著者は、原稿の博士Lynneaブラムボーの緊密な読書に感謝。この作品は、健康補助R01 EB000712、R01 EB008085、R01 CA134539、U54 CA136398、と5P60 DK02057933の国民の協会が後援した。教授李紅V.王はしかし、この作業をサポートしていませんでしたMicrophotoacoustics、（株）とEndra、株式会社、の経済的利害関係を持っています。

1。システムの構成<ol><li>光照射<ol><li>光照射源：固体パルスレーザー（INNOSLAB、Edgewave）と色素レーザー（CBR - D、Sirahを）ダイオード励起。 </li><li>出力レーザービームは、（パルス幅：7nsの）（P50C、Thorlabs）50μmのピンホールを通過して集光レンズ（LA1131、Thorlabs）で焦点を当てています。 </li><li>ピンホールは、効果的な空間フィルタリングのための基本モードビーム径のピンホールの直径に合わせて、集光レンズの焦点から少し離れて配置されている。 </li><li>フィルタ処理されたビームは、シングルモード光ファイバ（P1 - 460A - FC - 2、Thorlabs）に結合して中性の高密度フィルター（NDC - 50C - 2M、Thorlabs）と減衰されます。 </li><li>ファイバー出力は570nmの波長で〜2.6μmの回折限界の光学フォーカスを達成するために顕微鏡対物の背面開口部（RMS4X、Thorlabs）を埋めます。 </li></ol></li><li>超音波検出<ol><li>超音波トランスデューサー：50 MHzの中​​心周波数（V214 - BB - RM、オリンパスNDT）。 </li><li>超音波トランスデューサは、回折限界の光照射と同軸に整列される超音波検出、ために自家製音響光学ビームコンバイナ11に接続されている。 </li><li>球形空洞は音響レンズを生産するコンバイナの底から粉砕される。この音響レンズは水中で0.5の開口数を有し、50 MHzの中​​心周波数は43μmの音響焦点直径を与えます。 </li><li>光学および音響病巣は、検出感度を最大にするconfocally整列されます。 </li></ol></li><li>音響結合<ol><li>乾燥した超音波カップリングは、初期の光音響イメージングシステム12で使用された水、実験動物を水没を避けるために採用されている。 </li><li>イメージングウィンドウをシャーレ（直径9cm）の底部で開かれており、超音波および光学的に透明なポリエチレン膜で封止されている。 </li><li>ポリエチレン膜と画像化されたカップルの水没OR - PAMイメージングヘッドにシャーレ内のシャーレへのオブジェクトから生成された光音響波、および脱イオン水をさらに夫婦波になるオブジェクトの間に超音波ジェル（クリア画像、SonoTech） 。 </li></ol></li><li>エレクトロニクス<ol><li>超音波トランスデューサで検出された音響信号は、2つのカスケード接続アンプ（ZFL 500LN、Mini - Circuits社）によって増幅される</li><li>増幅された信号は、200 MS /秒のサンプリングレートで14ビットのデータ集録（DAQ）ボード（CompuScope 14200、ゲージ応用科学）によってデジタル化される</li></ol></li><li>スキャン方式<ol><li>ラスターは水平（XY）平面に沿って、または- PAMイメージングヘッドのスキャンを2次元（2 - D）は、DAQボードとポンプレーザの両方をトリガするパーソナルコンピュータによって制御されます。トリガ信号は、DAQボードからクロックアウト信号に同期しています。 </li><li> 2次元スキャナの高速軸は断面走査（Bスキャ​​ン）の方向として定義されています。 </li><li> B -スキャン画像のシーケンスは、直接3次元レンダリングでまたは2 - Dの最大振幅の投影（MAP）の画像のどちらか見ることができる体積のイメージを形成し、遅相軸に沿ってイメージングヘッドを翻訳することで取得することができます。 </li></ol></li></ol> 2。システムの配置<ol><li>音響焦点面の位置を（すなわち、最大パルスエコー超音波信号にトリガ信号からの遅延時間）を決定するためにパルス - エコーの超音波と超音波反射板を使用してください。このステップは、OR - PAMシステムを構築する際に一度だけ実行する必要がある。 </li><li>シングルモードファイバの結合効率を最大化します。 </li><li>光学的に吸収するオブジェクト（例えば、黒のテープの部分）の上に超音波ゲルを適用し、静かに脱イオン水で満たされたペトリ皿の中でイメージングウィンドウの下に取り付けます。 </li><li>水にイメージングヘッドを下ろし、そして音響レンズの下に閉じ込められた空気の泡を取り除く。 </li><li>吸収物体の光音響信号は、音響遅延から判断することができる音響焦点面、からになるまで、イメージングヘッドを調整します。 </li><li>平らなオブジェクトから生成された光音響信号の振幅を最大にするために顕微鏡対物レンズの垂直位置（すなわち、zの位置）を調整します。最大化した信号の振幅は、光焦点が垂直方向に音響を中心に揃っていることを示唆している。 </li><li>ターゲットから発生する光音響信号が対称的なパターンが表示されるまで顕微鏡対物の水平位置（すなわち、xとyの位置を）調整する。対称性は、光学フォーカスが水平方向に音響を中心に揃っていることを示唆している。 </li><li>光音響信号が対称と振幅の両方に最適化されるまでのステップ2.6と2.7を繰り返します。 </li></ol> 3。 SA<html> mple実験手続きのin vivo OR - PAMマウス耳管構造の<ol><li> このステップは、ヌードマウスは不要です 。カクテルの腹腔内注射[：;：0.1 ml/10グラム投与量1ミリリットルケタミン（100 mg / ml）を、0.1ミリリットルキシラジン（100 mg / ml）を、8.9 mlの生理食塩水レシピ]で動物を麻酔。耳の毛を剃って、さらにSurgiクリーム（カテゴリ＃：82565、アメリカンインターナショナルインダストリーズ）との残差の毛を脱毛する脱イオン水でクリーニングする前に。そのような脱毛が若干皮膚血管を刺激するかもしれないので、最善の計画的な実験の24時間前に行われることに注意してください。 </li><li>光音響レーザーシステムの電源を入れ、システムのアライメントを確認してください。 </li><li>吸入ガス（典型的な流量は、動物の体重に応じて、1.0〜1.5リットル/分である）でイソフルラン3％を気化して、マウスを麻酔し、実験を通してイソフルラン1％の麻酔を維持する。メディカルグレードの空気は、正常な生理状態でマウスを維持するために吸入ガスとして推奨されます。 </li><li>定位ステージにマウスを移し、そして加熱パッドで37℃の体温° Cを制御します。 </li><li>プラスチック板上にマウスの耳を平らにし、耳の上に超音波ゲルの層を適用する。ゲル内部の気泡をトラップすることは避けてください。その後、イメージングのウィンドウの下に耳を配置し、徐々に超音波ゲルコンタクトポリエチレン膜の底まで、動物の段階を上げる。膜に対して耳を押すと、耳に血流に影響を与える可能性があるので、ソフトコンタクトが必要です。 </li><li>その生理的状態を監視するためにマウスの足や尾部にパルスオキシメータをクランプし、乾燥やマウスの眼への偶発的なレーザーの損傷を防ぐために、目に軟膏を塗る。 </li><li>音響レンズを脱イオン水に浸漬されるまで、イメージングヘッドを下ろし、そして音響レンズの下に閉じ込められた空気の泡を取り除く。 </li><li>それは米国規格協会13のレーザーの安全基準内であることを確認するためにレーザフルエンスを確認してください。レーザーのフルエンスは、レーザビームが皮膚の表面下150μm以下に集束されたレーザパルスのエネルギーの80 NJに変換する20 MJ / cm 2の 、超えてはならない。 </li><li>外部トリガモードにレーザーを設定し、裁判のスキャンを開始します。最強の光音響信号が音響焦点面からになるまで、イメージングヘッドのZ位置を調整します。 </li><li>正しいスキャンパラメータを設定し、正式な画像取得を開始します。 </li><li>実験後、レーザーをオフにする、、マウスを離す脱イオン水でマウスの耳をきれいに、麻酔のシステムと温度コントローラをオフにし、マウスをアンロードするために動物の段階を下げ、脱イオン水のイメージングヘッドを持ち上げ定位のステージから。 </li><li>反復的なイメージングが必要な場合は、37℃で設定された環境温度℃でインキュベーターにマウスを置くそれが自然にウェイクアップ後の動物施設にマウスを返します。そうでなければ、euthanatizeとそれを処分するために動物のプロトコルに従ってください。 </li></ol> 4。機能的OR - PAM総濃度とヘモグロビンの酸素飽和度の<ol><li>オキシヘモグロビン（HBO 2） ​​とデオキシヘモグロビン（HBR）はヘモグロビンの二つの主要な形態、可視スペクトル領域で支配的な内因性光音響源です。 HBO 2とHBRは、別個の光吸収スペクトルを有し、したがってスペクトルヘモグロビン5の合計濃度（HBT）と酸素飽和度（SO 2）の両方を定量化するために区別することができます。ここでSO 2の測定のための適切な光波長を選択するのに役立つ2つのガイドラインは以下のとおりです。 </li><li>波長が十分な信号対雑音比と十分な浸透を確実にするためにヘモグロビンの吸収スペクトル（すなわち、550〜600 nm）のQ -バンドの中で選択する必要があります。 </li><li> HBO 2、HBRの吸収係数は顕著な違い（例えば、HBR -支配的な561 nmのとHBO 2 -支配的な578 nm）を持つ波長が推奨されます。 </li></ol> SO 2に加えて、HBTは追加[HBR]と[HBO 2]を一緒にして計算することができる、または、直接ヘモグロビンのモル吸光係数スペクトルの等吸収波長で測定することができます（例えば、530nmで、545 nmの、570nm以下、及びHBRとHBO 2は等しいモル吸光係数が584 nm）を14。 5。代表的な結果： 図1に示すように血管解剖とSO 2のデュアル波長（561および570 nm）のOR - PAMによって撮像された生きているヌードマウスの耳の中にあります。デュアル波長の典型的な画像取得時間は、関心のような地域のSO 2の測定（画像サイズ：10ミリメートル× 10ミリメートル、ステップサイズ：2.5μmの× 5μm）を〜80分です。 p_upload/2729/2729fig1.jpg&quot;altが=&quot;図1&quot;/&gt; 図1。 生体光学分解能光音響顕微鏡で 。血管の解剖学的構造を示す（A）総ヘモグロビン濃度（570 nmで買収）とヌードマウスの耳の中に（B）ヘモグロビンの酸素飽和度（561 nmと570 nmで取得した）のMAP画像。 （C）クローズアップパネルで囲み領域（）の。パネルのスケールバーは、（）両方（）と（B）に適用されます。

<ol>
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すべての実験動物の手順は、セントルイスのワシントン大学医学部動物実験委員会の学校で承認された実験動物のプロトコルに準じて実施した。

このビデオでは、我々は、システム構成、システムのアライメント、および典型的な実験手順を含むOR - PAMの実験的なプロトコルに関する詳細な指示を提供しています。ラベルのない、非侵襲的OR - PAMは、シングルキャピラリー的に微小血管機能と代謝の研究を有効にし、それにより微小循環に関連した生理学と病理学の理解を拡大する可能性を保持しています。 Microphotoacousticsは現在、このO...

自家製音響光学ビームコンバイナ： <ul><li>直角プリズム（NT32 - 545、エドモンドオプティクス） </li><li>菱形プリズム（NT49 - 419、エドモンドオプティクス） </li><li>シリコーンオイル（1000cSt、Clearco製品） </li><li> OR - PAMシステム（Microphotoacoustics） </li></ul>

three-dimensional optical-resolution photoacoustic microscopy

携帯とオルガネラレベルでの貴重な洞察を提供する光学顕微鏡は、、広く可能にする生物医学技術として認識されている。 in vivoでの柱として、3次元（3 - D）光学顕微鏡、single-/multi-photon蛍光顕微鏡および光コヒーレンストモグラフィは、（OCT）はそれぞれ、蛍光および光散乱コントラストに彼らの異常な感受性を示している。しかし、本質的な生理学的/病理学的情報をエンコードする生体組織の光吸収コントラストでは、、まだ課税をされていない。 生物医学photoacousticsの出現は、光照射が細胞内局在、たとえ1携帯電話または2レベルの横方向の解像度を達成するための回折限界に焦点を当てている光学顕微鏡の光学解像度光音響顕微鏡（OR - PAM）1の新しい枝につながっている。既存の光学顕微鏡技術に貴重な補完するものとして、OR - PAMは、少なくとも2つのノベルティにもたらします。まず、最も重要なことは、OR - PAMは、異常な感度（すなわち、100％）と光吸収のコントラストを検出します。蛍光顕微鏡3でまたは光散乱ベースの10月4日 （あるいはその両方を持つ）とOR - PAMを組み合わせることにより、生体組織の包括的な光学特性を提供します。第二に、OR - PAMは、蛍光顕微鏡およびOCTの純粋な光学過程とは対照的に、音響波に光吸収を符号化し、バックグラウンドのない検出を提供します。 OR - PAMの音響検出は、信号劣化の光散乱の影響を軽減し、自然に励起し、励起と蛍光スペクトルの重なりに起因する蛍光顕微鏡での共通の問題である検出、間に可能な限り干渉（すなわち、クロストーク）を排除。 光吸収イメージングのためのユニークな、OR - PAMは神経学5、6、眼科7、8、血管生物学9、皮膚科10、を含むがこれに限定されないが、その発明以来、広範な生物医学アプリケーションを、実証されています。このビデオでは、我々はシステム構成とOR - PAMの配置だけでなく、in vivoで機能的な微小血管イメージングのための実験手順を教える。

Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

Three-dimensional Optical-resolution Photoacoustic Microscopy

光学分解能光音響顕微鏡（OR - PAM）は、撮像光学吸収が可能な新たな技術は対照的です。<em&gt; in vivoで</em&gt;携帯電話の解像度と感度を持つ。ここで、我々は典型的なシステム構成、システムの配置、を含むOR - PAMの実験プロトコル、上に可視化命令を提供する<em&gt; in vivoで</em&gt;実験手順、および機能イメージングの手法。

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 ...

three-dimensional-optical-resolution-photoacoustic-microscopy

Research

JoVE Journal

Bioengineering

17.7K ビュー.  Washington University in St. Louis. 光学分解能光音響顕微鏡（OR - PAM）は、撮像光学吸収が可能な新たな技術は対照的です。 in vivoで携帯電話の解像度と感度を持つ。ここで、我々は典型的なシステム構成、システムの配置、を含むOR - PAMの実験プロトコル、上に可視化命令を提供する in vivoで実験手順、および機能イメージングの手法。

ビデオ: Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工学

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

導電性インクの平面および三次元印刷

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

光音響膀胱造影

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

三次元インクジェット印刷技術を用いて、ヒト軟骨組織の作製

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

の調製と分析<em&gt;インビトロ</em&gt;三次元乳癌サロゲート

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

イムノアッセイのための3次元紙ベースのマイクロ流体デバイスの作製

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.