이 연구는 생물학과 환경 연구의 사무실, 에너지 미국학과로와 버지니아 연방 보건 연구위원회에서 부여 기금을 후원합니다. 오크 리지 국립 연구소는 계약 번호 DE - AC05 - 00OR22725에 따라 에너지의 미국학과에 대한 UT - 바텔, LLC에 의해 관리됩니다.

1. 미카 준비 : <ol><li> AFM 현미경 (약 22 × 30mm)에 맞게 필요한 크기로 가위로 운모를 (전자 현미경 과학) 잘라. </li><li> 클리브가 양쪽에있는 미카가 일반적으로 바깥쪽 레이어를 제거하는 테이프를 사용하는 것은 부드럽고 깨지지 레이어가 남을 때까지. </li></ol> 2. 젤라틴 용액의 준비 : <ol><li> 실험실 병에 증류수 100 ML을 추가합니다. </li><li> 물이 끓기 시작할 때까지 전자 레인지에 병을 열. (전자 레인지를 사용할 수없는 경우, 물은 뜨거운 접시에 비커에 가열 수 있습니다.) </li><li> 젤라틴 0.5 g (를 시그마 G - 6144, G - 2625 또는 G - 2500)가 무게와 뜨거운 증류수에 추가할 수 있습니다. </li><li> 부드럽게 소용돌이는 젤라틴까지 병이 완전히 녹아있다. </li><li> 60-70 ° C.에 젤라틴 용액을 쿨 </li><li> 컷 죽습 미카가 담근 수있는에 작은 비커 (20 ML)에 약 15 ML 하거라. </li></ol> 3. 미카 코팅 : <ol><li> 예열 젤라틴 용액에 잠수함 미카 사각형과 운모가 (그림 1A) 코팅되어 신속하게 철회 등. </li><li> 코팅 후 (그림 1B의) 주변 공기의 건조에 종이 타월에 가장자리에 즉시 운모를 지원합니다. 젤라틴 코팅 운모는 최소 2 주 동안 사용할 수 있습니다. 초과 젤라틴 솔루션은 냉장 보관 간단 60-70 사이 ° C.에 대한 재고 솔루션을 재가열하여 약 한 달 동안 사용할 수 있습니다 케어는 젤라틴이 재가열하는 동안 삶은 아니라는 것을 보장하기 위해 이동해야합니다. </li></ol> 4. 젤라틴 코팅 운모에 박테리아를 장착 : <ol><li> 펠렛 세균성 문화 1 ML (0.5-600 nm의에서 1.0 OD) 800 4500 RCF에서. 세균에 따라 최소 RCF를 사용하는 것이 펠렛이 ~ 5 분 안에 세포 것입니다. </li><li> 필터링 탈이온수 또는 버퍼의 같은 볼륨에서 펠렛을 씻으십시오. </li><li> 원심 분리하여 다시 세포를 수집합니다. </li><li> nanopure 탈이온수 또는 버퍼 500 μL에 즉시 펠렛을 Resuspend. (세균성 정지 가시 AFM 이미징을위한 세포의 적절한 농도를하기 위해 흐린해야합니다.) </li><li> 피펫 팁 (그림 1C)를 사용하여 표면에 젤라틴 - 코팅 운모 및 확산에 세포 현탁액 (10-20 μL)를 적용합니다. 치료는 세균 솔루션을 적용하거나 유포하는 동안 물리적으로 피펫 팁로 젤라틴 표면을 만지지로 이동해야합니다. </li><li> 표면이 10 분 정도 휴식과 물 또는 버퍼 (그림 1 CD)의 흐름과 함께 씻어 수 있습니다. 절차 전반에 걸쳐, 샘플이 건조 허용되지 않도록하는데주의를 기울여야. </li></ol> 5. 대표 결과 : 그림 2는 젤라틴 - 코팅 운모에 장착된와 AFM에 의해 몇 군데있다 세균성 세포의 예제 이미지를 보여줍니다. 우리 실험실에서는 샘플은 일반적으로 100 μm의 검사 머리 복 PicoPlus 원자 힘 현미경을 (애질런트 테크놀로지, 템플, AZ)를 사용하여 몇 군데 있습니다. 악기는 0.5-1.0 라인 / s의의 스캔 속도로 라인 스캔 당 128-512 픽셀에서 운영하고 있습니다 일반적으로 사용되는 cantilevers는 0.01에서 0.1 NN / NM에 이르기까지 명목 스프링 상수와 Veeco 실리콘 질화물 프로브 (MLCT - AUHW, Veeco, 산타 바바라, CA)입니다. <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="그림 1"/> 그림 1. 60-70 ° C가 20 ML의 비커에 배치됩니다의 온도에서 다행. 족집게 한 켤레를 사용하면 갓 미카 사각형이 완전히 젤라틴 (A) 철회와 마이크로 원심 분리기 걸이 (B) 기대어 종이 타월에 직립 위치로 포장되어 아르 죽습. 세균성 시료의 비말은 X와 Y 방향 (C) 모두에서 pipet 팁있는 미카와 보급에 위치합니다. 샘플 적어도 10 분 동안 운모 표면에 품어 수 있도록 후, 증류수의 흐름에 샘플을 씻어. 고정이 효과가있다면 당신은 (D)의 오른쪽 패널에서 같이 미카에 불투명 자리를 볼 수 있습니다. 고정 문제가 해결되지 않았다면 당신은 왼쪽 패널에서와 같이 미카의 명확한 부분을 얻을 것이다. 비말에있는 박테리아의 농도에 미카 저울에 불투명 지점 젤라틴 처리된 운모에이 곳 저. <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="그림 2"/> E.의 그림 2. AFM 이미지 대장균. 패널 (A)에서 세포는 젤라틴 코팅 운모에 탑재하고 0.005 M PBS에서 몇 군데. 비교, 젤라틴 코팅 운모에 장착하고 공중에서 몇 군데 세균성 세포의 이미지를 패널 (B)로 표시됩니다. septa 분명로서 필리 (삽입된 페이지 패널 (B))의 존재로 표시 액체, 높은 해상도,에서 수집된 이미지에 표시되는 공기를 얻을 수 있습니다. 있지만

<ol>
	<li>Bernal, R., Pullarkat, P. A. <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+axons.">Mechanical properties of axons.</a> Phys Rev Lett. 99, 018301-018301 (2007).</li><li>Bray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+growth+in+response+to+experimentally+applied+mechanical+tension.">Axonal growth in response to experimentally applied mechanical tension.</a> Dev Biol. 102, 379-389 (1984).</li><li>Chetta, J., Kye, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+in+response+to+tensile+loading+of+mammalian+axons.">Cytoskeletal dynamics in response to tensile loading of mammalian axons.</a> Cytoskeleton (Hoboken). 67, 650-665 (2010).</li><li>Dennerll, T. J., Lamoureux, 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+cytomechanics+of+axonal+elongation+and+retraction.">The cytomechanics of axonal elongation and retraction.</a> J Cell Biol. 109, 3073-3083 (1989).</li><li>Fu, S. Y., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+and+molecular+basis+of+peripheral+nerve+regeneration.">The cellular and molecular basis of peripheral nerve regeneration.</a> Mol Neurobiol. 14, 1-2 (1997).</li><li>Gray, C., Hukkanen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+neural+growth:+calcitonin+gene-related+peptide+and+substance+P-+containing+nerves+attain+exceptional+growth+rates+in+regenerating+deer+antler.">Rapid neural growth: calcitonin gene-related peptide and substance P- containing nerves attain exceptional growth rates in regenerating deer antler.</a> Neuroscience. 50, 953-963 (1992).</li><li>Heidemann, S. R., Buxbaum, 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=Tension+as+a+regulator+and+integrator+of+axonal+growth.">Tension as a regulator and integrator of axonal growth.</a> Cell Motil Cytoskeleton. 17, 6-10 (1990).</li><li>Heidemann, S. R., Buxbaum, 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=Mechanical+tension+as+a+regulator+of+axonal+development.">Mechanical tension as a regulator of axonal development.</a> Neurotoxicology. 15, 95-107 (1994).</li><li>Heidemann, S. R., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomechanics+of+axonal+development.">Cytomechanics of axonal development.</a> Cell Biochem Biophys. 27, 135-155 (1995).</li><li>Iwata, A., Browne, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+outgrowth+of+mechanically+engineered+nervous+tissue+constructs+implanted+into+spinal+cord+lesions.">Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions.</a> Tissue Eng. 12, 101-110 (2006).</li><li>Lamoureux, P., Heidemann, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+elongation+within+and+along+the+axon.">Growth and elongation within and along the axon.</a> Dev Neurobiol. 70, 135-149 (2010).</li><li>Lamoureux, P., Zheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytomechanical+investigation+of+neurite+growth+on+different+culture+surfaces.">A cytomechanical investigation of neurite growth on different culture surfaces.</a> J Cell Biol. 118, 655-661 (1992).</li><li>Lindqvist, N., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+glial+(Muller)+cells:+sensing+and+responding+to+tissue+stretch.">Retinal glial (Muller) cells: sensing and responding to tissue stretch.</a> Invest Ophthalmol Vis Sci. 51, 1683-1690 (2010).</li><li>Loverde, J. R., Ozoka, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Axon+Stretch+Growth+in+Embryonic+and+Adult+Neurons.">Live Imaging of Axon Stretch Growth in Embryonic and Adult Neurons.</a> J. Neurotrauma. , (2011).</li><li>Lu, Y. B., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc Natl Acad Sci U S A. 103, 17759-17764 (2006).</li><li>O'Toole, M., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+model+of+axonal+elongation:+force,+viscosity,+and+adhesions+govern+the+mode+of+outgrowth.">A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.</a> Biophys J. 94, 2610-2620 (2008).</li><li>Pfister, B. J., Bonislawski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-grown+axons+retain+the+ability+to+transmit+active+electrical+signals.">Stretch-grown axons retain the ability to transmit active electrical signals.</a> FEBS Lett. 580, 3525-3531 (2006).</li><li>Pfister, B. J., Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+Engineering+Strategies+for+Peripheral+Nerve+Repair:+Surgical+Applications,+State+of+the+Art,+and+Future+Challenges.">Biomedical Engineering Strategies for Peripheral Nerve Repair: Surgical Applications, State of the Art, and Future Challenges.</a> Crit Rev Biomed Eng. 39, 81-124 (2011).</li><li>Pfister, B. J., Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extreme+stretch+growth+of+integrated+axons.">Extreme stretch growth of integrated axons.</a> J Neurosci. 24, 7978-7983 (2004).</li><li>Pfister, B. J., Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transplantable+nervous+tissue+constructs+comprised+of+stretch-grown+axons.">Development of transplantable nervous tissue constructs comprised of stretch-grown axons.</a> J Neurosci Methods. 153, 95-103 (2006).</li><li>Siechen, S., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+contributes+to+clustering+of+neurotransmitter+vesicles+at+presynaptic+terminals.">Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals.</a> Proc Natl Acad Sci U S A. 106, 12611-12616 (2009).</li><li>Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch+growth+of+integrated+axon+tracts:+extremes+and+exploitations.">Stretch growth of integrated axon tracts: extremes and exploitations.</a> Prog Neurobiol. 89, 231-239 (2009).</li><li>Smith, D. H., Wolf, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+to+produce+sustained+growth+of+central+nervous+system+axons:+continuous+mechanical+tension.">A new strategy to produce sustained growth of central nervous system axons: continuous mechanical tension.</a> Tissue Eng. 7, 131-139 (2001).</li><li>Weiss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+patterns:+The+mechanics+of+nerve+growth.">Nerve patterns: The mechanics of nerve growth.</a> Growth, Third Growth Symposium. 5, 163-203 (1941).</li><li>Zheng, J., Lamoureux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+regulation+of+axonal+elongation+and+initiation.">Tensile regulation of axonal elongation and initiation.</a> J Neurosci. 11, 1117-1125 (1991).</li></ol>

관심 없음 충돌 선언하지 않습니다.

다양한 요인에 의해 미생물의 AFM 장착 휴대폰 및 이미징에 영향을 미칠 수 있습니다. 코팅에 사용되는 젤라틴은 운모가 중요합니다. 상업 젤라틴은 물고기, 소, 돼지를 포함한 척추 동물의 숫자에서 격리됩니다. 기원과 처리 방법은 모두 고정 세균에 대한 젤라틴의 적합성을 결정합니다. 다양한 소스 및 젤라틴의 종류 immobilizing 박테리아에서 효과에 대한 평가되었다 [1]. 두 가지 가장 효과적인 gel...

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 젤라틴 </td><td> 시그마, 세인트 루이스, MO </td><td> G6144, G2625 또는 G2500 </td></tr><tr><td> PicoPlus 원자 포스 현미경 </td><td> 애질런트 테크놀로지 템피, AZ </td><td></td></tr><tr><td> AFM의 cantilevers </td><td> Veeco, 산타 바바라, CA </td><td> MLCT - AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

AFM은 이미징 도구를 기계적으로 프로브 표면 고해상도 (NM 규모)입니다. 그것은 화학 샘플을 치료하기 위해 필요없이, 액체 환경에서, 이미지 세포 biomolecules 수있는 능력이 있습니다. 이러한 목표를 달성하기 위해 예제는 충분히 스캔 AFM의 캔틸레버 팁 의해 끼쳤다 힘에 의해 제거를 방지하기 위해 장착 표면을 준수해야합니다. 많은 경우에, 성공적인 영상이 장착 표면에 시료의 고정에 따라 달라집니다. 최적 고정은 신진 대사 프로세스와 기능 특성이 손상되지 않습니다 같은 샘플을 법으로서해야합니다. 돼지의 (돼지) 젤라틴과 코팅 갓 죽습 운모 표면에 의해 부정 청구 박테리아가 표면에 고정화 수 있으며 AFM에 의해 액체에 몇 군데. 젤라틴 - 코팅 운모에 세균 세포의 고정은 부정 청구 세균과 긍정적인 혐의 젤라틴 사이의 정전 기적 상호 작용에 의한 가능성이 높습니다. 여러 가지 요인은 박테리아가 일시 중지되는 액체, 세균 긴장과 세균이 몇 군데있는 매체의 젤라틴 코팅 운모, 표면 특성에 박테리아의 배양 시간의 화학 성분을 포함하여 세균의 고정과 방해할 수 있습니다. 전반적으로, 젤라틴 코팅 운모의 사용은 이미지의 미생물 세포에 대해 일반적으로 적용할 수 발견됩니다.

Watch this Scientific Journal Video about Bacterial Immobilization for Imaging by Atomic Force Microscopy at JoVE.com

Bacterial Immobilization for Imaging by Atomic Force Microscopy

라이브 그람 음성 및 그람 양성 세균은 젤라틴 코팅 운모에 고정화 및 원자 힘 현미경 (AFM)을 사용하여 액체에 몇 군데 있습니다.

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 ...

bacterial-immobilization-for-imaging-by-atomic-force-microscopy

Research

JoVE Journal

Bioengineering

17.2K 조회수.  Oak Ridge National Laboratory. 라이브 그람 음성 및 그람 양성 세균은 젤라틴 코팅 운모에 고정화 및 원자 힘 현미경 (AFM)을 사용하여 액체에 몇 군데 있습니다.

비디오: Bacterial Immobilization for Imaging by Atomic Force Microscopy

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 ...

연구

생체공학

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

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

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 ...

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, ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

단일 분자 형광 현미경에 의한 수용체 역학의 고해상도 시공간 분석

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

루게릭 병에 TDP-43 변종 형태론 특정 시약의 분리에 대한 소설 원자 힘 현미경 기반 Biopanning

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

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 ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Calvarial 중요 한 뼈 결함 수리 다 현미경 Vivo에서 유전자 변형 3D-PLGA/nHAp 비 계에 의해 신생 시각화

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

회전 디스크-총 내부 반사 형광 현미경 검사 법 고급 시각화에 의해 세포 접착 형성

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

B 림프구 활성화를 연구하는 견인력 현미경 검사법

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

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 ...