우리는 표면에 바인딩 세포에 Mingzhai Sun에서 유용한 조언을 인정합니다. 우리는 귀중한 토론 네드 윈그린 및 Zemer Gitai 감사드립니다. 이 연구는 보건 부여 P50GM07150 국립 연구소, 국립 과학 재단 (National Science Foundation) 경력 보너스 PHY - 0844466과 알프레드 P. 슬로안 재단에 의해 지원되었다.

<ol><li> 지수 상 (OD가 = 0.2-0.4)로 Luria - Beltrani 국물 (LB) 매체 E. 대장균 세포를 성장. </li><li> filamentous 성장을 유도 15 분 세팔렉신 50 μg / ML과 보충 LB의 문화를 성장하고 원심 분리를 통해 5 번하여 문화를 집중. </li><li> 흐름 챔버로 물에 희석 1 % polyethylenimine을 흐르는하여 PEI - 코팅 coverslip을 확인하고, 5 분 부화 후 물로 씻으십시오. </li><li> 챔버에 집중 세포 배양을 흐름, 그리고 무소속의 세포를 제거하는 3 분 후 LB 및 세팔렉신 (50μg/mL)의 혼합물로 세탁. </li><li> 37 챔버를 품어 ° C가에 대한 연결된 세포 광학 트래핑 악기에 배치하기 전에기를 30 분 - 1 시간. </li><li> 30 분 물에 희석 0.1 % polylysine에서 0.5 - μm의 지름의 폴리스티렌 비즈 (뱅스 연구소)을 잠복기로 polylysine - 코팅 구슬을 만듭니다. 다음 비즈에게 3 번 씻은 후 물에 resuspend. </li><li> 세팔렉신 (50μg/mL)와 LB에 두 요소에 의해 비드 솔루션을 희석하고, 흐름 챔버로를 추가합니다. </li><li> 광학 부동 구슬을 함정과 세포의 자유로운 끝에 그것을 만져. (우리는 샘플의 움직임을 제어하는​​ 미친 도시 연구소 압전 단계를 사용합니다.) 적합한 전지 / 구슬 조합이 발견되면, 무소속의 구슬을 제거 LB (50μg/mL)에 세팔렉신와 챔버를 세척. </li><li> 셀 및 기록 힘 - 변위 데이터를 절곡 세력을 적용하는 맞춤 작성된 LabView 프로그램을 실행합니다. 프로그램 세부 정보 <ol class="lalpha"><li> 래스터는 감지 레이저 빔 및 녹화 3D PSD 전압 신호 내에 첨부된 비드를 검색하여 검출기 응답을 보정합니다. </li><li> 현미경 이미지의 세포 긴 축을 식별합니다. </li><li> 세포 긴 축에 직각 방향으로 단계에서 세포로 이동합니다. </li><li> 광학 트랩에서 거리 이동과 변위를 기록합니다. </li><li> 이전에 측정된 트랩 강성을 사용하여 강제로 적용 변위 변환 및 팁 변위 및 파일 강제 저장합니다. </li></ol></li></ol> 성공에 비밀 : 8 단계에서), 하나는 잘 정의된 붙어 끝에있는 세포를 찾을 수 있어야합니다. 일부 세포는 단지 다른 끝에 하나의 팁 및 굽힘 강제에 붙어있는 것은 전체 셀 pivoting보​​다는 굽힘 발생합니다. 적당한 쌍을은 조이스틱 제어 스테이지 모션을 사용하여 수동으로 신속하게 각각의 세포를 굽힘에 의해 발견됩니다. 대표 결과 : <img src="/files/ftp_upload/2012/2012fig1.jpg" alt="그림 1" /> 그림 1.이 그림은 하나의 세포에 대한 힘 - 변위 데이터를 보여줍니다. 이 라인의 경사가 세포의 굽힘 강성이다.

<ol>
	<li>Janmey, P. A., McCulloch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics:+integrating+cell+responses+to+mechanical+stimuli.">Cell mechanics: integrating cell responses to mechanical stimuli.</a> Annu Rev Biomed Eng. 9, 1-34 (2007).</li><li>Morris, D. M., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+biomechanical+understanding+of+whole+bacterial+cells.">Toward a biomechanical understanding of whole bacterial cells.</a> Annu Rev Biochem. 77, 583-613 (2008).</li></ol>

이 문서의 비디오 제작은 이러한 연구에 사용되는 악기를 생산 매드 도시 연구소에 의해 후원되었다.

여기에 제시 프로토콜은 양적 세균 세포의 굽힘 특성을 측정하기 위해 설계되었습니다. 실험 설정은 filamentously 성장을 만들 수있는 막대 모양의 세포에 적용할 수 있습니다. 우리는 성공적으로 E.의 굽힘 강성에 cytoskeletal 필라멘트의 효과를 조사하기 위해이 설정을 사용해야 대장균 세포. 이 같은 기술은 세포의 전반적인 굽힘 강성을 결정하는 압력, 셀 벽의 강성 및 기타 세포 구성 요소의 역할을 평가하는 데 사용할 수 있습니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>cephalexin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4895-5G</td>
<td>&nbsp;</td>
<tr>
<td>polyethylenimine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>181978-5G</td>
<td>&nbsp;</td>
<tr>
<td>polylysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>0.5-&mu;m-diameter polystyrene beads</td>
<td>&nbsp;</td>
<td>Bangs Laboratory</td>
<td>PS03N</td>
<td>&nbsp;</td>
<tr>
<td>Nano-LP Series nanopositioning system</td>
<td>&nbsp;</td>
<td>Mad City Labs</td>
<td>NanoLP series</td>
<td><a href="http://www.madcitylabs.com/nanolpseries.html">http://www.madcitylabs.com/nanolpseries.html</a></td>
</tr>		</tbody>
		</table>
		</div>

measuring the bending stiffness of bacterial cells using an optical trap

우리는 filamentous 막대 모양의 박테리아의 굽힘 강성을 측정하기위한 프로토콜을 개발했습니다. 미군은 광학 트랩, 높은 강도 레이저가 현미경의 목표 렌즈에 의해 아주 작은 지점에 초점을 맞춘 때 형성 빛이 만든 미세한 입체적인 온천과 함께 적용됩니다. 세포를 구부 위해, 우리는 먼저 화학 처리 coverslip에 사는 박테리아를 바인딩합니다. 이러한 세포의 성장에 따라, 세포의 중간 coverslip에 바인딩 남아 있지만, 성장 끝나는이 구속 무료입니다. 마약 세팔렉신와 filamentous 성장을 유도함으로써, 우리는 다른 끝이 무소속과 굽힘 세력의 영향을 유지하면서 세포의 한쪽 끝을은 표면에 붙어어진 세포를 식별할 수 있습니다. 굽​​힘 힘이 그러면 성장 세포의 팁에 polylysine - 코팅 비드를 바인딩하여 광학 트랩과 함께 적용됩니다. 힘 및 비드의 변위 모두 기록되고 세포의 굽힘 강성이 관계의 기울기집니다.

Watch this Scientific Journal Video about Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap at JoVE.com

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

우리는 세포 굽힘 강성을 측정 광학 트랩과 커버 - 미끄럼 방지 표면에 부착된 굽힘 filamentous 박테리아 세포에 대한 프로토콜을 제시한다.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

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

measuring-bending-stiffness-bacterial-cells-using-an-optical

Research

JoVE Journal

Biology

12.0K 조회수.  Princeton University. 우리는 세포 굽힘 강성을 측정 광학 트랩과 커버 - 미끄럼 방지 표면에 부착된 굽힘 filamentous 박테리아 세포에 대한 프로토콜을 제시한다.

비디오: Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

현미경의 올바른 관리 및 청소

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

연구

생물학

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

라이트 현미경의 주요 구성 요소

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

세균성 세포의 전자 Cryotomography

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

아크릴 아미드 기반 Hydrogels을 사용하여 세포 기능에 매트릭스 강성의 영향을 공부

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

싱글 생활 세포에서 mRNA의 전사의 속도론을 측정

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

에 박테리아의 효과를 측정<em&gt; C. 엘레</em계란 보존 분석을 사용하여&gt; 행동

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

삼투 물 투수 계수 (P 측정<sub&gt; F</sub&gt;) 구형 세포 : 예를 들어 식물 절연 원형질체

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

마우스 렌즈의 압축 강성을 평가하는 유리 Coverslips는 순차적으로 응용 프로그램 : 스트레인과 형태 학적 분석

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

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

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

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

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

의 강성 측정<em&gt; 전의 VIVO</em원자 힘 현미경을 사용하여&gt; 마우스 대동맥

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