Name: atomic force microscopy imaging and force spectroscopy of supported lipid bilayers
Uploaded: 2015-07-22T14:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers at JoVE.com

עבודה זו נתמכה על ידי אגודת מקס פלנק, המרכז הגרמני לחקר הסרטן, אוניברסיטת טובינגן, וBundesministerium für Bildung und Forschung (מענק לא. 0,312,040). אנו מודים אדוארד הרמן שעזרו לנו להפוך את ניתוח נתוני כוח עקומת וד&quot;ר יעקב Suckale לקריאה זהירה של כתב היד הזה.

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המחברים מצהירים שאין להם אינטרסים כלכליים מתחרים.

SLBs המורכב מdOPC: SM: חול (2: 2: 1) נוצרו על נציץ לאחר ספיחת שלפוחית ​​וקרע שנגרם על ידי סידן כלורי. הרכב שומנים בדם זה מופרד לשלבי ד L וo L. שלב o L מועשר בsphingomyelin וכולסטרול, והוא / (איור 1 א) נוזל צמיג פחות יותר מאשר שלב ד L 11. הפרדת o L משל?...

מיקרוסקופית כוח אטומי (AFM) מייצר תמונה של פני השטח על ידי סריקה על פני שטח של המדגם באמצעות שלוחה עם קצה חד מאוד 1. התנועה של שלוחה חוקרת את הטופולוגיה של מדגם המשטח. AFM כבר מיושם באופן נרחב למולקולות ביולוגיות - כוללים חלבונים, DNA, וקרומים, בשל צדדיות שלה בניתוח דגימות קבועות באוויר או מצב כמעט טבעי בנוזל 2-5. מלבד יכולת ההדמיה ברזולוציה הגבוהה שלה בטווח ננומטר, שלוחה AFM פועלת כקפיץ לחקור כוחות אינטראקציה (הידבקות ודחייה) ותכונות מכאניות של המדגם 5,6. התופעה זו ידועה בכח ספקטרוסקופיה. במצב זה, החללית מתקרבת ראשונה המדגם ומפעילה כוח על זה, ואז הוא חזר עד שהוא מאבד קשר עם המדגם (איור 1 א). עקומות שנוצרו להראות כוח כפונקציה של מרחק של שלוחה לשתי האפליקציהמקק והכחשה. מספר נכסים כוללים מודולוס האלסטיות למדוד את הנוקשות של חומר, וניתן לגזור כוחות ההידבקות. bilayers שומנים הנתמכים הם קרומים ביולוגיים מודל שוכבים על גבי תמיכה מוצקה - בדרך כלל נציץ,, סיליקה הזכוכית בורוסיליקט התמזגה, או חמצון סיליקון 7. הם מוכנים תוך שימוש בטכניקות שונות כמו בתצהיר שלפוחית, שיטת אנגמיור-באגט וספין-ציפוי 8,9. ההדמיה AFM נעשתה שימוש כדי לעקוב אחר היווצרות bilayers הנתמכות הבאות 10, ולחקור מבנים שונים שהוקמו על ידי קרומים של קומפוזיציות שונות 11-15. ביצוע כוח ספקטרוסקופיה על תוצאות bilayers נתמכות בשיא בעקומת הגישה. שיא זה מצביע על הכוח הדרוש כדי לחדור את bilayer, והוא נקרא כוח פריצת דרך. עובי bilayer גם ניתן למדוד באמצעות כוח העקומה 6. כוח הפריצה האופייני של bilayersטווח שבין 1-50 ננ 6. מאפיינים אלה תלויים באריזת שומנים (שלב נוזלי או ג&#39;ל) ומבנה (אורך שרשרת acyl ומידת unsaturation) ושונה על ידי סוכני קרום פעיל 16. התאוריה מאחורי הקרע הוסברה 17 ופרמטרים ניסיוניים אחרים כגון רכות שלוחה, רדיוס קצה ומהירות גישה משפיעים גם על כוח פריצת הדרך 15,16,18. ספקטרוסקופיה הכוח נעשתה שימוש כדי לנתח את התכונות של שלבים שונים שומנים 11,19, שינויי הרכב תלוי 12,20, כמו גם השפעות של מולקולות ביולוגיות אחרות, כמו פפטידים, ביציבות של הקרום 21. הנטייה השטוחה של bilayers נתמכת היא יתרון עבור שילוב AFM עם שיטות אחרות כגון מיקרוסקופיה תהודת 22 וקרינת המשטח plasmon 11,19 לאפיין מבנה ותכונות של ממברנות טובות יותר. פרוטו וידאו מפורט זהcol נועד להכין bilayers שומנים נתמכים באמצעות תצהיר שלפוחית ​​ולנתח אותם עם AFM והכח ספקטרוסקופיה. בעוד שלפוחית ​​בגדלים שונים ניתן להשתמש כדי להכין bilayers, פרוטוקול זה מתמקד בשלפוחית ​​unilamellar קטנה וגדולה. bilayers נתמכת ששלב להפריד לנוזל הורה (o L) ושלבים נוזליים סדר (ד L) התאפיינה 11,15. הקרום מורכב מdi-oleoyl-phosphatidylcholine (dOPC), sphingomyelin (SM), וכולסטרול (חול) בשעה 2: יחס 1: 2. דגמי הרכב זה רפסודות השומנים בדם, אשר הציעו להתנהג כפלטפורמות חשובות לסחר בחלבון ומיון, איתות תא ותהליכים תאיים אחרים 23,24.

<table><tbody><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine</td><td>Avanti Polar Lipids, Inc.</td><td>850375P</td><td>Comes as lyophilized powder in sealed vials. Dissolve all powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20&amp;nbsp;&amp;deg;C. Visit &lt;a href=&quot;http://www.avantilipids.com/index.php?option=com_content&amp;amp;view=article&amp;amp;id=1679&amp;amp;Itemid=398&quot;&gt;here&lt;/a&gt; for more information on storage and handling.</td></tr><tr><td>Sphingomyelin (Brain, Porcine)</td><td>Avanti Polar Lipids, Inc.</td><td>860062P</td><td>Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20&amp;nbsp;&amp;deg;C. Visit &lt;a href=&quot;http://www.avantilipids.com/index.php?option=com_content&amp;amp;view=article&amp;amp;id=1679&amp;amp;Itemid=398&quot;&gt;here&lt;/a&gt; for more information on storage and handling.</td></tr><tr><td>Cholesterol</td><td>Avanti Polar Lipids, Inc.</td><td>700000P</td><td>Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20&amp;nbsp;&amp;deg;C. Visit &lt;a href=&quot;http://www.avantilipids.com/index.php?option=com_content&amp;amp;view=article&amp;amp;id=1679&amp;amp;Itemid=398&quot;&gt;here&lt;/a&gt; for more information on storage and handling.</td></tr><tr><td>Sodium chloride (NaCl), 99.8%</td><td>Carl Roth GmbH + Co. KG</td><td>9265.1</td><td></td></tr><tr><td>Potassium chloride (KCl), 88%</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Sodium hydrogenphosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;), &gt;99%</td><td>AppliChem GmbH</td><td>A1046</td><td></td></tr><tr><td>Potassium dihydrogenphosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;), 99%</td><td>Carl Roth GmbH + Co. KG</td><td>3904.1</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;), molecular biology grade</td><td>AppliChem GmbH</td><td>A4689</td><td></td></tr><tr><td>HEPES, molecular biology grade</td><td>AppliChem GmbH</td><td>A3724</td><td></td></tr><tr><td>Glass coverslip, 24&amp;nbsp;x&amp;nbsp;60&amp;nbsp;mm, 1&amp;nbsp;mm thickness</td><td>Duran Group</td><td>2355036</td><td></td></tr><tr><td>Punch and Die Set</td><td>Precision Brand Products, Inc</td><td>40105</td><td></td></tr><tr><td>Optical Adhesive</td><td>Norland Products, Inc.</td><td>NOA 88</td><td>Liquid adhesive that hardens when cured under long wavelength UV light.&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Mica blocks</td><td>NSC Mica Exports Ltd.</td><td></td><td>These are mica pieces at least 1 sq. inches in area and thickness ranging from 0.006 inches to 0.016 inches. They are cut to a specific size by the company for shipping. Small mica discs can be punched from the mica blocks using the punch and die set.&amp;nbsp; Always handle mica with gloves or tweezers.</td></tr><tr><td>Laboratory Equipment Grease</td><td>Borer Chemie AG</td><td>Glisseal N</td><td></td></tr><tr><td>Liposome Extruder</td><td>Avestin</td><td>LiposoFast-Basic</td><td>As an alternative one can also look at offers from Northern Lipids, Inc.</td></tr><tr><td>Adhesive Tape</td><td>3M</td><td>Scotch(R) Magic (TM) Tape 810 (1-inch)</td><td></td></tr><tr><td>Bath Sonicator</td><td>Bandelin Sonorex Digitec</td><td>DT-31</td><td>No heating, Frequency: 35 kHz, Ultrasonic Peak Output: 160 W, HF Power: 40 W. (&lt;a href=&quot;http://www.bandelin.com/datenblaetter/dt/DT_31_H_1798d_DE_GB_FR_BANDELIN.pdf&quot;&gt;Data sheet&lt;/a&gt;)</td></tr><tr><td>Silicon Nitride AFM Cantilever&amp;nbsp;</td><td>Bruker AFM Probes</td><td>DNP-10</td><td>Each cantilever has four tips and their nominal tip radius is 20 nm (with possible maximum at 60&amp;nbsp;nm). Based on the specifications, we use tip D with resonance frequency of 18 kHz, and nominal spring constant of 0.06 N/m.</td></tr><tr><td>AFM</td><td>JPK</td><td>JPK Nanowizard II mounted on Zeiss Axiovert 200</td><td></td></tr></tbody></table>

atomic force microscopy imaging and force spectroscopy of supported lipid bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

bilayers נתמכת שומנים מורכבים מdOPC: SM: חול (2: 2: 1) היו צילם בAFM (איור 2 AC). בגלל הרכב השומנים בדם, o L SM / חול עשירה ושלבי ד L dOPC העשירים נצפו. פרופיל הגובה מהדמית AFM יכול לספק מידע חשוב על מבנה הקרום. על ידי הסתכלות על פרופיל הגובה, עובי bilayer ניתן למדוד בנוכחות של...

Watch this Scientific Journal Video about Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers at JoVE.com

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

כוח האטומי מיקרוסקופית הדמיה וחיל ספקטרוסקופיה של bilayers שומנים הנתמכים

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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Research

JoVE Journal

Bioengineering

14.6K Views.  Interfaculty Institute for Biochemistry. We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Video: Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

כוח ספקטרוסקופיה של מולקולות חלבון יחיד באמצעות מיקרוסקופ כוח אטומי

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Biochemistry

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

חוקר מולקולה בודדת הדבקה על ידי הכוח האטומי ספקטרוסקופיה

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Engineering

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

הדמיה ברזולוציה משנה ננומטר עם מיקרוסקופית כוח אטומי משרעת אפנון בנוזל

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

חוקר מערכות קולטן ליגנד של Cellulosome עם המולקולה בודדת מבוססת AFM חיל ספקטרוסקופיה

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

הכנת מיכה נתמכת יפידים bilayers לרזולוציה גבוהה מיקרוסקופיה אופטית הדמיה

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

המולקולה בודדת מיקרוסקופ פלואורסצנטי במישורים נתמך bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Visualizing Solution Structure at Solid-Liquid Interfaces using Three-Dimensional Fast Force Mapping

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the solution conditions such as ion concentrations, pH, ligands, and trace additives, as well as the underlying crystallography and chemistry. In this context, three-dimensional fast force mapping (3D FFM) has emerged as a promising tool for investigating solution structure at interfaces. This capability is based on atomic force microscopy (AFM) and allows the direct visualization of interfacial regions in three spatial dimensions with sub-nanometer resolution. Here we provide a detailed description of the experimental protocol for acquiring 3D FFM data. The main considerations for optimizing the operating parameters depending on the sample and application are discussed. Moreover, the basic methods for data processing and analysis are discussed, including the transformation of the measured instrument observables into tip-sample force maps that can be linked to the local solution structure. Finally, we shed light on some of the outstanding questions related to 3D FFM data interpretation and how this technique can become a central tool in the repertoire of surface science.

Here, we present a protocol for using three-dimensional fast force mapping - an atomic force microscopy technique - for visualizing solution structure at solid-liquid interfaces with the subnanometer resolution by mapping the tip-sample interactions within the interfacial region.

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the ...

Chemistry

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.

מיקרוסקופ כוח אטומי ננו-הזחה מבוססת Cantilever: מדידות תכונות מכניות בקנה מידה ננומטרי באוויר ובנוזל

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

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

הרכבה של חיקוי תאים מודלים נתמכים ומושעים של בולי עץ לחקר אינטראקציות מולקולריות

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

בליטה מיקרוסקופ כוח: שיטה לכמת כוחות שפותחה על ידי בליטות תא

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...