Name: characterizing multiscale mechanical properties of brain tissue using atomic force microscopy, impact indentation, and rheometry
Uploaded: 2016-09-06T12:30:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry at JoVE.com

We acknowledge support of this work by the National Multiple Sclerosis Society and Simons Center for the Social Brain. BQ acknowledges support from the U.S. National Defense Science &amp; Engineering Graduate Fellowship program.

<ol>
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The authors have nothing to disclose.

本論文で各技術は、脳組織の機械的性質の異なる側面を測定します。クリープコンプライアンスと応力緩和弾性率は時間に依存する機械的特性の尺度です。貯蔵弾性率および損失弾性率は、速度に依存する機械的特性を表します。インパクトインデントも速度に依存する機械的特性を測定したが、エネルギー散逸のコンテキストインチ組織の機械的特性を特徴づけるとき、両方のAFM対応イン?...

生物学的器官を含むほとんどの軟組織石灰化した骨または工学材料と比較して機械的及び構造的に複雑な、低剛性であり、非線形および時間依存の変形を示します。体内の他の組織と比較して、脳組織は1Paで数百程度の弾性率をEと、格段に準拠しています。脳組織は、異なるとの構造的不均一性を示し、また、機能的に異なるグレーと白質領域を互いにかみ合っ。脳組織の力学を理解することは、傷害の間に脳の応答を模倣する材料と計算モデルの設計に役立つ機械的損傷の予測を容易にし、保護戦略の設計を可能にします。さらに、このような情報は、組織再生のための設計目標を考慮するために使用することができ、より良好な多発性硬化症や自閉症などの疾患と関連している脳組織の構造的変化を理解します。 HERE、我々はメソ、ミクロで、脳組織を含む機械的に準拠した組織の粘弾性特性を特徴づけるために利用可能ないくつかの実験的アプローチ、およびマクロスケールを説明し、実証します。 マイクロスケールで、我々はクリープコンプライアンスを行い、原子間力顕微鏡（AFM）対応のインデントを使用して緩和実験を強制します。典型的に、AFM対応くぼみは、試料2-4の弾性率（または瞬間剛性）を推定するために使用されます。しかし、同一の器具はまた特性5-10（タイムまたはレートに依存）マイクロ粘弾性を測定することができます。 図1に示すこれらの実験の原理は、時間の経過とともに、脳組織内にプローブを片持ちAFMをインデント力や押し込み深さの特定の大きさを維持し、それぞれ押し込み深さと力の対応する変化を測定することです。これらのデータを用いて、クリープCOMPを算出することができますそれぞれliance J Cと緩和弾性率G R、。 メソスケールで、我々は振り子ベースのインストルメントナノインデンターを使用して、組織構造と水和レベルを維持する流体に浸漬条件における衝撃インデントの実験を行いました。実験は、 図2に示されている。振り子が組織と接触するように揺動するように、振動振り子が組織内に静止するまでの変位を時間の関数として記録されているプローブ。プローブの結果として生じる減衰高調波振動運動から、我々は、組織11,12の（エネルギー散逸率に関する）の最大侵入深さx MAX、エネルギー散逸能力K、及び散逸品質係数Qを算出することができます。 マクロスケールで、我々は、周波数依存のせん断弾性率を定量化するために、平行平板レオメーターを使用しました。組織の貯蔵弾性率G &#39;及び損失弾性率G &quot;を 、と呼ばれるレオメトリーのこのタイプでは、高調波角度歪みを適用する（剪断歪みに対応する）既知の振幅と周波数で及びreactionalトルクを測定（及びせん断応力に対応します） 図3に示すように、測定されたトルクの結果として得られる振幅と位相遅れおよびシステムの幾何学的な変数から、我々は興味13,14の印加周波数で&quot;G &#39;及びGを計算することができます。

<table><tbody><tr><td>Xylazine</td><td>Lloyd Laboratoried</td><td></td><td>perscription drug</td></tr><tr><td>Ketamine</td><td>AnaSed Injections</td><td></td><td>perscription drug</td></tr><tr><td>Vibratome (Vibrating blade microtome)</td><td>Leica</td><td>VT1200</td><td></td></tr><tr><td>Hibernate-A Medium</td><td>Gibco</td><td>A1247501</td><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent neural medium for adult tissue</td></tr><tr><td>Atomic Force Microscope, MFP-3D-BIO</td><td>Asylum Research</td><td>-</td><td></td></tr><tr><td>Petri Dish Heater</td><td>Asylum Research</td><td>-</td><td></td></tr><tr><td>AFM Probe, 0.03 N/m, 10&amp;nbsp;&amp;micro;m radius borosilicate sphere</td><td>Novascan</td><td>PT.GS</td><td></td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>mussel-derived bioadhesive</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>alternate suppliers can be used</td></tr><tr><td>Sodium Hydroxide, 1 N</td><td>Sigma-Aldrich</td><td>59223C</td><td>alternate suppliers can be used</td></tr><tr><td>Instrumented Indenter, NanoTest Vantage</td><td>Micro Materials Ltd.</td><td>-</td><td>probe tip needs to be machined (steel flat punch, 1&amp;nbsp;mm diameter, 4-5&amp;nbsp;mm length)</td></tr><tr><td>NanoTest Liquid Cell</td><td>Micro Materials Ltd.</td><td>-</td><td></td></tr><tr><td>Parallel Plate Rheometer MCR501</td><td>Anton-Parr</td><td>-</td><td></td></tr><tr><td>PP25&amp;nbsp;</td><td>Anton-Parr</td><td>-</td><td>25 mm diameter flat measurement plate</td></tr><tr><td>Adhesive Sandpaper</td><td>McMaster-Carr</td><td>4184A48</td><td>alternate suppliers can be used</td></tr><tr><td>Loctite 4013 Instant Adhesive</td><td>Henkel</td><td>20268</td><td>alternate suppliers can be used</td></tr></tbody></table>

characterizing multiscale mechanical properties of brain tissue using atomic force microscopy, impact indentation, and rheometry

機械的な模擬または組織再生研究のためかどうか、脳の性質に触発された材料を設計エンジニアには、脳組織自体がよく、様々な長さと時間スケールで特徴づけされなければなりません。多くの生物学的組織のように、脳組織は、複雑な階層構造を示します。しかし、大部分の他の組織とは対照的に、脳はPaで数百のオーダーのヤング弾性率をEと、非常に低い機械的剛性のものである。この低剛性は、キーの機械的特性の実験的特性評価に課題を提示することができます。ここでは、異なる長さスケールおよび負荷速度で、例えば、脳組織などの水和、コンプライアントの生物学的材料の弾性および粘弾性特性を測定するように適応されているいくつかの機械的特性評価技術を実証します。マイクロスケールで、我々は原子間力顕微鏡対応のインデントを使用して、クリープコンプライアンスと力緩和実験を行います。 mesosでCALEは、我々は振り子ベースのインストルメント圧子を使用して、衝撃インデント実験を行います。マクロスケールで、我々は周波数に依存するせん断弾性率を定量化するために平行プレートレオメトリーを行っています。また、それぞれの方法に伴う課題や制限について説明します。一緒にこれらの技術は、より良い脳の構造を理解するために、バイオ風の材料を操作するために使用することができます脳組織の徹底的な機械的特性評価を可能にします。

図4は 、それぞれ適用された力あるいは押し込み深さ（ 図4A、D）、与えられた、クリープコンプライアンスのための時間応答（ 図4B、E）対代表インデントと力を示し、リラクゼーション実験を強制します。これらのデータおよびシステムの幾何学的形状を使用して、クリープコンプライアンスJの C（t）と緩和弾?...

Watch this Scientific Journal Video about Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry at JoVE.com

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

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 ...

characterizing-multiscale-mechanical-properties-brain-tissue-using

Research

JoVE Journal

Neuroscience

12.3K ビュー.  Massachusetts Institute of Technology. We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

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

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

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.

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 ...

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 ...

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 ...

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 ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

ライブセル上の近くの同時レーザー走査共焦点と原子間力顕微鏡(コンポカル)

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

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 ...

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young&#39;s modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young&#39;s modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young&#39;s moduli, and generation of Young&#39;s modulus maps. Finally, it shows how to determine and isolate Young&#39;s modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This protocol visualizes how to prepare cryosections of murine lung tissue, perform atomic force microscopy force map experiments, and analyze the data to determine Young's modulus of the native murine pulmonary basement membrane.

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

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