Yazarlar finansman desteği NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / NCRR S10RR026645, (SPH) ve Koruyucu ve Restoratif Diş Bilimleri Bölümleri kabul ve orofasial Bilimler, ucsf. Ayrıca, yazarlar Xradia Lisans Bursu (AJ), Xradia Inc, Pleasanton, CA kabul. Yazarlar verilerinin sonradan işleme ile ona yardım için Dr Kathryn Grandfield, ucsf ederim; Drs. Stephen Weiner ve Gili Naveh, Fen Rehovot&#39;taki, İsrail Weizmann Enstitüsü; situ yükleme cihaza özgü onların anlayışlı tartışmalar Dr Ron Shahar, Kudüs Hebrew Üniversitesi, İsrail. Yazarlar ayrıca Micro XBT kullanımı ve in situ yükleme cihazı için ucsf de Biomateryaller ve Biyomühendislik mikroBT Görüntüleme Tesisi teşekkür etmek istiyorum.

Hayvan gövdesi ve ötenazi: Bu tasvirde kullanılan bütün hayvanlar Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) ve Ulusal Sağlık Enstitüsü (NIH) kurallarına uygun olarak patojen içermeyen koşullar altında tutuldu. Standart sabit-pelet sıçan yemi ve suyla birlikte hayvanları sağlayın. IACUC tarafından onaylanmış gibi ucsf standart protokolüne uygun karbon dioksit boğulma, bilateral torakotomi bir iki aşamalı yöntemi ile hayvanlar Euthanize. Dokusu bozulmasını önlemek için, hayvan kurban 24 saat içinde biyomekanik test gerçekleştir. 1.. Hazırlanması ve Rat Altçene&#39;ye veya Maksillada diseksiyonu <ol><li> Koronoid ve kondiler süreci (Şekil 1) 15 de dahil olmak üzere tüm mandibula, koruyarak nazikçe membranöz doku ve kas dokusu ekleri koparılması ile sıçan altçeneleri çıkarın. </li><li> Arabayla Ayrı hemimandiblesefully bir neşter ile mandibular symphysis&#39;in fibröz doku kesme. Not: fiziksel 2. molar biyomekanik test engel ise koroner ve kondiler süreçler ve mandibulanın ramus (Şekil 1) çıkarılmalıdır. </li><li> Molar yüklenmesini engellemeyecek şekilde hamuru oda maruz kalmadan kesicilerin kesti. </li></ol> 2. Yerinde Basınç Yükleniyor için Numune Hazırlama (Şekil 2) <ol><li> Önce in situ yükleme cihazı (Şekil 2A) bir in yüklemeden için deney numunesinin önemli ölçüde daha sert olan bir malzeme kullanılarak bir çelik parçası üzerinde numune hareketsiz. Not: polimetilmetakrilat (PMMA), eğer varsa, bir diş Explorer kullanılarak uzaklaştırılmıştır, bu çalışma ve fazla miktarda numune hareketsiz hale getirmek için kullanılmıştır. </li><li> Hem de bir düz kenarı kullanarak AFM metal numune disk ile faiz paralel molar (ler) oklüzal yüzeyini hizalayınuçaklar (yani mesial-distal ve yanak-lingual). </li><li> Azılarını çevreleyen bir cisimle bir çukur oluşturun. Not: Bu alan aşırı sıvı içeren ve yerinde yükleme sırasında doku hidrasyon korumak için bir &quot;hendek&quot; olarak hizmet etmelidir. </li><li> Bir diş kompozit kullanılarak eş (Şekil 2B) ya da eksantrik (Şekil 2C) yükleme oluşturmak için diş yüzeyini hazırlayın. 15 saniye için oklüzal yüzeyi üzerinde% 35 fosforik asit jeli ile, ilgi konusu dişin yüzeyini aşındırma. </li><li> Iyonu giderilmiş su ile iyice yıkayın ve pürüzlendirici bir hava / su şırınga ya da bir basınçlı hava spreyi kullanarak yüzeyi kurutun. Bir kaşif ile, ince bir tabaka halinde açık başlangıç ​​çizgileri içine bağlama maddesinin bir damla yayıldı. Dental bir ışık ile bileşik Cure. Not: Tüm adımlar içeren kompozit bir lamba doğrudan ışık olmadan gerçekleştirilmelidir. Bu koşullar, istenmeyen bir polimerizasyon sürecini hızlandırmak ve coul olurd kompozitin doğru yerleştirilmesini engeller. Oda aydınlatma kabul edilebilir. </li><li> Ince bir neşter veya jilet ile komşu dişlerin aşırı bonding ajanı çıkarın. </li><li> Yüzeyin hazırlanması sonra yüzeyi üzerinde akışkan dental bileşik yerleştirin ve bir diş explorer kullanarak, ilgi konusu molar (ler) olukları içine yayıldı. </li><li> 30 saniye boyunca diş kür ışık kompozit Açığa. </li><li> Ilgi molarlık (lar) ve 30 saniye boyunca ışık tedavi oklüzal düzleminden, bir dental reçine bileşik kullanılarak, yaklaşık 3-4 mm&#39;lik bir oklüzal oluşumunu kalıp. </li><li> Bir cetvel ve bir yüksek hızlı el parça kullanılarak tüm örneklerde tutarlı bir yükleme düzeni sağlamak için düz bir yüzeye paralel kompozit birikimini üst azaltın. Not: Biyomekanik Testler sırasında, diğer örnekleri 50 mg / ml penisilin tris-fosfat tamponlu çözelti (TBS) içerisinde saklanır ve streptomisin 15 olmalıdır. </li></ol> 3. Yükleme Cihazı Drift veSertlik, Material Property Farklılaşan Yetenek, Lifli Joint in situ Yükleniyor <ol><li> Şekil 2B&#39;de gösterildiği gibi, tek tip yükleme için yükleme aşamasının örs üzerinde kompozit birikimi ile örnek ve bir test edin. </li><li> Eş merkezli veya dış merkezli yükleme (Şekil 2B ve 2C) kontrol etmek için sınırlı bir yük için numune yükleme ardından kompozitin yüzeyi üzerinde bir eklem kağıt yerleştirin. </li><li> Numune hidrasyon sağlamak için numune etrafında TBS-batırılmış Kimwipe yerleştirin. Numune etrafında bir oluk yapmak ve görüntüleme sırasında nemli organı tutmak için TBS ile doldurun. </li><li> Hemimandibula hareketsizliği izleyen bir deplasman oranda istenen pik yük molar sıkıştırmak için Deben yazılımı içine girdi pik yük ve deplasman oranı. Not: Malzeme yükü dönüştürücü duyarlılık = 0.1 (zaman içinde sıkıştırılır gibi tipik okumalar gerici yükü içermelidirN). Yük zaman ve yer değiştirme-zaman, sıkıştırılmış malzeme için yük-deplasman eğrisi 16-18 alınmalıdır. Yükleme döngüsü toplanan verileri kullanarak, çeşitli ortak özellikleri de belirlenebilir. Eklemin sertliği doğrusal bölümü yer değiştirme eğrisinin 19 karşı yükün yükleme fazı (verinin yaklaşık olarak en son% 30) eğimini alınarak hesaplanır. </li></ol> 4. Fosfotungstik Asit (PTA) ile Yumuşak Doku, PDL boyanması, Not: X-ışını zayıflama kontrastı arttırmak için, PDL% 5 PTA çözeltisi 20 ile boyanmış olmalıdır. <ol><li> Dolgu PTA boyama, temiz bir 1.8 ml&#39;lik bir cam karpülün içine çözeltisi ve şırıngaya yüklendi karpül yerleştirin. </li><li> Ilgi azıdişi çevreleyen periodontal dokular için yapısal hasarı önlemek için komşu dişlerin PDL-uzaya solüsyonu yavaş yavaş (5 dk / karpül) enjekte edilir. Not: Yukarıdaki adımlar b gerekirçözeltisi yaklaşık 5 tam carpules (9 mi) e kadar tekrar enjekte edilir ve çevredeki dokulara akmasına izin verilir. Prepped örnekleri de kalan PTA çözeltisi (8 saat) içinde gece boyunca ıslatılmış olabilir. </li></ol> 5. Önerilen μ-XCT Tarama Ayarları Aşağıdaki tarama ayarları ile m-XCT gerçekleştirin: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Amaç Büyütme </td><td> 4X, 10X </td></tr><tr><td></td><td> 1.800 görüntüleri </td></tr><tr><td> X-ışını tüpü gerilim </td><td> 75 kVp (PTA lekeli örnekler için 50 kVp) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> Pozlama Saati </td><td> ~ 8-25 sn * </td></tr><tr><td></td><td> ~ 4 mikron (4X objektif), ~ 2 mikron (10X objektif) ** </td></tr></tbody></table> * Maruz kalma süresi, geometri ve numune ve X-ışını tüpü vo optik yoğunluğuna bağlı olarak değişebilirltage. ** Gerçek piksel çözünürlük biraz kaynak, numune ve dedektör yapılandırmasına göre farklı olacaktır.

<ol>
	<li>Popowics, T. E., Rensberger, J. M., Herring, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel+microstructure+and+microstrain+in+the+fracture+of+human+and+pig+molar+cusps.">Enamel microstructure and microstrain in the fracture of human and pig molar cusps.</a> Arch. Oral Biol. 49, 595-605 (2004).</li><li>Jantarat, J., Palamara, J. E., Messer, 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=An+investigation+of+cuspal+deformation+and+delayed+recovery+after+occlusal+loading.">An investigation of cuspal deformation and delayed recovery after occlusal loading.</a> J. Dent. 29, 363-370 (2001).</li><li>Jantarat, J., Panitvisai, P., Palamara, J. E., Messer, 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=Comparison+of+methods+for+measuring+cuspal+deformation+in+teeth.">Comparison of methods for measuring cuspal deformation in teeth.</a> J. Dent. 29, 75-82 (2001).</li><li>Asundi, A., Kishen, 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+strain+gauge+and+photoelastic+analysis+of+in+vivo+strain+and+in+vitro+stress+distribution+in+human+dental+supporting+structures.">A strain gauge and photoelastic analysis of in vivo strain and in vitro stress distribution in human dental supporting structures.</a> Arch. Oral Biol. 45, 543-550 (2000).</li><li>Asundi, A., Kishen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+digital+photoelastic+investigations+on+the+tooth-bone+interface.">Advanced digital photoelastic investigations on the tooth-bone interface.</a> J. Biomed. Opt. 6, 224-230 (2001).</li><li>Wang, R. Z., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-structure+relations+in+human+teeth+using+Moire+fringes.">Strain-structure relations in human teeth using Moire fringes.</a> J. Biomech. 31, 135-141 (1998).</li><li>Wood, J. D., Wang, R., Weiner, S., Pashley, 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=Mapping+of+tooth+deformation+caused+by+moisture+change+using+moire+interferometry.">Mapping of tooth deformation caused by moisture change using moire interferometry.</a> Dent. Mater. 19, 159-166 (2003).</li><li>Dong-Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulus+of+elasticity+of+human+periodontal+ligament+by+optical+measurement+and+numerical+simulation.">Modulus of elasticity of human periodontal ligament by optical measurement and numerical simulation.</a> Angle Orthod. 81, 229-236 (2011).</li><li>Li, J., Li, H., Fok, A. S., Watts, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+correlations+of+material+parameters+of+light-cured+dental+composites.">Multiple correlations of material parameters of light-cured dental composites.</a> 25, 829-836 (2009).</li><li>Zhang, D., Arola, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+digital+image+correlation+to+biological+tissues.">Applications of digital image correlation to biological tissues.</a> J. Biomed. Opt. 9, 691-699 (2004).</li><li>Zhang, D., Mao, S., Lu, C., Romberg, E., Arola, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dehydration+and+the+dynamic+dimensional+changes+within+dentin+and+and+enamel.">Dehydration and the dynamic dimensional changes within dentin and and enamel.</a> Dent. Mater. 25, 937-945 (2009).</li><li>Qian, L., Todo, M., Morita, Y., Matsushita, Y., Koyano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformation+analysis+of+the+periodontium+considering+the+viscoelasticity+of+the+periodontal.">Deformation analysis of the periodontium considering the viscoelasticity of the periodontal.</a> 25, 1285-1292 (2009).</li><li>Lin, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+a+bone-periodontal+ligament-tooth+fibrous+joint.">Biomechanics of a bone-periodontal ligament-tooth fibrous joint.</a> J. Biomech. , (2012).</li><li>Qian, L., Todo, M., Morita, Y., Matsushita, Y., Koyano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformation+analysis+of+the+periodontium+considering+the+viscoelasticity+of+the+periodontal.">Deformation analysis of the periodontium considering the viscoelasticity of the periodontal.</a> 25, 1285-1292 (2009).</li><li>Huelke, D. F., Castelli, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+supply+of+the+rat+mandible.">The blood supply of the rat mandible.</a> Anat. Rec. 153, 335-341 (1965).</li><li>Chiba, M., Komatsu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+responses+of+the+periodontal+ligament+in+the+transverse+section+of+the+rat+mandibular+incisor+at+various+velocities+of+loading+in+vitro.">Mechanical responses of the periodontal ligament in the transverse section of the rat mandibular incisor at various velocities of loading in vitro.</a> J. Biomech. 26, 561-570 (1993).</li><li>Natali, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visco-hyperelastic-damage+constitutive+model+for+the+analysis+of+the+biomechanical+response+of+the+periodontal+ligament.">A visco-hyperelastic-damage constitutive model for the analysis of the biomechanical response of the periodontal ligament.</a> J. Biomech. Eng. 130, (2008).</li><li>Naveh, G. R., Shahar, R., Brumfeld, V., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+movements+are+guided+by+specific+contact+areas+between+the+tooth+root+and+the+jaw+bone:+A+dynamic+3D+microCT+study+of+the+rat+molar.">Tooth movements are guided by specific contact areas between the tooth root and the jaw bone: A dynamic 3D microCT study of the rat molar.</a> J. Struct. Biol. 177, 477-483 (2012).</li><li>Lin, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+a+bone-periodontal+ligament-tooth+fibrous+joint.">Biomechanics of a bone-periodontal ligament-tooth fibrous joint.</a> J. Biomech. 46, 443-449 (2013).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+comparative+morphology:+simple+staining+methods+allow+high-contrast+3D+imaging+of+diverse+non-mineralized+animal+tissues.">MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.</a> BMC Physiol. 9, 11 (2009).</li><li>Carrillo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+of+polydimethylsiloxane+elastomers:+Effect+of+crosslinking,+work+of+adhesion,+and+fluid+environment+on+elastic+modulus+(vol+20,+pg+2820).">Nanoindentation of polydimethylsiloxane elastomers: Effect of crosslinking, work of adhesion, and fluid environment on elastic modulus (vol 20, pg 2820).</a> J. Mater. Res. 21, 535-537 (2006).</li><li>Hiiemae, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Masticatory+function+in+the+mammals.">Masticatory function in the mammals.</a> J. Dent. Res. 46, 883-893 (1967).</li><li>Hunt, H. R., Rosen, S., Hoppert, 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=Morphology+of+molar+teeth+and+occlusion+in+young+rats.">Morphology of molar teeth and occlusion in young rats.</a> J. Dent. Res. 49, 508-514 (1970).</li><li>Komatsu, K., Sanctuary, C., Shibata, T., Shimada, A., Botsis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-relaxation+and+microscopic+dynamics+of+rabbit+periodontal+ligament.">Stress-relaxation and microscopic dynamics of rabbit periodontal ligament.</a> J. Biomech. 40, 634-644 (2007).</li><li>Lin, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+a+bone-periodontal+ligament-tooth+fibrous+joint.">Biomechanics of a bone-periodontal ligament-tooth fibrous joint.</a> J. Biomech. 46, 443-449 (2013).</li><li>Quintarelli, G., Zito, R., Cifonelli, 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=On+phosphotungstic+acid+staining.">On phosphotungstic acid staining.</a> I. J. Histochem. Cytochem. 19, 641-647 (1971).</li><li>Quintarelli, G., Cifonelli, J. A., Zito, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phosphotungstic+acid+staining.">On phosphotungstic acid staining.</a> II. J. Histochem. Cytochem. 19, 648-653 (1971).</li><li>Quintarelli, G., Bellocci, M., Geremia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phosphotungstic+acid+staining.+IV.+Selectivity+of+the+staining+reaction.">On phosphotungstic acid staining. IV. Selectivity of the staining reaction.</a> J. Histochem. Cytochem. 21, 155-160 (1973).</li><li>Crabtree, W. N., Murphy, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+ethanol+as+a+fixative+in+urinary+cytology.">The value of ethanol as a fixative in urinary cytology.</a> Acta Cytol. 24, 452-455 (1980).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu protokolü kurulmasında ilk adım rijit bir gövde kullanarak yükleme çerçevenin sertliğini değerlendirmek içeriyordu. Sonuçlara göre, önemli ölçüde daha düşük sertlik sertlik değerleri ile örneklerin daha fazla test için yükleme tertibatının kullanılmasını sağlayan önemli ölçüde daha yüksek idi. İkinci adım, rijit bir gövde, farklı çapraz yoğunlukları PDMS malzeme ve lifli eklemleri kullanarak oluşturulan yükleme-boşaltma eğrisinin iki fazını kullanarak farklı sertlik de?...

Çeşitli deneysel yöntemler diarthrodial ve fibröz eklemlerin biyomekanik araştırmak için kullanılmaya devam. Diş organı biyomekanik özgü yöntemler gerginlik ölçme 1-3, photoelasticity yöntemleri 4, 5, Harelenme interferometri 6, 7, elektronik benek desen interferometri 8, ve dijital görüntü korelasyon (DIC) 9-14 kullanımını içerir. Bu çalışmada, yenilikçi bir yaklaşım, bir elyaflı mafsalın iç yapıları ortaya çıkarmak için X-ışınları kullanılarak (yumuşak bölgeler aşağıdakilerden oluşan mineralize dokuları ve arayüzleri ve bu gibi bağ dokularının arayüz) in vivo koşullara denk yüklerde invazif olmayan görüntülemeyi içerir. Bir mikro-X-ray mikroskop bağlanmış bir in situ yükleme cihazı kullanılacaktır. Yük-zaman ve yük-deplasman eğrileri taze hasat sıçan hemi-mandibulanın içinde ilgi molar yüklü olarak tahsil edilecektir. M1) Hiçbir yük ve yüklenen ve zaman 2) eş ve eksantrik yüklü: Bu çalışmada sunulan yaklaşım ain hedefi en şartlarını karşılaştırarak diş kemiğin üç boyutlu morfoloji etkisini vurgulamaktır. Kesim örnekleri için ihtiyacı ortadan kaldırarak, ve ıslak koşullarda bütün dokunulmamış organlar üzerinde deney yapmak için 3D stres durumunda maksimum koruma sağlayacak. Bu çeşitli yükleme senaryolar altında karmaşık dinamik süreçleri anlamada soruşturma yeni bir alan açar. Bu çalışmada, Sprague Dawley sıçan sağlam bir lifli eklem içinde test PDL biyomekanik için yöntem, bir optimum bioengineering bir model sistem olarak kabul ortak bir detaylı olarak açıklanacaktır. Deneyler organ düzeyinde biyomekanik ile ilgili olarak eklemin üç önemli özelliklerini vurgulamak amacıyla hidratlı koşullar altında çiğneme yüklerinin simülasyon içerecektir. Üç puan içerecektir: yerinden vs 1) gerici kuvvet:diş alveolar yuva içinde yer değiştirme ve yükleme gerici tepki, 2) üç boyutlu (3D) uzamsal konfigürasyon ve morfometrik: alveolar yuva ile dişin geometrik ilişki nedeniyle bir değişiklik için okumalar 1 ve 2 3) değişiklikleri konsantrik eksantrik yüklerin yani yükleme ekseni. Önerilen teknik, üç temel okumalar nedeniyle fonksiyonel taleplerindeki değişikliklere ve / veya hastalığa omurgalı ya da eklemlerin adaptif doğasını araştırmak için uygulanabilir. Anılan okumalar değişiklikler, farklı yükleme hızlarında özellikle deplasman gerici yükler arasındaki korelasyon ve gerici yük-zaman ve yük-deplasman eğrileri çıkan ortak biyomekanik genel değişiklikleri vurgulamak için uygulanabilir. Önerilen protokol etkinliği 3D Morfometri ve eklem genel biyomekanik mekanik test okumalarını birleştirilmesi tarafından değerlendirilecektir.

<table border="0" cellpadding="0" cellspacing="0" width="652">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Bard Parker Blade</td>
			<td style="width:101px;">BD</td>
			<td style="width:123px;">MEDC-001054</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM metal disk</td>
			<td style="width:101px;">Ted Pella</td>
			<td style="width:123px;">16218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Polymethyl methacrylate&#160;</td>
			<td style="width:101px;">GC America</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Uni-Etch</td>
			<td style="width:101px;">Bisco</td>
			<td style="width:123px;">E5502EBM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Optibond Solo Plus</td>
			<td style="width:101px;">Kerr Corp</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Filtek Flow</td>
			<td style="width:101px;">3M</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Hurculite Ultra</td>
			<td style="width:101px;">Kerr</td>
			<td style="width:123px;">34346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris buffer</td>
			<td style="width:101px;">Mediatech Inc.</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Articulating paper</td>
			<td style="width:101px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Phosphotungstic Acid</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">HT152</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint

Bu çalışma, yeni bir biyomekanik test protokol göstermektedir. Bu protokolün bir avantajı bu şekilde simüle fizyolojik yükler ve ıslak koşullarda, iç yapı elemanları görselleştirme sağlayan, yüksek çözünürlüklü bir X-ışını mikroskop kuple yerinde yükleme cihazı olarak bir kullanımını içerir. Deneysel örnekler bozulmamış kemik-periodontal ligament (PDL)-diş lifli eklemler içerecektir. 1) deplasman vs gerici güç: diş deplasman alveol soket içinde ve yükleme, 2) üç boyutlu (3D) mekansal konfigürasyonu kendi gerici tepki sonuçları, organ düzeyinde biyomekanik uygulanabilir gibi protokol üç önemli özellikleri gözler önüne serecektir ve morfometrik: geometrik alveolar soketi ile diş arasında ilişki nedeniyle, yani eksensel eş merkezli ikinci yükleme ekseninde bir değişiklik için okumalar 1 ve 2 &#39;de 3) değişir. Önerilen protokolün etkinliği mekanik te birleştirilmesi tarafından değerlendirilecektir3D Morfometri ve eklem genel biyomekaniğine acı okumaları. Buna ek olarak, bu tekniği özellikle gerici yükler önce lifli eklem tomogram edinme, deneysel koşullar dengelenmeye ihtiyacı vurgulamak olacaktır. Bu önerilen protokol ex vivo koşullar altında numunelerin test sınırlıdır ve yumuşak doku mekanik yanıtını görselleştirmek için kontrast maddelerin kullanımının doku ve organ düzeyinde biyomekanik hakkında hatalı sonuçlara yol açabileceği unutulmamalıdır.

Yükleme cihazı &quot;tepki&quot; sürekli bir yük altında, &quot;pushback&quot;, sertlik, ve sistem sürüklenme Tahmini Boşluk: döngüsünün yükleme ve boşaltma bölümleri arasında gerçek boşaltma numune uzakta üst çene (Şekil 3) çeker, yani olduğu gibi, başlamadan önce motorun içinde ters vites sırasında 3 saniyelik bir duraklama mevcuttur. Bu süre sistem çenelerinin açılması kapanış geçmek için ça...

Watch this Scientific Journal Video about In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint at JoVE.com

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

Bu çalışmada, lifli ortak biyomekanik için mikro-X-ışını bilgisayarlı tomografi ile birlikte yerinde yükleme cihazının bir kullanımının tartışılacaktır. Ortak biyomekanik bir genel değişim ile tanımlanabilir deneysel okumalar içerecektir: 1) gerici deplasman vs kuvvet, alveol soket içinde yani diş deplasman ve yükleme onun gerici tepki, 2) üç boyutlu (3D) mekansal yapılandırma ve morfometrik, yani geometrik nedeniyle yükleme ekseni örneğin, konsentrik ya da eksantrik yükler bir değişiklik için alveol yuva ile diş ve okumalar 1 ve 2 &#39;de 3) değişikliklerin ilişkisi.

Yerinde Basınç Yükleme ve Kemik-periodontal ligament-diş Lifli Joint Korelatif noninvaziv Görüntüleme

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

in-situ-compressive-loading-correlative-noninvasive-imaging-bone

Research

JoVE Journal

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

13.2K Görüntüleme.  University of California San Francisco.  Bu çalışmada, lifli ortak biyomekanik için mikro-X-ışını bilgisayarlı tomografi ile birlikte yerinde yükleme cihazının bir kullanımının tartışılacaktır. Ortak biyomekanik bir genel değişim ile tanımlanabilir deneysel okumalar içerecektir: 1) gerici deplasman vs kuvvet, alveol soket içinde yani diş deplasman ve yükleme onun gerici tepki, 2) üç boyutlu (3D) mekansal yapılandırma ve morfometrik, yani geometri...

Video: In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Biyomühendislik

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

Fibröz Proteinler gerin ve Tedbir Roman Yerçekimi Kuvveti Spektrometresi kullanır gösterilmesi

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Yerli, Fibröz Uzun Aralığı ve Bölümlere Uzun Aralığı Kollajen in vitro sentezi olarak

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Doku Mühendisliği için iki eksenli Mekanik Yükleniyor Biyoreaktör tasarımı

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

Noninvaziv metabolik profil FIBS etkin

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Lifli iskelelerinin içine Microspheres ibra Elektroeğirme Büyüme Faktörü

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Korelatif Mikroskopisi kullanılarak sağlam hücrelerde Epidermal Büyüme Faktörü Alıcısının stokiyometrisinden incelenmesi

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

En iyi duruma getirilmiş kurulum ve manyetik etki alanı görüntüleme ile Situ histeresis ölçüm için iletişim kuralı

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors için noninvaziv tanı koyma proteaz aktivitesi Vivo tespit

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Cryo-immobilize hücreler yapılarda bağdaşık ışık elektron izleme ve Viral Protein Imaging mikroskobu (CLEM) ilişkili

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...