AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.

<ol>
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The authors have nothing to disclose.

AFMのインデントは、細胞および組織の剛性（弾性率）を特徴付けるために使用することができます。本稿では、マウスで下行大動脈および大動脈弓を隔離し、これらの動脈領域のex vivoでの弾性係数を決定するための詳細なステップバイステップのプロトコルを提供します。私たちは今、要約し、この論文に記載された方法の技術的な問題と制限事項について説明します。 いくつかの技術的な問題は、彼らの小型、薄型の性質を与えられたマウスの大動脈の単離と解析で発生することができます。洗浄動脈は縦方向に開かれている場合には、注意が弾性率のデータが収集された内膜（腔面）を破壊することないように注意する必要があります。最終サンプル皿中の過剰のPBSが皿に動脈の適切な接着を防止し、これは接着の前に動脈に触れることなく、ティッシュペーパーを使用して慎重に除去する必要があります。分離された動脈が紙組織シベニクに固執することができること、しかし、注意してくださいルは、過剰なPBSを除去しようとすると、これはサンプルが損傷することがあります。培養皿に動脈を接着するが、動脈が伸びていないことは非常に重要であるとのみ接着剤の少量の動脈の両端にビット・バイ・ビット印加されます。 AFMインデンテーションを行う場合には、接着剤が塗布された動脈の端部にプロービング回避することが重要です。新たに単離した動脈は弾性率の測定のために使用されるので、最後に、時間が最小化されるべき測定を収穫からの経過しました。 本研究で用いた球状の先端が500nmの半径を有しているので、最大500nmの押し込み深さを正確ヘルツモデルを用いて分析することができます。この押し込み深さは、内膜21に位置する内皮細胞の最上層と相関するであろう。動脈血サンプルのサブ内皮層のメカニズムを調べるために、より大きなコロイドプローブ（10 - 直径20μm）を使用することができますインデントと押し込み深さと計算された剛性の変化のために22,23を決定することができます。生物学的サンプルは通常、粘弾性ではなく純粋に弾性のため、また、（一定の押し込み深さで適用される力の減衰を監視する）応力緩和の測定は、動脈の機械的特性24,25の粘性成分を決定するために使用することができます。 AFM分析の潜在的な制限は、組織の小さな領域を測定スキャンサイズ、です。動脈細胞を含むほとんどの生物学的試料は、地形的および生体力学的に均質ではなく、この不均一性は、潜在的に圧痕部位の平滑性、押し込み深さと、試料に加えられた力の量に応じて弾性率の人為大きな変動をもたらすことができます表面。全体の弾性率の代表的な平均値を得るためには、反復AFMインデンテーションAを実行することが重要です以前の研究は、鋭いピラミッド状先端を有するAFMインデンテーションは、弾性率の高い見積もりをもたらし、ソフトな損傷を受けたことを明らかにしたとして、図4に示すように、動脈を通じてトン複数のランダムな場所は、。また、我々は、適切なサイズの球状チップを使用します生物学的サンプル26,27。 動脈硬化の我々のex vivoでの AFM測定は2補強平滑筋細胞の分子マーカーとよく一致するように見えるにもかかわらず、これらの動脈サンプルを機械的にロードされないことを理解することが重要であり、機械的負荷は、全体に重要な役割を果たす可能性が高いです動脈力学。 AFM、筋運動記録法（機械的負荷との互換性のex vivo法）28とパルス波速度（動脈硬化のin vivoで測定）29によって決定されるような動脈硬化の比較は可能性が高いarterの最も包括的な理解を提供しますIAL力学。 我々は、マウスの動脈のex vivoでの剛性を測定する接触モードでAFMを記載しています。この方法は、ランダムに大動脈の複数の領域にインデントによって平均組織剛性を推定します。組織の大きな領域からの剛性の空間分布を調べるために、最近の論文は、力ボリュームモード30でAFMを使用しています。このアプローチは、同時に剛性の変化のマップおよび組織の地形表面の高解像度画像が得られます。組織サンプルの硬さを測定することに加えて、力ボリュームモードのAFMは、細胞内の剛性10,31の空間分布をモニターするために細胞内で使用することができます。

The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol. 
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.

<table><tbody><tr><td>BioScope Catalyst AFM system</td><td>Bruker</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE 200 inverted microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Silicon nitride AFM probe</td><td>Novascan Technologies</td><td>PT.SI02.SN.1</td><td>0.06 N/m cantilever; 1 &amp;micro;m SiO&lt;sub&gt;2&lt;/sub&gt; particle</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>See section 1.4</td></tr><tr><td>Dumont #5SF forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>See section 1.8</td></tr><tr><td>Fine Scissors-ToughCut</td><td>Fine Science Tools</td><td>14058-11</td><td>See section 1.4 (medium sized)</td></tr><tr><td>Vannas-T&amp;uuml;bingen spring scissors</td><td>Fine Science Tools</td><td>15008-08</td><td>See section 1.6 (small sized)</td></tr><tr><td>60 mm TC-treated cell culture dish</td><td>Corning</td><td>353004</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, 1x</td><td>Corning</td><td>21-031-CM</td><td>Without calcium and magnesium</td></tr><tr><td>Krazy Glue instant all purpose liquid</td><td>Krazy Glue</td><td>KG58548R</td><td>See section 2.2</td></tr><tr><td>Gel-loading tips, 1 - 200 &amp;micro;l</td><td>Fisher</td><td>02-707-139</td><td>See section 2.2</td></tr><tr><td>Tip Tweezers</td><td>Electron Microscopy Sciences</td><td>78092-CP</td><td>See section 3.2</td></tr><tr><td>50-mm, clear wall glass bottom dishes</td><td>TED PELLA</td><td>14027-20</td><td>See section 4.4</td></tr></tbody></table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

動脈硬化は、心血管疾患や老化の顕著な特徴のための重要な危険因子とバイオマーカーです。原子間力顕微鏡（AFM）（ 等のプラスチック、ガラス、金属、）ハードに至るまで様々な材料のための粘弾性の機械的性質を特徴付けるための多目的な分析ツールである任意の基板上のセルに表面。広く、細胞の剛性を測定するために使用されるが、あまり頻繁に大動脈の剛性を測定するために使用されてきました。本稿では、無負荷マウスの動脈のex vivoでの弾性率を測定するために、接触モードでAFMを使用するための手順について説明します。我々は、マウスの大動脈の単離のために私たちの手順を説明し、その後、AFM分析のための詳細な情報を提供します。これは、レーザービームのアライメント、ばね定数とAFMプローブの偏向感度のキャリブレーション、および力曲線を取得するためのステップバイステップの手順が含まれています。また、データANALYための詳細なプロトコルを提供します力曲線のsisの。

<html> 図5Aは、6ヶ月齢、雄のC57BL / 6マウスから降順（胸部）大動脈の位相コントラスト画像を示しています。 AFMカンチレバーは、直接組織上記の場所にあり、インデントのための準備ができました。 図5Bおよび5Cは、コンタクトモードでのAFMインデントによって得られた代表的な力曲線を示しています。 図5B及び図5Cに示す緑色の線は、球用ヘルツモデルを用いて得られたベストフィット曲線を表します。本文中に記載されているように図5Dに、下行大動脈および大動脈弓の平均剛性は、3つの個々のマウスから決定しました。下行大動脈および大動脈弓はそれぞれ、層状と乱流の領域を表すことに注意してください。それにもかかわらず、彼らの（機械的にアンロード）弾性率は、AFMによって決定されるように似ています。 0 / 54630fig5.jpg &quot;/&gt; 図5：大動脈ex vivoで降順にマウスオーバーAFMカンチレバーを示す力-押込み曲線と剛性データ分析 （A）位相差顕微鏡写真。スケールバーは100μm。（BC）（B）下行大動脈および（C）大動脈弓のために取得された2つの代表的な力インデンテーション曲線のセット。（D）平均マウスの下行大動脈の剛性および大動脈弓のように決定しました。上述。エラーバーは、平均+ SEMを示します。 n = 3の独立した生物学的複製。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54630/54630fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

の剛性を測定します<em&gt; ex vivoで</em原子間力顕微鏡を使用&gt;マウス大動脈

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

measuring-stiffness-ex-vivo-mouse-aortas-using-atomic-force

Universidad San Sebastian

Research

JoVE Journal

Biology

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

10.5K ビュー.  University of Pennsylvania. We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

ビデオ: Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

ex vivoでのウイルス形質導入を用いた毛包におけるGFPの標的発現

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

生物学

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

原子間力顕微鏡を用いた生細胞の機械的特性を測定する

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

生物工学

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

昇順大動脈剛性の測定<em&gt;インビボ</em&gt;超音波​​を使用したマウスにおける

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

医学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

光トラップを用いた細菌細胞の曲げ剛性の測定

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

原子間力顕微鏡を用いた肺組織のマイクロメカニカル特性

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.
To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young&#39;s (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.
A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

原子間力顕微鏡と偏光顕微鏡を組み合わせた肝臓のこわばり測定

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

マウス網膜毛細血管および内皮下マトリックスの硬さの測定

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

著者スポットライト:早期糖尿病性網膜症における血管の硬直と炎症の調査

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

の剛性を測定します

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