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.

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The authors have nothing to disclose.

본 논문에서는 각 기술은 뇌 조직의 기계적 성질의 다른 측면을 측정합니다. 크리프 준수 및 응력 완화 계수는 시간에 따른 기계적 특성의 측정이다. 저장 및 손실 모듈러스는 속도 의존 기계적 특성을 나타낸다. 충격 압입은 속도 - 종속 기계적 특성을 측정하지만, 에너지 소비의 맥락이다. 조직의 기계적 특성을 특성화 할 때, 모두 AFM 사용 들여 쓰기 및 유변학은 일반적으로 방법을 사용한다. 전...

생물학적 장기를 포함하는 대부분의 부드러운 조직 광물 뼈 재료 또는 설계에 비하여 기계적 및 구조적으로 복잡하고, 저 강성이고, 비선형 및 시간에 따른 변형을 나타낸다. 신체의 다른 조직에 비해 뇌 조직은 아빠 1의 100 단위 정도의 탄성 계수를 E로, 현저하게 준수합니다. 뇌 조직은 독특하고 깍지 회색과도 기능적으로 차이가 백질 영역과 구조적 이질성을 나타낸다. 이해 뇌 조직 역학 부상시 뇌의 반응을 모방하는 기계적 손상의 예측을 용이하게하고, 보호 전략 공학 있도록 재료 및 계산 모델의 설계에 도움이된다. 또한, 이러한 정보는 조직의 재생을위한 설계 목표를 고려​​하는 것이 사용될 수 있고, 더 다중 경화증 및 자폐증과 같은 질병과 관련된 뇌 조직의 구조적 변화를 이해. H의 전에, 우리는 설명하고 중간 -, 마이크로에서 뇌 조직을 포함하여 기계적으로 준수하는 조직의 점탄성 특성을 특성화 할 수있는 여러 가지 실험 방법을 설명하고, 매크로 규모. 마이크로 스케일에서, 우리는 크리프 컴플라이언스를 실시 및 원자 현미경 (AFM)을 이용하여 사용이 가능한 압입 완화 실험 강제. 일반적으로, AFM 기반 압입는 시료 2-4의 탄성률 (강성 또는 순시)를 추정하는데 사용된다. 그러나, 같은 기기는 또한 마이크로 점탄성 (또는 시간 - 속도 - 의존적) 특성 5-10를 측정하는데 사용될 수있다. 도 1에 도시 한 실험의 원리는 시간이 지남에 따라, 뇌 조직에 프로브 외팔보 AFM에 들여 힘 또는 압입 깊이의 특정 크기를 유지하고, 각각 압입 깊이와 힘의 대응 변화를 측정하는 것이다. 이러한 데이터를 사용하여, 우리는 크리프 샘플 콘텐츠를 계산할 수각각 liance J C 및 휴식 계수 G R,. 중규모, 우리는 진자 기반 인스트루먼트 나노 인 덴터를 이용하여 조직 구조 및 수분 레벨을 유지하는 유체 침지 조건에 영향을 들여 실험을 수행 하였다. 실험 장치는도 2에 도시되어있다. 진자가 조직과 접촉하여 요동으로 진동 진자 조직 내 쉴 때까지의 변위가 시간의 함수로서 기록된다 프로브. 프로브 얻어진 감쇠 고조파 진동 운동에서, 우리는 조직 (11, 12)의 (에너지 손실의 속도에 관한)의 최대 침투 깊이 X 최대 에너지 소산 용량 K 및 소산 품질 인자 Q를 계산할 수있다. 매크로 스케일에서, 우리는, 주파수 의존 전단 탄성률을 정량화하는 평행 판 레오 미터를 사용하여. 조직의 저장 탄성률 G &#39;와 손실 탄성률 G &quot;를 지칭 유변학이 유형에서는, 고조파 각 균주를 적용 (전단 변형을 대응하는) 공지의 진폭과 주파수 및 reactional 토크 측정 (및 전단 응력에 대응) 도 3에 도시 된 바와 같이. 상기 측정 된 토크의 결과 진폭 및 위상 지연과 시스템의 기하학적 변수부터는 관심 13,14의인가 주파수 &#39;G&#39;와 G를 계산할 수있다.

<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

기계 모조품 또는 조직 재생 연구에 대한 여부, 뇌의 특성에 의해 영감 재료를 설계 및 엔지니어, 뇌 조직 자체는 물론 다양한 길이와 시간 규모에서 특징해야합니다. 많은 생물학적 조직과 마찬가지로 뇌 조직은 복잡한 계층 구조를 나타낸다. 그러나 대부분의 다른 조직과는 대조적으로, 뇌는 아빠의 100 단위 정도의 영의 탄성 계수를 E로, 매우 낮은 기계적 강성이다.이 낮은 강성 키 기계적 특성의 실험적 특성에 문제를 표시 할 수 있습니다. 여기, 우리는 서로 다른 길이 스케일과 로딩 속도에서 뇌 조직으로 수화, 준수 생물학적 재료의 탄성 및 점탄성 특성을 측정하도록되어 여러 가지 기계적 특성 분석 기법을 보여줍니다. 마이크로 스케일에서, 우리는 원자 힘 현미경을 사용 들여 쓰기를 사용하여 크리프 준수 및 강제 휴식 실험을 실시하고 있습니다. 메소에서케일, 우리는 진자 기반 계측 압자를 사용하여 충격 압입 실험을 수행합니다. 거시적, 우리는 주파수에 의존 전단 탄성 계수를 정량화하는 평행 판 레오 메타를 실시하고 있습니다. 우리는 또한 각 방법과 관련된 문제 및 제한 사항에 대해 설명합니다. 이 두 기술은 더 뇌의 구조를 이해하는 바이오 고무 물질을 설계하는 데 사용될 수 뇌 조직의 충분히 기계적 특성을 가능하게한다.

그림 4는 각각 가해진 힘 또는 압입 깊이 (그림 4A, D), 부여, 크리프 준수에 대한 시간 응답 (그림 4B, E) 대 대표 들여 쓰기와 힘을 보여줍니다 및 휴식 실험을 강제로. 이러한 데이터는 시스템의 구조를 사용하여 크리프 컴플라이언스 J의 C (t)와 이완 계수 G R (t)에 힘이 뇌 (도 4C, F)의 상이한 영?...

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.

원자 힘 현미경, 충격 들여 쓰기 및 유변학를 사용하여 뇌 조직의 멀티 스케일 기계 속성을 특성화

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 조회수.  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.

비디오: 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).

원자 힘 현미경을 사용하여 살아있는 세포의 기계적 특성을 측정

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

연구

생체공학

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.

계장 AFM-들여 쓰기에 의해 정량 경도 측정

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

공학

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.

다층 물고기 비늘 (의 특성<em&gt; Atractosteus 주걱</em&gt;) 나노 인 덴 테이션, X-선 CT, FTIR, 및 SEM을 사용하여

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.

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.

원자 힘 현미경을 사용하여 폐 조직의 미세 기계적 특성

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

생물학

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).

조기 골관절염을 검출하기 위한 원자력 현미경 검사법 적용

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.

라이브 셀에서 동시 레이저 스캐닝 공초점 및 원자력 현미경 검사(Conpokal) 근처

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.

연질 물질 나노인덴테이션을 위한 실험 및 데이터 분석 워크플로우

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.

인간 관절 연골 외식물에 대한 원자력 현미경 기반 미세 압입의 실제 문제 해결

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.