Name: a high-performance compact photoacoustic tomography system for in vivo small-animal brain imaging
Uploaded: 2017-06-21T14:00:00.000+00:00
Duration: 5 min 32 s
Description: Watch this Scientific Journal Video about A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging at JoVE.com

この研究は、シンガポール教育省（ARC2 / 15：M4020238）とシンガポール保健省国立医学研究評議会（NMRC / OFIRG / 0005/2016：M4062012）が資金を提供するTier 2助成金によって支えられています。作者は機械ショップの助けを借りてChow Wai Hoong Bobbyに感謝したいと思います。

すべての動物実験は、シンガポール南陽技術大学の動物実験および使用委員会（Animal Protocol Number ARF-SBS / NIE-A0263）によって承認されたガイドラインおよび規制に従って行われた。 1.システムの説明<ol><li> 図1aに示すように、PLDを円形スキャナの内部に取り付けます。 PLDをレーザードライバユニット（LDU）に接続します。 注記：PLDは〜803 nmの波長で〜136 nsのパルスを提供し、最大パルスエネルギーは約1.42 mJ、繰返し速度は最大7 kHzです。レーザ駆動部（LDU）は、温度コントローラと、可変電源と、電源（12V）と、ファンクションジェネレータとを備え、材料の表を参照してください。可変電源はレーザパワーを制御するために使用され、ファンクションジェネレータはPLDの繰り返しレートを変更するために使用されます。 </li><li> PLDレーザーのスイッチを入れます。 PLDの繰り返しレートを &quot;7,000&quot;に設定するHzをLDU内の関数ジェネレータを使用して実行します。可変電源の電圧を「3.1」Vに設定することにより、パルスエネルギーを1.42mJに増加させる。 </li><li> 図1aに示すように、PLD出射窓の前に光学ディフューザー（OD）を取り付けて、出力ビームを均一にします 。 注：細かいグリット（ すなわち、 1,500グリットの磨き）のディフューザーを使用してください。 </li><li> 図1aに示すように、USTホルダーに焦点を合わせたUSTをスキャン領域の中央に向けて取り付けます 。 注：USTの中心周波数は2.25 MHzで、焦点距離は1.9インチです。 </li><li> 図1aに示すように、超音波検出器をアクリルタンクの中に置きます 。 USTが完全に浸水するようにタンクを水で満たしてください。 注：水媒体は、脳（サンプル）から光音響信号をUSTに結合するために使用されます。アクリル水タンク（WT;材料表参照）はカスタム小動物イメージング用に設計されています。水タンクの設計図は図1bに示されています 。 </li><li>パルサー/レシーバーユニット（PRU; 材料表を参照）を使用して、サンプルからのPAシグナルを確認します。 注：これらの信号は、100 MS / sのサンプリングレートで12ビットのDAQカード（ 表の表を参照）でデジタル化され、コンピュータに保存されました。 </li></ol>ラット脳画像化のための動物の準備注記：上記のPLD-PATシステムは、小型動物の脳を画像化するために実証された。これらの実験では、健康な雌ラット（ 表の表を参照）を使用した。 <ol><li> 2mLのケタミン、1mLのキシラジン、および1mLの生理食塩水（0.2mL / 100gの用量）のカクテルを腹腔内注射することによって動物を麻酔する。 </li><li>ヘアクリッパーを使って動物の頭皮の毛皮を除去する。優しく剃毛した毛髪に脱毛クリームを塗る毛皮のさらなる枯渇のための領域。 <ol><li>綿棒で4〜5分後に塗布したクリームを除去する。 </li><li>動物の目に人工涙軟膏を塗布し、麻酔およびレーザー照射による乾燥を防ぐ。 </li></ol></li><li>ラボジャック上の呼吸用マスク（ 表の表を参照）が装備されたカスタムメイドの動物ホルダー（ 表の表を参照）を取り付けます。 </li><li>動物をホルダー上で倒れやすい場所に置きます。イメージング中に動物の動きを避けるために、外科用テープを使用してホルダーに固定します。 </li><li>呼吸マスクがラットの鼻と口を覆って、吸入麻酔薬を送達するようにしてください。 </li></ol> 3. インビボラット脳画像化<ol><li> 呼吸マスクを麻酔機に接続します。麻酔装置のスイッチを入れ、0.75％イソフルランで酸素を1.0L /分供給するように設定します。 <ol><li>パルスオキシメータをクランプしてください動物の生理学的状態を監視するために使用される。 </li></ol></li><li> ラットの頭皮に無色の超音波ゲルの層を塗布する。ラボジャックの位置をスキャナの中心に合わせます。呼吸マスクは、イメージングウィンドウに合わせてカスタマイズされています。市販のノーズコーンの10％を切断し、手袋に接続する。 <ol><li>ラボジャックの高さを手動で調整して、イメージング面がUSTの焦点にくるようにします。 </li></ol></li><li>必要に応じて、データ収集ソフトウェアのパラメータを設定します（材料表を参照）。データ収集ソフトウェアプログラムを実行して、収集（ すなわち、画像化）を開始する。 注：このプログラムは、USTを回転させてAラインPA信号を収集するために使用されます。収集されたAラインはコンピュータに保存されます。 </li><li>撮影期間全体で動物を観察し、撮影が完了した後にPAT再構成に進む。 </li><li>データ取得が終了したら、再構成ソフトウェアプログラムを使用して、A線から断面脳画像を再構成する。 </li><li>麻酔システムをオフにして、ステージから動物を取り出し、ケージに戻し、意識が回復するまでモニターします。 注：たとえば、USTを5秒間回転させると、PLDは35,000（= 5 x 7,000）パルスを出力し、USTは35,000のAラインを収集します。 70,000を超える信号を平均化することによって（Aライン= 35,000 / 70 = 500の平均化後）、35,000のAラインが500に低減される。 図1cは、レーザパルスおよびAライン収集の照明を示す。遅延和和逆投影アルゴリズムに基づく再構成プログラムを使用すべきである。 </li></ol><img alt="図1" class="xfigimg" src="/files/ftp_upload/55811/55811fig1.jpg" /> 図1： PLD-PATシステムの回路 図 （ a ）PLD-PATの略図。 PLD：パルスレーザダイオード、CSP：サークルAM：麻酔機、M：モータ、MPU：モータプーリユニット、LDU：レーザドライバユニット、PRU：パルサ/レシーバユニット、UST：超音波トランスデューサ、WT：水タンク、PF：ポリマーフィルム、DAQ：データ取得カード。 （ b ） インビボ小動物脳画像化のための水槽の概略図、上面図（1）および断面図（2）。 A：メトリックネジ、B：アクリル環状プレート、C：シリコーンOリング、D：厚さ100μmの透明ポリエチレンカバー。このタンクはその底部に直径9cmの穴を有し、超音波で光学的に透明な100μm厚のポリエチレン膜で封止された。 （ c ）PLDおよびAラインからのレーザパルスの照射の概略図であり、5秒の連続走査時間で収集される。 <a href="http://ecsource.jove.com/files/ftp_upload/55811/55811fig1large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a>

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著者は、原稿に関連する金銭的関心はなく、開示する可能性のある他の利益相反はない。

この研究は、PLD-PATシステムを用いてラットのインビボ脳イメージングを行うためのプロトコールを提示する。このプロトコルには、イメージングシステムとそのアラインメントの詳細な説明と、ラットの脳イメージングのイラストが含まれています。既存のOPOベースのPATシステムは高価で大型であり、5〜10分で1つの断面画像を提供することができる。 PLD-PATシステムは、コンパクトで...

<html>光音響トモグラフィ（PAT）は、臨床および前臨床試験1,2,3,4,5の両方において多くの用途を有するハイブリッド撮像モダリティである。 PATでは、ナノ秒のレーザーパルスが生体組織を照射します。組織発色団による入射光の吸収は、局部的な温度上昇をもたらし、次に音波が音波の形態で放出される。超音波検出器は、試料周辺の様々な位置で光音響信号を収集する。光音響（PA）信号は、光音響画像を生成するために様々なアルゴリズム（遅延 - 和アルゴリズムなど） 6を使用して再構成される。 このハイブリッドイメージングモダリティは、高解像度、深部組織イメージングおよび高い光吸収コントラスト7を提供し 、class = &quot;xref&quot;&gt; 8。最近では、より長い波長（約1,064nm）およびリン系フタロシアニンと呼ばれる外因性造影剤の助けを借りて、鶏の乳房組織において約12cmの画像深度9が達成された。この深度感度は、共焦点蛍光顕微鏡法、2光子蛍光顕微鏡法、 10光干渉断層法、 11 等の他の光学法の深度感度よりもはるかに高い.PATは、2つ以上の波長を使用して、器官における構造的および機能的変化。多くの人間の病気について、小動物のモデルは十分に確立されている12,13,14,15。小動物の画像化のために、いくつかの様式が実証されている。これらのアプローチのうち、PAイメージングは​​、上記の利点のためにかなり迅速に注目を集めています。 PATは、小動物4,16,17,18の組織および器官（ すなわち、心臓、肺、肝臓、眼、脾臓、脳、皮膚、脊髄、腎臓など ）における血管造影の可能性を示している。 PATは、小動物脳画像の確立されたモダリティである。 PA波は発色団による光吸収のために生成されるので、多重波長PATは総ヘモグロビン濃度（HbT）と酸素飽和度（SO 2 ）のマッピングを可能にする19,20,21,22。脳神経血管造影は、外因性造影剤12,23,24の助けを借りて達成された。 PAモダリティは脳の健康をよりよく理解するのに役立ちます分子レベルおよび遺伝子レベルで情報を提供する。 小動物イメージングのために、Nd：YAG / OPOレーザーがPAT励起源として広く使用されている。これらのレーザーは〜10 nsの繰返し率で約5 nsの近赤外パルスをエネルギー（OPO出力ウィンドウで約100 mJ）で照射します。このようなレーザを備えたPAシステムは、高価でかさばり、レーザ光源の繰り返し率が低いため、単一素子超音波トランスデューサ（UST）を用いた低速イメージングを可能にする。そのようなPAシステムにおける典型的なA線獲得時間は、断面25当たり約5分である。このような長い測定時間を有するイメージングシステムは、全身イメージング、時間分解能イメージングなどのための生理学的パラメータを制御することが困難であるため、小動物イメージングには理想的ではない。複数の単一素子USTを採用することによって、アレイベースのUST、または高繰返し率のレーザーを使用すると、PAのイメージング速度を高めることができますシステム。サンプルの周りのすべてのPA信号を収集するために1つの単一素子USTのみを使用することにより、システムのイメージング速度が制限される。高速または高感度のイメージング技術のために、円形または半円形の幾何学配置で配置された複数の単一素子USTが実証されています。線形、半円形、円形、および容積配列などのアレイベースのUST26は、リアルタイムイメージングにうまく使用されています1 。これらのアレイベースのUSTはイメージングスピードを増加させ、測定感度を低下させるが、高価である。しかし、アレイベースのUSTを使用するPAシステムのイメージング速度は、依然としてレーザの繰り返し速度によって制限されています。 パルスレーザ技術は、高繰返し率のパルスレーザダイオード（PLD）を製造するために進歩した。 7,000フレーム/秒臨床超音波プラットフォーム27を使用してPLDでBスキャン光音響イメージングが実証された。このようなPLDは、単一要素のUST円形走査形状であっても、PATシステムである。単一要素のUSTは、アレイベースのUSTとは異なり、安価で高感度です。過去10年間で、PAイメージングの励起源として高繰返し率PLDの使用に関する研究はほとんど報告されていませんでした。ファントム28の PAイメージングのためにファイバベースの近赤外PLDが実証された。低エネルギーPLDを使用して、人間の皮膚より1mm低い深さの血管のインビボイメージングが実証された29 。 PLDベースの光学解像度光音響顕微鏡（ORPAM）が報告された。 PLDを使用して、0.43Hzのフレームレートで〜1.5cmの深さのイメージングが実証されました30 。非常に最近、PLD-PATシステムが報告され、生物組織25,31において〜3秒程度の短時間および〜2cmの画像深度で画像が提供された。この研究は、このような低コストでコンパクトなシステムが高クオ高速でも画像を表示できます。 PLD-PATシステムは、高フレームレート（7,000fps）光音響イメージング、表面血管イメージング、指関節イメージング、2cm深部組織イメージング、小動物脳イメージングなどに使用することができる。 PLDからの低パルスエネルギーパルスは、その適用をマルチスペクトルおよび深部組織イメージングに限定する。実験は、前臨床応用に使用されたのと同じPLD-PATシステムを用いて小動物に対して行われている。この研究の目的は、小型動物のインビボ 2D断面脳画像化のためのPLD-PATシステムの視覚化実験デモを提供することである。

<table><tbody><tr><td>Pulsed laser diode</td><td>Quantel, France</td><td>QD-Q1910-SA-TEC</td><td>It is the excitation laser source with specifications 803 nm, 1.4 mJ per pulse, 136 ns pulse, 7 kHz maximum, dimentions : 11.0 x 6.0 x 3.6 cm, weight: ~150 gm</td></tr><tr><td>Stepper motor with gearbox</td><td>LIN Engineering (Servo Dynamics)</td><td>Motor: CO-5718-01P, Gearbox: DPL64/1, I = 10 for NEMA 23; power supply PW100-48</td><td>To move the detector holder in a circular geometry. Torque: 2.08 N-m, Rotor inertia: 2.6 kg-cm2</td></tr><tr><td>Ultrasonic pulser/receiver</td><td>Olympus</td><td>5072PR</td><td>To receive, filter and ampligy the PA signal from UST. Its bandwidth is 35 MHz, and gain is &amp;plusmn;59 dB.</td></tr><tr><td>Ultrasound Transducer</td><td>Olympus</td><td>V306-SU-NK-CF1.9IN/Q4200069</td><td>Ultrasonic sensors used for photoacoustic detection. Central freqency 2.25 MHz, 0.5 in, Cylindrical focus 1.9 inch</td></tr><tr><td>PCIe DAQ (Data acquisition) Card</td><td>GaGe</td><td>CSE4227/ A6000610/B0E00610</td><td>12 bit, 100 Ms/s, 2 channels, 1 Gs on board memory, PCIe x16 interface</td></tr><tr><td>Rats</td><td>In Vivos Pte Ltd, Singapore</td><td>NTac:SD, Sprague Dawley / SD</td><td>Female, weight 100 &amp;plusmn;10g</td></tr><tr><td>Acrylic water tank&amp;nbsp;</td><td>NTU workshop</td><td>Custom-made</td><td>It contains the water that acts as an acoustic coupling medium between brain and detector</td></tr><tr><td>Circular Scanner&amp;nbsp;</td><td>NTU workshop</td><td>Custom-made</td><td>Scanner is made out of Alluminum&amp;nbsp;</td></tr><tr><td>Anesthetic Machine</td><td>medical plus pte ltd</td><td>Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td><td>Supplies oxygen and isoflurane to animal</td></tr><tr><td>Pulse Oxymeter portable</td><td>Medtronic</td><td>PM10N with veterinary sensor</td><td>Monitors the pulse oxymetry of the animal</td></tr><tr><td>Ultrasound gel</td><td>Progress/parker acquasonic gel</td><td>PA-GEL-CLEA-5000</td><td>Clear ultrasound gel</td></tr><tr><td>Data acqusison software</td><td>National Instruments Corporation,Austin,TX,USA)</td><td>NI LabVIEW 2015 SP1</td><td>LabVIEW based program was developed in our laboratory for controlling the stepper motor and acquring the PA singnals from the detector</td></tr><tr><td>Data processing software</td><td>Matlab (Mathworks, Natick, MA, USA)</td><td>Matlab R2012b</td><td>Matlab code for reconstruction of PA images was developed in our lab</td></tr><tr><td>Temperature controller&amp;nbsp;</td><td>LaridTech, MO,USA</td><td>MTTC1410</td><td>It will constantly control temperature of the PLD&amp;nbsp;</td></tr><tr><td>12 V power supply&amp;nbsp;</td><td>Voltcraft&amp;nbsp;</td><td>PPS-11810</td><td>To supply operating voltage for PLD</td></tr><tr><td>Variable power supply&amp;nbsp;</td><td>BASETech</td><td>BT-153</td><td>To change the laser output power</td></tr><tr><td>Funtion generator&amp;nbsp;</td><td>Funktionsgenerator</td><td>FG250D</td><td>To change the repetetion rate of the PLD. It will provide TTL signal to synchronize the DAQ with the laser excitation.</td></tr><tr><td>Animal distributor</td><td>In Vivos Pte Ltd, Singapore</td><td></td><td>Animal distributor that supplies small animals for research purpose.</td></tr><tr><td>Animal holder</td><td>NTU workshop</td><td>Custom-made</td><td>Used for holding the animal on its abdomen</td></tr><tr><td>Breathing mask</td><td>NTU workshop</td><td>Custom-made</td><td>Used along with animal holder to supply anesthesia mixture to the animal</td></tr><tr><td>Pentobarbital sodium</td><td>Valabarb</td><td></td><td>Used for euthanizing the animal after the expeirment.</td></tr><tr><td>Optical diffuser</td><td>Thorlabs</td><td>DG10-1500</td><td>Used to to make the laser beam homogeneous</td></tr></tbody></table>

a high-performance compact photoacoustic tomography system for in vivo small-animal brain imaging

インビボ小動物イメージングは​​、前臨床研究において重要な役割を果たす。光音響トモグラフィー（PAT）は、前臨床および臨床の両方の用途に大きな可能性を示す新興ハイブリッドイメージングモダリティです。従来の光パラメトリック発振器ベースのPAT（OPO-PAT）システムは、かさばって高価であり、高速イメージングを提供することができない。近年、PATの代替励起源として、パルスレーザダイオード（PLD）が成功裏に実証されている。パルスレーザダイオードPAT（PLD-PAT）は、光音響ファントムや生体組織の高速イメージングにも成功しています。この研究は、PLD-PATを用いたインビボ脳イメージングのための視覚化された実験プロトコルを提供する。このプロトコルには、コンパクトなPLD-PATシステム構成とその説明、脳イメージングのための動物の準備、および2D断面ラット脳画像の典型的な実験手順が含まれる。 PLD-PATシステムはコンパクトでコスト効率が良い高速で高品質な画像を提供することができます。 インビボで様々なスキャン速度で収集された脳画像が提示される。

記載されたPLD-PATシステムの能力を実証するインビボ脳イメージング結果がこのセクションに示されている。 PLD-PATシステムの高速イメージング能力を実証するために、2つの異なる健康なラットのインビボ脳イメージングを行った。 図2は、様々なスキャン速度での雌ラット（93g）の脳画像を示す。 図2aおよびb...

Watch this Scientific Journal Video about A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging at JoVE.com

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

小型動物での高速インビボ脳イメージングのためのコンパクトパルスレーザダイオード光音響トモグラフィ（PLD-PAT）システムが実証されています。

高性能小型光音響トモグラフィーシステム<em&gt;インビボ</em&gt;小動物脳イメージング

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

a-high-performance-compact-photoacoustic-tomography-system-for-vivo

Research

JoVE Journal

Bioengineering

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

10.4K ビュー.  Nanyang Technological University. 小型動物での高速インビボ脳イメージングのためのコンパクトパルスレーザダイオード光音響トモグラフィ（PLD-PAT）システムが実証されています。 

ビデオ: A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三次元光学分解能光音響顕微鏡

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

生物工学

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

癌バイオマーカーの局在の小型動物のCTガイド下時間領域拡散蛍光トモグラフィー

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

医学

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 ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

非侵襲的実質細胞、血管および代謝高周波超音波および光音響ラット脳深部イメージング

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

神経科学

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

切り替え可能な音響光学分解能光音響顕微鏡<em&gt;インビボ</em&gt;小動物の血管系イメージング

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

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, ...

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

ラット皮質脈管構造での洗浄・インおよび染料の洗浄を監視するためのパルスレーザーダイオードベースのデスクトップ光音響断層撮影

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

血管可視化のための二重ラスター走査光音響小動物イメージャー

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

げっ歯類における血液脳関門開口のためのハイスループット画像誘導定型神経ナビゲーションと集中超音波システム

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

虚血性脳卒中の非侵襲的統合PAUSAT全脳イメージング

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

著者スポットライト:虚血性脳卒中の非侵襲的全脳イメージングのための統合光音響、超音波、血管造影断層撮影法(PAUSAT)  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

虚血性脳卒中の非侵襲的全脳イメージングのための統合光音響、超音波、および血管造影断層撮影(PAUSAT)

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

高性能小型光音響トモグラフィーシステム

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