저자는이 프로젝트와의 도움을 엘레나 Lomakina, 리처드 Bauserman, 마가렛 젊은, 셰이 Vaknin, 제시카 스나이더, 크리스 Striemer, Nakul Nataraj, 숙취 리 정, Tejas Khire, 그리고 에릭 램을 감사드립니다. 이 프로젝트는 NIH # PO1 HL 018,208로 운영되었습니다.

1. 방법 1.1 셀 흐름 상공 회의소 세포가 1.0 파 (10 dyn / cm 2)의 전단에 따라 성장 후 정신 병원 MFP3D AFM (산타 바바라, CA)에 직접 전달 될 ​​수 있도록 그림 1에 표시된 흐름 챔버는, 건설되었습니다. <ol><li> 15 분에 한 후 증류수로를 세척 : 흐름 챔버 먼저 피라냐 솔루션 (H 2 O 2 3시 1분 H 2 SO 4) 유리 슬라이드를 청소하여 실험을 준비했습니다. 그들은 그 다음에 구운 된 건조하고 진공 증착 챔버에서 실란 aminopropyl의 triethoxy (APTES)을 코팅. </li><li> 실리콘 가스켓은 실루엣 SD 절단 도구를 사용하여 절단되었습니다. 이것은 우리가 잘게 세포의 성장 동안 유량 및 전단 응력의 제어를위한 흐름 챔버 크기를 제어 할 수있었습니다. 일반적으로 채널은 0.4 mm의 실리콘의 시트에서 긴 19mm에 폭 6.4 mm를 잘라되었습니다. 장군에 필요한 유량테 1.0 파 (10 dyn / cm 2)의 전단 응력은 방정식과 사각형 채널에 판상 흐름을 가정 계산됩니다 : </li></ol><img alt="수식 1" fo:content-width="0.737in" fo:src="/files/ftp_upload/50163/50163eq1highres.jpg" src="/files/ftp_upload/50163/50163eq1.jpg" /> Q는 유량이고, τ는 전단 응력이며, μ는 매체의 점도이다 1.0 MPA (0.01 dyn * 초 / cm 2)로 여기 가정, H는 높이이며, w는 유동 챔버의 폭입니다 . <ol start="3"><li> 흐름 챔버의 상단 부분은 세포 배양 접시에있는 가스켓과 정렬과 자기 링으로 고정되었습니다. 조립체는 살균을 위해 이소 프로필 알코올 (IPA)로 가득했다. </li><li> 전체 흐름 시스템은 조립했습니다. 세포 배양 접시의 흐름 포트는 세 방향 밸브에 연결되었습니다. 밸브는 30 ML의 주사기를 엽니 다 연결되었습니다s입니다. IPA 그 다음 4퍼센트 태아 송아지 혈청 (FCS)와 맥코이의 중간의 30 ML에 세척 된 시스템을 통해 플러시되었습니다. 이 시스템은 다음 VEC 기술 세포의 성장 매체 20 ML로 가득했다. 표지는 주사기의 정상에 위치했다. 캐치 저장소의 주사기 캡 피드 저수지로 매체를 이동하는 장소에서 바늘했다. 멸균 0.2 μm 필터는 시스템의 오염을 방지하기 위해 덮개의 공기 유입구에 부착되었다. 흐름 챔버는 세포 심는 준비했습니다. </li></ol> 1.2 세포 배양 <ol><li> 인간 배꼽 정맥 내피 세포 (HUVEC의) 성장 매체는 VEC 기술에서 구입 (Rensselaer, NY)과 T25 플라스크에 합류로 성장했다. </li><li> 성장 매체는 술병과 2.5 %의 트립신 2 ML을 발표 세포 monolayer에서 제거되었습니다. 세포가 솔루션에 있던되면 trypsinization은 플라스크에 세포 배양 매체의 10 ML를 추가로 침묵했다. </li><li> 일전자 세포 현탁액은 5 분 동안 centrifuged되었고 표면에 뜨는이 삭제되었습니다. 세포는 유동 챔버 내로 분사에 대한 세포 배양 매체 (혈청을 포함) 1 ML에 resuspended되었습니다. </li><li> 세포 현탁액은 (0.5 ML ~ 50,000 세포) 주사기에로드하고 세 방향 밸브를 통해 흐름 챔버에 주입했다. </li><li> 세포는 흐름이 시작되기 전에 2 시간에 정착하고 유리 기판을 준수 할 수 있었다. 세포는 37 보육의 흐름에 따라 성장했습니다 ° 1-5일에 대한 C 합류 때까지. </li></ol> 1.3 캔틸레버 준비 및 셀 들여 쓰기 <ol><li> Tipless AFM의 cantilevers (NanoWorld, 스위스)은 5 분에 질산에 세척하고 기상 증착 챔버에서 실란 aminopropyl의 triethoxy (APTES)와 기능화되었습니다. </li><li> 5 행크의 버퍼 소금 솔루션에 (HBSS) 준비되었습니다 무게 NHS-sulfo-LC-비오틴의 MG / ML의 솔루션입니다. cantilevers는 conju 15 분 동안 솔루션에 잠겨 있었다게이트 N-Hydroxysuccinimide (NHS) 화학으로 실란. </li><li> 비오틴 무료 매체의 솔루션은 12 시간에 streptavadin 구슬 200 μl와 VEC 기술 세포 배양 매체 (혈청 포함)의 20 ML을 잠​​복기에 의해 만들어졌습니다. 구슬은 자석 매체에서 제거되었고 매체가 0.22 μm 살균 필터를 통해 필터링되었습니다. </li><li> 흐름 챔버는 세포 배양 접시에서 제거되었고 셀은 37 ° C 비오틴 - 무료 매체에 세척했다. </li><li> 비오틴 무료 매체 1 ML에 streptavidin으로 코팅 2.4 μm 구슬 1 μl의 주식 솔루션을 준비했고, 주식의 100 μl은 세포 배양 접시에 추가되었습니다. </li><li> Streptavidin 비즈는 구슬, retracting 옆에있는 유리 표면에 팁을 착륙 비드 위에 캔틸레버의 꼭대기를 위치 한 다음 구슬과에 휴식 몇 초에 캔틸레버를 눌러 캔틸레버를 포착했다. </li><li> 캔틸레버의 감도 측정했습니다맨 유리의 지역에 압입 및 전압의 함수로 팁 편향을 설정하는 곡선의 기울기를 사용하여 D. </li><li> 캔틸레버의 봄 상수 다음 MFP3D 소프트웨어의 열 보정으로부터 계산되었다. </li><li> 그림 2와 같이 보정 된 캔틸레버는 다음 샘플을 들여 쓰기하는 데 사용되었다. 이 2.4 μm의 구슬은 부드러운 glycocalyx 층의 기계적 성질이 감지 할 수 있도록 세포 표면에 큰 접촉 면적을 제공합니다. 캔틸레버는 세포 핵 및 세포로 끝이 약 3 μm 세포 표면 위의 캔틸레버 높이를 설정하는 데 사용되었다 부드러운 접근 방식 근처에있는 셀 위에 위치했다. 소프트웨어는 7 NN의 최대 힘 1 μm / 초의 속도로 20 반복 들여 쓰기로 설정되었습니다. 약 6 초는 연속 연락처 사이에 경과. 그것은 glycocalyx의 시간에 따라 달라 속성 테스트 들여 쓰기의 다양한 속도를 사용할 수 있지만이 초기에<html> 실험은 단일 들여 쓰기 속도는 (1 μm / 초)가 사용되었습니다. </li></ol> 2. 들여 쓰기 이론 반경 R의 영역과 반 공간 탄성에 들여 쓰기는 들여 쓰기의 힘, F가 방정식에 의해 주어집니다 헤르츠 이론을 사용하여 설명 될 수있다 : <img alt="수식이" fo:content-width="1.157in" fo:src="/files/ftp_upload/50163/50163eq2highres.jpg" src="/files/ftp_upload/50163/50163eq2.jpg" /> δ는 어디 들여 쓰기 깊이와 E *는 테스트중인 자료 (그림 3)의 감소 계수입니다. 균일 한 탄성 반 공간을 impinging 끝도없이 치열한 indenter의 경우, E의 *는 방정식에 의해 주어집니다 : 3/50163eq3.jpg &quot;/&gt; E가 어디 탄성 계수와 ν는 재료의 푸 아송 비율입니다. 폴리머 필름과 최근의 작업은 박막 1의 계수와 두께를 결정하기위한 두 레이어 모델의 개발에 영감을하고 있습니다. 우리는 전지 본체의 표면에 균일 한 박막 부드러운 필름 glycocalyx 치료하여 세포 생물학에이 모델을 적용하고 있습니다. 이 모델을 사용하여 시스템의 감소 계수가된다 : <img alt="수식 4" fo:content-width="2.85in" fo:src="/files/ftp_upload/50163/50163eq4highres.jpg" src="/files/ftp_upload/50163/50163eq4.jpg" /> E GC는 glycocalyx의 계수이고, E 셀의 전지 본체의 계수이며, P, Q와 N은 경험적으로 폴리머에 맞는에서 결정되었으며, 및 z는 방정식에 의해 주어집니다 상수 : <p class="jove_content" fo:keep- together.within 페이지 = &quot;항상&quot;&gt; <img alt="수식 5" fo:content-width="1.257in" fo:src="/files/ftp_upload/50163/50163eq5highres.jpg" src="/files/ftp_upload/50163/50163eq5.jpg" /> t는 glycocalyx 층의 두께입니다. 이러한 매개 변수의 개략적 인은 그림 3에 표시됩니다. 모델은 강성 기판 (1)에 박막의 계수와 두께를 결정하는 정확한 방법 표시되었습니다. 그림 4와 같이이 방정식은, 내피 glycocalyx의 계수와 두께를 결정하는 셀에 들여 쓰기에서 얻은 곡선에 맞게 사용할 수 있습니다.

<ol>
	<li>Clifford, C., Seah, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+measurement+of+young's+modulus+for+compliant+layers+on+stiffer+substrates+including+the+effect+of+poisson's+ratios.">Nanoindentation measurement of young's modulus for compliant layers on stiffer substrates including the effect of poisson's ratios.</a> Nanotechnology. , (2009).</li><li>Gouverneur, M., Spaan, J. A. E., Pannekoek, H., Fontijn, R. D., Vink, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shear+stress+stimulates+incorporation+of+hyaluronan+into+endothelial+cell+glycocalyx.">Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx.</a> Am. J. Physiol. Heart. Circ. Physiol. 290 (1), 458-452 (2006).</li><li>Hocde, S. A., Hyrien, O., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+distribution+relative+to+neutrophil+surface+topography+assessed+by+tirfm.">Cell adhesion molecule distribution relative to neutrophil surface topography assessed by tirfm.</a> Biophysical Journal. 97 (1), 379-387 (2009).</li><li>Lipowski, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+as+a+barrier+to+leukocyte+adhesion+and+its+mediation+by+extracellular+proteases.">The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.</a> Annals of biomedical engineering. 40 (4), 840-848 (2012).</li><li>Lu, L., Oswald, S. J., Ngu, H., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+actin+stress+fibers+in+living+cells.">Mechanical properties of actin stress fibers in living cells.</a> Biophysical Journal. 95 (12), 6060-6071 (2008).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+surface+layer.">The endothelial surface layer.</a> Pflugers Archiv. European Journal of Physiology. 440 (5), 653-666 (2000).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophysical Journal. 87 (6), 4237-4245 (2004).</li><li>vanden Berg, B. M., Vink, H., Spaan, J. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx+protects+against+myocardial+edema.">The endothelial glycocalyx protects against myocardial edema.</a> Circulation Research. 92 (6), 592-594 (2003).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+impact+of+membrane+microtopology+on+effective+two-dimensional+affinity.">Quantifying the impact of membrane microtopology on effective two-dimensional affinity.</a> J. Biol. Chem. 276 (16), 13283-138 (2001).</li><li>Vink, H., Duling, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circulation Research. 79, 581-589 (1996).</li></ol>

관심 없음 충돌이 선언 없습니다.

우리는 두 레이어 모델과 내피 벽에 혈액에 순환 백혈구의 상호 작용을 모델링 할 수 헤르츠 이론에서 계산 값을 사용했습니다. 우리는 10 PN 부하에 따라 50 nm의 직경이 백혈구에 microvillus가 glycocalyx에 약 150 nm의 들여 쓰기 것이라는 총 두께의 일부만 계산했습니다. 이이 실험에서 측정 된 특성을 가진 glycocalyx은, 세포 세포의 상호 작용에 중요한 장벽이며 세포가 백혈구 유착 동안 부착 캐스케이드...

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 맥코이의 중간 </td><td> Gibco </td><td> 16600-082 </td><td></td></tr><tr><td> 태아 혈청 </td><td> Hyclone </td><td> SH30070 </td><td></td></tr><tr><td> 내피 세포 성장 매체 </td><td> VEC 기술 </td><td> MCDB-131 </td><td></td></tr><tr><td> 풀링 된 인간 배꼽 정맥 내피 세포 </td><td> VEC 기술 </td><td> PHUVEC/T-25 </td><td></td></tr><tr><td> 황산 </td><td> JT 베이커 </td><td> 9681-02 </td><td></td></tr><tr><td> 과산화수소 </td><td> VWR </td><td> BDH3742-1 </td><td></td></tr><tr><td> (3-aminopropyl) triethoxysilane </td><td> 올드리치 </td><td> 440140-100ML </td><td></td></tr><tr><td> 이소 프로필 알코올 </td><td> VWR </td><td> BDH8999-4 </td><td></td></tr><tr><td> 트립신 </td><td> Cellgro </td><td> 25-054-C1 </td><td></td></tr><tr><td> 행크의 버퍼 소금 솔루션 </td><td> Gibco </td><td> 14175-095 </td><td></td></tr><tr><td> sulfo-NHS-LC-비오틴 </td><td> 열 과학 </td><td> 21,335 </td><td></td></tr><tr><td> Streptavadin 비즈 </td><td> Dynabeads </td><td> 112.06D </td><td></td></tr><tr><td> MFP - 3D AFM </td><td> 망명 연구 </td><td></td><td></td></tr><tr><td> Tipless Cantilevers </td><td> Nanoworld </td><td> ARROW-TL1-50 </td><td></td></tr><tr><td> 실루엣 SD </td><td> Quickutz </td><td> 실루엣-SD </td><td></td></tr><tr><td> 실리콘 고무 </td><td> Stockwell Elastomerics </td><td> SE50-RS </td><td></td></tr><tr><td> 30 ML의 주사기 </TD&gt; <td> 벤톤 디킨슨 </td><td> 309650 </td><td></td></tr><tr><td> 18 게이지 바늘 </td><td> 벤톤 디킨슨 </td><td> 305196 </td><td></td></tr><tr><td> 확장 세트 </td><td> Hospira </td><td> 4429-48 </td><td></td></tr><tr><td> 4 방향 밸브 </td><td> Teleflex </td><td> W21372 </td><td></td></tr><tr><td> 여성 / 남성 포트 캡 </td><td> 스미스의 의료 </td><td> MX491B </td><td></td></tr><tr><td> 연동 운동의 펌프 </td><td> 왓슨-Marlow </td><td> 401U / D </td><td></td></tr><tr><td> 연동 운동의 배관 </td><td> 왓슨-Marlow </td><td> 903.0016.016 </td><td></td></tr><tr><td> 무균 필터 </td><td> 막 생활 과학 </td><td> 4652 </td><td></td></tr></tbody></table>

quantifying the mechanical properties of the endothelial glycocalyx with atomic force microscopy

백혈구 및 백혈구 캡처하는 동안 혈관 벽의 상호 작용에 대한 우리의 이해는 내피 표면 층의 기계적 성질의 불완전한 이해에 의해 제한됩니다. 이것은 백혈구의 접착 분자가 표면 지형 3 상대적으로 비 균일하게 분포되어 알려져 있습니다, 그 지형이 다른 표면 9 접착제 결합 형성을 제한, 그 생리 접촉 힘 (≈ 5.0 - microvillus 당 10.0 PN)는 등으로 microvilli을 압축 할 수 있습니다 반대 표면 3, 7 분자의 접근성을 증가 자신의 휴식 길이의 세 번째로 조금. 우리는 두 계층 구조, 상대적으로 강성 전지 본체뿐만 아니라 glycocalyx, luminal 표면 6 부드러운 보호 설탕 코팅으로 내피를 고려합니다. 그것은 glycocalyx는 내피 표면 4 백혈구의 접착을 줄이기 위해 장벽의 역할을 할 수 것으로 나타되었습니다.이 보고서에서 우리는 내피 기계 강성이 결합 형성에 영향을 미칠 수 방법을 이해하기 위해 내피 표면의 deformability를 해결하기 시작합니다. 정적 문화에 성장 내피 세포는 강력한 glycocalyx을 표현할 수 없지만, 생리 흐름 조건에서 성장 세포가 생체 2 관찰 대략 glycocalyx에 시작한다. 내피 세포 본체의 계수는 약 5-20 kPa 5로 원자 힘 현미경 (AFM)을 사용하여 측정되었습니다. glycocalyx의 두께와 구조는 전자 현미경 8을 사용 연구되었으며, glycocalyx의 계수는 간접적 인 방법을 사용하여 추정되었지만, 우리의 지식으로, 살아있는 세포에 glycocalyx 계수의 직접 측정 대한 출판 보고서가 없었습니다 . 본 연구에서는, 우리는 mA 자신의 glycocalyx 표현을 극대화 할 조건에서 배양 된 세포에 새로운 AFM 프로브로 만든 들여 쓰기 실험을 제시내피 glycocalyx의 계수와 두께 KE 직접 측정.

전형적인 실험에서, 20 힘 - 대 - 거리 곡선은 근처 있지만 핵 (~ 2 μm 이내)에, 일반적으로 perinuclear 지역에서, 세포의 특정 지역에서 얻었다. 곡선은 측정 기간 동안 모든 샘플 드리프트에 대한 계정에 정렬 한 후 그림 4에 도시 된 바와 같이, 캔틸레버 노이즈를 제거하는 평균했습니다. 곡선 분석하고 얇은 폴리머 필름 (1)의 계수와 두께를 결정하기 위해 개발되었습니다 두 개의...

Watch this Scientific Journal Video about Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy at JoVE.com

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

내피 glycocalyx의 기계적 특성은 AFM의 cantilevers에 마이크론 크기의 영역을 사용하여 들여 쓰기에 의해 측정되었다. 내피 세포는 glycocalyx 식을 유도하기 위해 생리 흐름 조건에서 사용자 정의 챔버에 배양 하였다. 데이터는 glycocalyx 두께와 탄성 계수를 결정하기 위해 박막 모델을 사용 분석 하였다.

원자 힘 현미경과 내피 Glycocalyx의 기계적 성질을 정량화

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

quantifying-mechanical-properties-endothelial-glycocalyx-with-atomic

Research

JoVE Journal

Bioengineering

13.1K 조회수.  University of Rochester. 내피 glycocalyx의 기계적 특성은 AFM의 cantilevers에 마이크론 크기의 영역을 사용하여 들여 쓰기에 의해 측정되었다. 내피 세포는 glycocalyx 식을 유도하기 위해 생리 흐름 조건에서 사용자 정의 챔버에 배양 하였다. 데이터는 glycocalyx 두께와 탄성 계수를 결정하기 위해 박막 모델을 사용 분석 하였다.

비디오: Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

원자 힘 현미경에 의해 영상에 대한 세균 고정

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

연구

생체공학

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

원자 힘 현미경을 사용하여 재조합 DNA와 단백질 단지의 시각화

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

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, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

견인 힘 현미경의 세포 배양 표면 근처 지역화 기자 형광 구슬을위한 새로운 방법

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

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.

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

원자 힘 현미경 이미징 및 지원 지질 이중층의 힘 분광학

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

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

입자 추적 Microrheology에 의한 바이오 필름의 기계적 성질의 제자리 매핑에서

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

물 정화를위한 막으로 방오 특성을 가진 히드로 겔의 합성

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxial 기계 Characterizations 것 심장 밸브의

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

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 ...