useful to the operator to improve lesion delivery. 
Shear wave imaging and acoustic radiation force impulse can both characterize the elasticity of the tissue by measuring acoustic radiation force-induced displacements 7,8. However, in both methods, the HIFU treatment is typically interrupted to monitor the ablation. Our group has developed a technique called Harmonic Motion Imaging for Focus Ultrasound (HMIFU) which can monitor the HIFU treatment with ultrasound without stopping the ablation9,10. Briefly, a HIFU transducer sends an amplitude-modulated wave to the region to ablate while simultaneously generating an oscillatory motion in the focal region. A co-axially aligned ultrasound transducer is used to image this oscillation. The magnitude of the induced motion is related to the stiffness of the tissue.
To ensure proper lesion delivery, the temporal resolution of real-time monitoring is of key interest in ablation guidance. Recently, our group has shown real-time streaming of displacement at a frame rate up to 15 Hz, imaged with diverging waves in a narrow field of view and using a fast image reconstruction method 11. Several beamforming techniques can be used to image the displacements. A large field of view can be obtained with diverging wave imaging by changing the delay profile but the axial direction is not aligned with the HIFU beam on the lateral regions and the wave is attenuated due to geometric spreading in the lateral direction, which can affect the quality of the displacement estimation. In contrast, the lateral field of view for plane wave is upper bounded by the active aperture but the axial direction is aligned with the HIFU beam at the focus and there is no geometric spreading in the lateral direction. Depending on the type of application, one or the other imaging method can be selected. The objectives of this protocol are to show how plane wave imaging can provide real-time streaming of displacements images using HMIFU during ablation and to compare the quality of the motion estimation between diverging and plane wave imaging.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>P4-2 Phased array</td>
			<td>ATL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H-178 HIFU transducer</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-D positioner</td>
			<td>Velmex Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AT33522A function generator</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 ultrasound system</td>
			<td>Verasonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3100L RF amplifier</td>
			<td>ENI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matching network</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Degasing system</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Programming software</td>
			<td>Matlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jacket software package</td>
			<td>Accelereyes</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring of high intensity focused ultrasound (hifu) ablation of in vitro canine livers using harmonic motion imaging for focused ultrasound (hmifu)

集束超音波（HMIFU）のための調和運動イメージングは​​、高強度の超音波（HIFU）アブレーションを集中実行し、監視することができる技術です。振動運動は、関数発生器を用いて、25 Hzの振幅変調信号を印加することにより、93素子と4.5 MHzの中​​心周波数HIFUトランスデューサの焦点で生成されます。 68kPaのピーク圧力で、64素子と2.5 MHzの撮像トランスデューサは、共焦点無線周波数（RF）チャネルのデータを取得するためにHIFUトランスデューサの中心に配置されています。このプロトコルでは、 インビトロでのイヌの肝臓に7 Wの音響パワーとHIFUを用いた熱アブレーションのリアルタイム監視が記載されています。 HIFU治療は、2分の間に組織に適用され、アブレーション領域1000フレーム/秒に発散または平面波イメージングを使用してリアルタイムで画像化されます。 RFチャネルデータの行列は、画像再構成のための疎行列乗算されます。ビューの再構築されたフィールドは、WAを発散するために90°でありますVEの平面波イメージングのための20ミリ、データが80 MHzでサンプリングされます。再構成は、4.5の表示フレームレートでリアルタイムに画像化するためにグラフィック処理装置（GPU）上で実行されます。再構成されたRFデータの1次元の正規化相互相関は、焦点領域内の軸方向の変位を推定するために使用されます。焦点深度でのピーク・ツー・ピーク変位の大きさが原因病変の形成組織の硬化を意味熱アブレーションの間に減少します。平面波のための焦点領域における変位信号対雑音比（SNR d）はその平面波イメージングは波撮像発散よりHMIFUための優れた変位マップの品質を生成するために表示される表示波発散よりも1.4倍高かったです。

HIFU切除中にHMI変位のリアルタイムストリーミングが発散し、平面波イメージングを用いて得ることができる。 図2は、HIFU切除中にインビトロイヌ肝臓における平面波イメージングを用いた音響放射力誘起される変位のリアルタイム表示を示すビデオ画面キャプチャであります。変位は4.5ヘルツの表示フレームレートでコンピュータの画面上でリアルタイムにストリーミン...

Watch this Scientific Journal Video about Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU a

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

高強度のリアルタイムモニタリングの超音波（HIFU）アブレーションを重視しました<em&gt;インビトロ</em集束超音波のための調和運動イメージングの使用&gt;犬の肝臓（HMIFU）

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

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Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

9.9K ビュー.  Columbia University. This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

ビデオ: Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

コントラストの超音波は、薬剤ベアリングナノ粒子デリバリーと微小血管アブレーションを経由してマウスの神経膠腫の治療を対象と

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

生物工学

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

全肝臓の細胞化とRecellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

低強度の超音波を用いた接着依存性のシグナルの誘導

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

医療画像処理アプリケーションのための超音波の広帯域光検出器

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

ディープティシュ人間血管造影および機能前臨床研究のためのユニバーサルハンドヘルド三次元光音響イメージングプローブリアルタイムで

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

遺伝的にコードされたFRETベースの胆汁酸センサを用いた細胞内胆汁酸動態のリアルタイムモニタリング

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

適切なポジショニングと骨のマイクロ アーキテクチャを使用して体内のマイクロ-コンピューター断層撮影の焦点の高分解能イメージングのためのラットの後肢の拘束

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持ちの臨床音響小動物の非侵襲的な実時間イメージングによる画像計測

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

マイクロ電極へのエッチングナノアーキテクチャへの集束イオンビームリソグラフィー

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...