Name: quantitative hardness measurement by instrumented afm-indentation
Uploaded: 2016-11-22T14:00:00.000+00:00
Duration: 8 min 21 s
Description: Watch this Scientific Journal Video about Quantitative Hardness Measurement by Instrumented AFM-indentation at JoVE.com

A.C. is grateful to KoreaTech for financial support.

1.インストゥルメンタルセットアップとキャリブレーション<ol><li> インストゥルメンタルセットアップ <ol><li> 0,1≥180 kHzの、品質係数Q≥300、および曲げ剛性k≥40 N / mのfは最初のフリー共振周波数にタイプDT-NCLRまたはCDT-NCLRの硬いダイヤモンド被覆カンチレバーを使用してください。 </li><li> AFMの製造元から提供されているクランプホルダーに選択されたカンチレバーをマウントします。その長軸がAFMの高速走査方向に対して垂直になるようにカンチレバーを配置するために特別な注意を払ってください。また、二成分エポキシ接着剤を使用して、AFMの製造元から提供されているカンチレバーホルダーにカンチレバーを接着。 </li><li> AFMヘッド上にカンチレバーホルダーをマウントし、AFMカンチレバーに集中するAFMシステムで通常利用可能な光学顕微鏡を使用しています。カンチレバーの長軸が高速走査方向に垂直であることを再確認してください。ない場合は、バックに行きますセクション1.1.2。 </li><li>それは、カンチレバーの最後に反映されるように、レーザービームの位置を合わせます。フォトダイオードの電圧の和を監視し、和信号を最大化するために微調整を行っています。典型的な和信号の値が2 [V]の範囲であります</li><li>縦と横方向の変位に対応する電圧がほぼゼロであるフォトダイオードの中心に反射されたレーザスポットを持参するように、ミラーの水平方向と垂直方向の傾斜角度を調整します。 </li></ol></li><li> 較正 <ol><li>カンチレバーの0,1 fの最初のフリーの曲げ共振を決定するために、周波数掃引を実行します。 </li><li> 19に従って計算カンチレバーkの曲げ剛性を決定します （1） <img alt="式（1）" src="/files/ftp_upload/54706/54706eq1.jpg" /> Eはヤング率でありcantilの幅wは、Lは 、カンチレバーの長さでありますこれまで、およびtは厚さです。この目的のために、よりよい精度のために、光学顕微鏡または走査型電子顕微鏡によるカンチレバーの長さと幅を測定します。によると、0,1 fはその最初のフリーの曲げ共振周波数からカンチレバーの厚さを計算します （2） <img alt="式（2）" src="/files/ftp_upload/54706/54706eq2.jpg" /> ここで、ρは質量密度です。 </li><li> AFMの設定メニューで実験に使用される特定のカンチレバー型用のフォトダイオードの感度のデフォルト値を選択します。 アプローチ]ボタンをクリックすることで、荷重Fで基準サンプルと接触さカンチレバー先端を持参のn = 10 nNの。 </li><li> AFMソフトウェアの力分光法メニューを開き、50 nmおよび0.3ミクロン/秒のzスキャナ後退/拡張にz軸スキャナの相対的な後退と拡張子を設定します。そう、力 - 距離曲線の記録はなります離れた試料表面から50 nmまでのzスキャナの後退の最初で構成し、同じ距離のアプローチや陥没の一連の。 </li><li>試料の変形の影響を回避するために、そのようなナノ結晶ダイヤモンドやサファイアなどの滑らかで非準拠表面上1.2.4で提案セットパラメータと力距離曲線を記録します。これを行うには、AFMソフトウェアの力分光法メニューの取得]ボタンをクリックします。 </li><li> AFMソフトウェアのキャリブレーションメニューで、線形関数と力 - 距離曲線の反発部分を取り付けます。フィッティングラインの逆勾配は、フォトダイオードの感度Sに相当します。 実行キャリブレーション]ボタンをクリックすることで、AFMソフトウェアのキャリブレーションメニューで、機器・ソフトウェアのデフォルト値に決定された値を代入します。 </li></ol></li></ol> 2.試料の調製注：THIで測定されたサンプルsの実験は、物理蒸着法によって雲母上に成長させた厚さ100nm、原子的に平滑な金（111）薄膜で構成されています。 <ol><li>両面カーボンテープを用いて、機器の製造元から提供されている磁気サンプルホルダーにサンプルをマウントします。カーボンテープをリラックスさせるように、測定中の試料のドリフトを回避するために、測定の前に1日のサンプルをマウントします。また、通常は数分以内に乾燥銀ペイント、とホルダーにサンプルをマウントします。 </li><li> X / Yスキャナ上に磁気試料ホルダーをマウントします。 </li></ol> 3.測定手順<ol><li>オフ共鳴し、これらの値が自動的にこの特定のカンチレバーのための機器・ソフトウェアによって設定されていることをA = 20 nmのノートでの振動振幅（この実験では、F = 190.67 kHz単位で）わずかに発振周波数を設定します。 セットポイント = 5 nmで手動で発振セットポイントを設定します。 </li><li>ドローAFMのステップモータを使用して、試料表面に向かってカンチレバー。力センサは、試料表面と衝突しないことを確認してください。粗アプローチの間に焦点が合ってカンチレバーを維持し、試料表面が完全な焦点になる前に粗大なアプローチを停止します。 </li><li>自動的にアプローチ]ボタンをクリックすることで力センサに近づきます。振動の振幅が設定値に達すると、先端は試料表面のトポグラフィーを走査する準備ができています。 </li><li> 5×5から1×1μm²に至るまでの領域上の地形の一連の画像を記録する（利用可能な場合は、X / Y-スキャナを傾けることによって地形信号の傾きを調整します）。同じ領域の連続した画像は、ドリフトの兆候を示さないこと、およびzスキャナの位置はほぼ一定に保たれていることを確認します。そうでない場合は、システムが安定するまで、撮影を続けます。 </li><li>システムが安定しており、滑らかな1×1μm²領域が発見されると、Fを引っ込めますORCEは後退ボタンをクリックすることで、試料表面から数マイクロメートルのセンサー。 </li><li>楽器メニューの力分光法モードを選択し、10nmの力セットポイントで、予め選択された1×1μm²エリアの中央に力センサを移動させます。それが一定になるまでのzスキャナの位置を監視します。 </li><li>中央予め選択された1×1μm²エリアの中心に対応する点の2×2のグリッドを選択します。 500 nmの2次隣接する点間の距離を設定します。 </li><li> 300ナノメートル/秒の速度で0から150 nmまで変化させると、同じ距離を同じ速度で後退さ相対スキャナ距離を設定します。試料表面に関してカンチレバーの傾斜角を考えると、φは、傾斜角20である垂直スキャナ拡張Z、中に日焼けφ×Zで横スキャナを移動させることでチルト補正を適用します。 注：いくつかのinstにrumentsは、彼らの力分光法やインデントモードでは、カンチレバーの傾きを占めます。これは、この研究で使用したAFMの場合です。 </li><li> AFMのインデントデータの取得を開始するために、機器のソフトウェアで開始ボタンを押してください。 </li><li> AFMの押込測定が完了した後、数マイクロメートル離れて試料表面からの力センサを後退させます。 </li><li>機器のソフトウェアメニューの非接触AFMモードイメージングを選択し、セクション3.1および3.2に記載されている手順を繰り返します。 </li><li>インデントの正確な位置を特定するために、3.3節と同様の1×1μm²表面積にわたってスキャンを実行します。 500×500nm²表面領域上にさらに表面スキャンは、より詳細に画像に残っているインデントを行うことができます。 </li></ol> 4.データ解析<ol><li> 画像処理 <ol><li>高速スキャンディレクトリ内の行を揃えるように記録された地形の画像を処理中央値の差に基づいてection。 Gwyddionの組み込み関数を使用してください。 </li></ol></li><li> Gwyddionのインデント解析機能を使用して、投影面積にインデントのPを計算します。 </li><li> Gwyddionの先端解析機能を用いてインデントのトポグラフィー画像からAFMチップの形状を推定します。そして、先端形状の画像を平均化し、平均先端形状の半開角αを測定します。 </li><li>力-距離曲線は、先端の変位を計算するδ13に従ってにより力-変位曲線に変換します （3） <img alt="式3" src="/files/ftp_upload/54706/54706eq3.jpg" /> ここで、Zは、相対的なスキャナの位置です。 </li><li>さて、先端の変位対力をプロットします。結果の曲線は、通常、原子論的可塑性のイベントに対応するいくつかの100午後の範囲の長さ、で、いわゆるポップインが表示されます。 thesというの最初を使用電子ポップインはエル 4δ弾性限界で先端の変位を決定します。 </li><li>ヘルツ機能21と力-変位曲線の弾性部分を取り付けます。 （4） <img alt="式4" src="/files/ftp_upload/54706/54706eq4.jpg" /> Rは、先端半径であり、E &#39;*はで与え、弾力性の減少係数であります<img alt="式（5）" src="/files/ftp_upload/54706/54706eq5.jpg" /> 、M S、Tはそれぞれ、サンプルの先端の押し込み弾性率であると。この場合には、適合パラメータであります<img alt="式6" src="/files/ftp_upload/54706/54706eq6.jpg" /> 。 </li><li>フィット関数および実験曲線21の間の面積の差から塑性Wの可塑性の仕事を計算するように塑性政権にフィット機能を拡張します。 </li><li> 、1に係る試料の硬度を算出する2 （5） <img alt="式（7）" src="/files/ftp_upload/54706/54706eq7.jpg" /> そして （6） <img alt="式8" src="/files/ftp_upload/54706/54706eq8.jpg" /> F nは、maxは最大荷重がある場合、pは 4.2節で計算されたインデントの投影面積は、αは、4.3節で計算された先端の半開角であり、δ エルは、最初の可塑性における先端変位でありますイベントは、δmaxは最大の先端変位（4.4節を参照）です。 </li></ol>

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The authors have nothing to disclose.

この方法は、ダイヤモンドコーティングされたAFMチップのAu（111）薄膜の表面上に一連のくぼみを行うために提示されています。非接触AFM画像及びAFMのインデントは、同一の力センサを用いて行きました。非接触イメージングのための要件は0,1≥180キロヘルツとAFMのインデントに高い品質係数Q≥300 fが高い第一の自由共振周波数であり、適用される垂直方向の力は...

For the last 150 years, hardness values have been used to characterize the mechanical behavior of materials and to predict their performance under various loading conditions. The hardness of a material describes the resistance of its surface to the penetration of a harder indenter. For metallic materials, the hardness relates to resistance to plastic flow.
Although the implementation of hardness testing has proven to be relatively easy, the yielded results have long been rather empirical, making them unable to describe the intrinsic properties of the investigated material. The empirical nature of hardness testing results has been a consequence of the use of various indenter geometries, each of which has a different established hardness scale. However, all empirical hardness scales, such as the Brinell hardness (HB) test for spherical indenters and the Vickers (HV) and Knoop (HK) hardness tests for different types of four-sided pyramids, have one thing in common: the hardness number is determined from the ratio of the applied load to the developed area of the remaining indent. In an attempt to relate the hardness of metals to their fundamental mechanical properties, the hardness measured with a spherical indenter has been defined as the ratio of the applied load to the projected area of the remaining indent. This hardness value, also known as the Meyer's hardness (H), is equal to the mean contact pressure and directly relates to the yield strength of non-work-hardening metals1. 
Hardness testing has long been limited to the macro-scale, in which case the sizes of indents have been measured by optical means. The development of instrumented indentation, where force-displacement curves are recorded, has been recognized as a valuable alternative for determining the hardness of a material, although the size of the remaining indent may be too small to be accurately imaged. In this case, the projected area of the indent is calculated from the tip displacement according to a so-called indenter-area function2. Due to this development, the analysis of the curvature of force-displacement curves and of the occurrence of distinct plasticity events in the shape of pop-ins is now widely used to study the mechanisms of plastic deformation at the micrometer scale, such as by means of nanoindentation3.
It is well accepted that the carriers of plastic deformation in metals, i.e., dislocations, operate at the nanometer scale. To understand their modes of operation at the atomic level, new experimental techniques with atomic resolution are needed. Initial investigations of atomistic plasticity events at the interfaces between nanometer-sized single asperities and single crystalline Au surfaces of different orientations have been carried out by interfacial force microscopy (IFM)4, 5. Atomic force microscopy (AFM) indentation has been applied to observe the nucleation and gliding of single dislocations in KBr(100) single crystals6, Cu(100)7, and Au(111)8, 9. There, atomistic plasticity events were observed in the shape of pop-ins, with lengths in the range of 1 &#197;. AFM indentation has also been used to measure the nano-hardness and -elasticity of gold nano-island grown on mica10. In this work, the nano-hardness was found to be smaller than the bulk value and was observed to depend linearly on the indentation area. Also, a detailed measurement protocol has been proposed to determine the hardness of hard surfaces, such as fused quartz and silicon, by AFM indentation with a diamond-tipped sapphire cantilever11. In particular, this method takes into account the non-linearity of the photo-sensitive deflection detectors for large cantilever deflection12. More recently, AFM indentation and non-contact AFM imaging have been combined to quantitatively determine the hardness and the fundamental mechanisms of plastic deformation of a Pt(111) single crystalline surface and Pt-based metallic glass13. For Pt(111), plastic deformation mechanisms at the nanometer scale were found to be consistent with the discrete mechanisms established for larger scales. Further, the nanometer-scale plastic deformation of the metallic glass was found to be not discrete, but rather continuous and highly localized around the indenting tip. These results revealed a lower size limit for metallic glasses, below which shear transformation mechanisms are not activated by indentation. AFM indentation has also been used to determine the hardness values of biological samples (such as collagen fibrils)14, polymers15, and colloidal crystals16.
In the present work, a careful experimental procedure is described that includes the force sensor selection, its calibration, the calibration of the cantilever deflection detection system, and the minimization of instrumental drift for accurate and reproducible force-distance measurements. Also, a method for the data analysis is presented that allows the extraction of force-penetration curves from recorded force-distance curves. Further representative results are shown and discussed in the light of recent findings in the field of plastic deformation of small volumes.
For this experiment, an atomic force microscope (AFM) was used. The cornerstone of an AFM is its micro-fabricated force sensor (usually a cantilever beam), with a sharp tip at the end whose radius is in the range of several nanometers. In the particular configuration of the instrument used for this experiment, the cantilever is mounted onto a piezo-electric z-scanner, while the sample to be investigated is mounted onto a piezo-electric x/y-scanner. During AFM imaging, the force sensor tip is scanned over a sample to register interaction forces with the sample surface that result in a deflection of the cantilever according to Hooke's law. The static or dynamic deflection of the cantilever can be measured with an optical beam deflection detector, consisting of a laser diode, a set of mirrors, and a photodiode that converts the cantilever deflection into a voltage. During imaging, the signal at the photodiode is controlled by a feedback loop so as to keep the interaction forces at the sample surface; this results in adjustments of the z-scanner position, which are recorded and displayed as a topography image.
Prerequisites for the experiment described below are that the piezo-electric scanners of the atomic force microscope are well-calibrated and that the instrument stays in the laboratory at a constant temperature and humidity level. The reader should be aware that, depending on the atomic force microscope model, some of the experimental procedure steps may have to be modified. In particular, all measurements are performed after setting the scanners' ranges to "small"; this function allows for the reduction of the x/y-scanner range to 5 x 5 &#181;m&#178; and the z-scanner range to 4 &#181;m, which ensures a resolution at small scales. This function is not available on all commercial AFM and is not mentioned in the remainder of the text.
For data analysis, the use of the free SPM data analysis software Gwyddion17 and the Matlab software package18 are recommended.
This protocol gives a description of the experimental procedure to be followed in order to perform instrumented hardness measurements by AFM. Since the handling of different commercial AFMs may differ from one model to the other, the reader should refer to the manual provided by the AFM manufacturer for detailed setting procedures and software information. In the following text, in order to reproduce the described experiment, it is assumed that the reader is familiar with the handling of the particular AFM used here.

<table><tbody><tr><td>AFM XE-100</td><td>Park Instruments</td><td>discontinued</td><td>Atomic force microscope</td></tr><tr><td>CDT-NCLR</td><td>NanoSensors</td><td>CDT-NCLR</td><td>Conductive diamond coated non-contact lever</td></tr><tr><td>100 nm thick Au(111) thin film on Mica</td><td>Phasis</td><td>20020011</td><td>atomically smooth gold thin film</td></tr></tbody></table>

quantitative hardness measurement by instrumented afm-indentation

In this work, a combination of amplitude-modulated non-contact atomic force microscopy and atomic force spectroscopy is applied for instrumented hardness measurements on an Au(111) surface with atomistic resolution of single plasticity events. A careful experimental procedure is described that includes the force sensor selection, its calibration, the calibration of the cantilever deflection detection system, and the minimization of instrumental drift for accurate and reproducible force-distance measurements. Also, a method for the data analysis is presented that allows the extraction of force-penetration curves from recorded force-distance curves. A typical curve displays a clear elastic deformation regime up to the first plasticity event, or pop-in, with a length in the range of one to two Burger's vectors. Later plasticity events exhibit the same magnitude. The work of plasticity is further extracted from the measurements. Finally, the hardness is determined in combination with the indentation curve using non-contact atomic force microscopy images of the remaining indents.

この研究では、カンチレバーkの曲げ剛性は、幾何学的なビーム理論19に従って算出しました。この作業で使用される特定のダイヤモンド被覆カンチレバーのために、我々はK = 55.69 N / mのを見つけました。我々はダイヤモンドコーティングを無視していることに注意してください。ダイヤモンドコーティングの厚さは、カンチレバーの厚さよりも?...

Watch this Scientific Journal Video about Quantitative Hardness Measurement by Instrumented AFM-indentation at JoVE.com

Quantitative Hardness Measurement by Instrumented AFM-indentation

This experimental protocol describes how atomic force microscopy can be used to measure hardness at the true nanometer scale and to detect single atomistic plasticity events.

計装AFMインデンテーションによる定量硬度測定

In this work, a combination of amplitude-modulated non-contact atomic force microscopy and atomic force spectroscopy is applied for instrumented hardness ...

quantitative-hardness-measurement-by-instrumented-afm-indentation

Research

JoVE Journal

Engineering

9.6K ビュー.  KoreaTech, Korea University of Technology and Education. This experimental protocol describes how atomic force microscopy can be used to measure hardness at the true nanometer scale and to detect single atomistic plasticity events. 

ビデオ: Quantitative Hardness Measurement by Instrumented AFM-indentation

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

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

多層魚の鱗の特性（<em&gt; Atractosteusヘラ</em&gt;）ナノインデンテーション、X線CT、FTIR、およびSEMを用いて

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

原子間力顕微鏡を用いて植物細胞と植物組織の機械的特性とターゴール圧を測定

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

発生生物学

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

AFMベースの力分光法のための単一分子の共有結合

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

化学

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子間力顕微鏡カンチレバーを用いたナノインデンテーション:空気および流体中のナノスケールでの機械的特性測定

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

工学

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

原子間力顕微鏡、インパクトインデント、およびレオを使用した脳組織のマルチスケール力学的性質を特徴づけます

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

神経科学

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 ...

生物学

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

早期変形性関節症の検出に対する原子間力顕微鏡の応用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

ソフトマターナノインデンテーションの実験・データ解析ワークフロー

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

原子間力顕微鏡を用いたヒト関節軟骨外植片のマイクロインデンテーションにおける実用的課題への対処

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...