Die Autoren erkennen an finanzieller Unterstützung NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / NCRR S10RR026645, (SPH) und die Abteilung für Präventive und Restaurative Zahnwissenschaften und Orofacial Wissenschaften, UCSF. Darüber hinaus sind die Autoren erkennen Xradia Graduate Fellowship (AJ), Xradia Inc., Pleasanton, CA. Die Autoren danken Dr. Kathryn Grand, UCSF für ihre Unterstützung bei der Nachbearbeitung von Daten; Drs. Stephen Weiner und Gili Naveh, Weizmann Institute of Science, Rehovot, Israel, Dr. Ron Shahar, The Hebrew University of Jerusalem, Israel für ihre speziell für die in-situ-Ladevorrichtung aufschlussreiche Diskussionen. Die Autoren möchten auch Biomaterialien und Bioengineering MicroCT Imaging Facility an der UCSF für den Einsatz von Mikro-XCT-und in-situ-Ladevorrichtung danken.

Tier Gehäuse und Euthanasie: Alle Tiere in dieser Demonstration verwendet wurden unter keimfreien Bedingungen in Übereinstimmung mit den Richtlinien der Institutional Animal Care und Verwenden Committee (IACUC) und dem National Institute of Health (NIH) untergebracht. Geben Tiere mit Standard-Fest-Pellet Rattenfutter und Wasser ad lib. Euthanize Tiere über eine Zwei-Schritt-Verfahren zur Erstickung mit Kohlendioxid, bilaterale Thorakotomie gemäß dem Standardprotokoll von UCSF durch IACUC genehmigt. Führen biomechanischen Tests innerhalb von 24 Stunden Tieropfer zu Gewebeabbau zu vermeiden. 1. Vorbereitung und Präparation der Ratte eine Unterkiefer-oder Oberkiefer <ol><li> Entfernen Ratte Mandibeln durch vorsichtiges Abtrennen häutigen Gewebe und Muskelgewebe Anhänge und gleichzeitig die gesamte Unterkiefer, einschließlich des Processus coronoideus und dem Gelenkfort (Abbildung 1) 15. </li><li> Separate hemimandibles mit dem Autoefully Schneiden des Fasergewebe der Symphyse mit einem Skalpell. Hinweis: Die Koronar-und Gelenkfort und Ramus des Unterkiefers (1) zu entfernen, wenn sie physisch behindern biomechanischen Tests der 2. molaren werden. </li><li> Schneiden Sie die Schneidezähne, ohne dass die Zellstoffkammer nicht zu Belastung der Molaren zu behindern. </li></ol> 2. Probenpräparation für die in-situ Druckbelastung (Abbildung 2) <ol><li> Immobilisierung der Probe auf einem Stahlstichleitung durch Verwendung eines Materials, das wesentlich steifer als die experimentelle Probe vor dem Laden in ein in situ Ladevorrichtung (2A). Hinweis: Polymethylmethacrylat (PMMA) verwendet, um die Probe in dieser Studie und überschüssiges immobilisieren, falls vorhanden, wurde unter Verwendung eines zahnärztlichen Sonde entfernt. </li><li> Richten Sie die Kaufläche der Molaren (e) von Interesse parallel mit der AFM-Metall-Objektplatte mit einer geraden Kante in beidenEbenen (dh mesial-distal und bukkal-lingual). </li><li> Erstellen Sie ein Trog mit einem stumpfen Instrument rund um die Backenzähne. Hinweis: Dieser Raum sollte als &quot;Burggraben&quot; um überschüssige Flüssigkeit zu enthalten und zu pflegen Gewebehydration während der in-situ-Lade dienen. </li><li> Bereiten Sie die Zahnoberfläche zum Aufbau für konzentrische (2B) oder exzentrisch (2C) Befüllung mit einem Dental-Komposit. Ätzen der Oberfläche des Zahns von Interesse mit 35% iger Phosphorsäure-Gel auf Kaufläche für 15 sek. </li><li> Mit VE-Wasser spülen Ätzmittel gründlich und trocknen Sie die Oberfläche mit einer Luft / Wasser-Spritze oder einen Druckluftbehälter. Mit einem Explorer, verbreiten einen Tropfen des Bindemittels in offene Höcker in einer dünnen Schicht. Heilung der Verbund mit einer Zahn fixieren. Hinweis: Alle Schritte mit Verbundwerkstoffe sollten ohne direkte Licht einer Lampe durchgeführt werden. Solche Bedingungen würden unerwünscht beschleunigen die Polymerisation und could verhindern eine ordnungsgemäße Platzierung der Verbund. Raumbeleuchtung ist akzeptabel. </li><li> Überschüssiges Bindemittel von benachbarten Zähnen mit einem feinen Skalpell oder einer Rasierklinge. </li><li> Zeigen fließfähige Zahnverbund auf der Oberfläche nach der Herstellung der Oberfläche und verteilt ihn in Nuten der Molaren (e) von Interesse mit einem Zahn Explorer. </li><li> Expose der Verbundzahn Heilung Licht für 30 Sekunden. </li><li> Schimmel eine okklusale Aufbau von etwa 3-4 mm unter Verwendung eines Dentalharz-Verbund, von der okklusalen Ebene der Molaren (n) von Interesse und Licht Heilung für 30 Sekunden. </li><li> Reduzieren Sie die Oberseite der Verbundaufbau zu einer flachen Oberfläche parallel eine einheitliche Ladeschema in allen Proben durch Verwendung eines geraden Kante und eine Hochgeschwindigkeits-Handstück zu ermöglichen. Anmerkung: Während der biomechanischen Tests sollten andere Proben in Tris-Phosphat-Pufferlösung (TBS) mit 50 mg / ml Penicillin gespeichert und Streptomycin 15. </li></ol> 3. Lädt Geräte Drift undSteifigkeit, Materialeigenschaften Differenzierungsfähigkeit in situ Laden des Faser Joint <ol><li> Sichern die Probe mit dem Verbundaufbau auf dem Amboß der Ladephase und Test für eine gleichmäßige Belastung, wie in Fig. 2B gezeigt. </li><li> Legen Sie eine Artikulationspapier auf der Oberfläche des Verbund gefolgt von dem Laden der Probe auf eine endliche Last für konzentrische oder exzentrische Belastung (2B und 2C) zu überprüfen. </li><li> Zeigen TBS-getränkten Kimwipe um die Probe zu Probe Hydratation zu gewährleisten. Stellen Sie eine Mulde auf der Probe und füllen Sie es mit TBS, die Orgel während der Bildgebung mit Feuchtigkeit versorgt. </li><li> Eingangs-und Spitzenlastverschiebungsrate in den Deben Software, um die Molaren zu einer gewünschten Spitzenlast bei einer Verschiebung Rate nach Ruhigstellung des Kieferhälfte zu komprimieren. Hinweis: Typische Anzeigen sollte eine reaktionäre Last enthalten, wie das Material im Laufe der Zeit zusammengedrückt (Lastsensorempfindlichkeit = 0,1N). Von Last-Zeit-und Weg-Zeit sollte ein Last-Verschiebungs-Kurve für das Druckmaterial erhalten 16-18 werden. Verwendung der von dem Ladezyklen gesammelten Daten können verschiedene Eigenschaften der Verbindung bestimmt werden. Die Steifheit des Gelenks ist, indem die Steigung des linearen Teils (ungefähr die letzten 30% der Daten) der Ladephase der Last-Weg-Kurve 19 berechnet werden. </li></ol> 4. Die Färbung der Soft Tissue, der PDL, mit Phosphorwolframsäure (PTA) Hinweis: Um Röntgenschwächungs Kontrast zu erhöhen, sollte die PDL mit 5% PTA-Lösung 20 gefärbt werden. <ol><li> Backfill PTA-Färbelösung in ein sauberes 1,8 ml Glascarpule und legen geladen Carpule in die Spritze. </li><li> Inject Lösung langsam (5 min / Glaszylinder) in die PDL-Raum der benachbarten Zähne, um strukturelle Schäden an parodontalen Gewebe um Molaren von Interesse zu verhindern. Hinweis: Die oben genannten Schritte sollte be wiederholt, bis etwa 5 Vollglaszylinder (9 ml)-Lösung werden injiziert und in die umgebenden Gewebe zu strömen. Die Proben vorbereitet kann auch über Nacht in der verbleibenden PTA-Lösung (8 Stunden) eingeweicht werden. </li></ol> 5. Empfohlene Scan-Einstellungen μ-XCT Führen m-XCT mit den folgenden Scan-Einstellungen: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Ziel Vergrößerung </td><td> 4X, 10X </td></tr><tr><td></td><td> 1800 Bilder </td></tr><tr><td> Röntgenröhrenspannung </td><td> 75 kVp (50 kVp für PTA gefärbten Proben) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> Belichtungszeit </td><td> Zwischen 8-25 sec * </td></tr><tr><td></td><td> ~ 4 um (4X Ziel), ~ 2 um (10X-Objektiv) ** </td></tr></tbody></table> * Belichtungszeit basierend auf der Geometrie und der optischen Dichte der Probe und der Röntgenröhre variieren voltage. ** Tatsächlichen Pixelauflösung leicht basierend auf der Konfiguration der Quelle, Probe und Detektor unterscheiden.

<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>

Die Autoren haben nichts zu offenbaren.

Der erste Schritt bei der Schaffung dieses Protokoll beteiligt Bewertung der Steifigkeit der Laderahmen mit Hilfe eines starren Körpers. Basierend auf den Ergebnissen wurde die Steifigkeit signifikant höher, die die Verwendung der Ladevorrichtung zum weiteren Testen von Proben mit deutlich geringeren Steifigkeitswerte. Der zweite Schritt markiert die Fähigkeit des Instrumentes, um verschiedene Steifigkeitswerte unter Verwendung von zwei Phasen der Lade-Entlade-Kurve, die durch einen starren Körper, PDMS-Materialien ...

Mehrere experimentelle Methoden weiterhin verwendet werden, um die Biomechanik des diarthrodialen und Faserverbindungen zu untersuchen. Spezifisch für die Zahnorgan Biomechanik Verfahren umfassen die Verwendung von Dehnungsmessstreifen 1-3, Spannungsoptik Verfahren 4, 5, Moiré-Interferometrie 6, 7, elektronischen Specklemuster-Interferometrie 8 und digitale Bildkorrelation (DIC) 9-14. In dieser Studie beinhaltet das innovative Konzept nichtinvasiven Bildgebung mit Röntgenstrahlen, um die inneren Strukturen eines Fasergelenk freizulegen (mineralisierten Geweben und deren Schnittstellen aus weicheren Zonen und Schnittstellen Gewebe wie Bänder) bei Belastung entspricht in vivo-Bedingungen. Ein in situ-Ladevorrichtung mit einem Mikroröntgenmikroskops gekoppelt verwendet. Die Last-Zeit-und Kraft-Verschiebungskurven wird als Mol-Interesse in einem frisch geernteten Ratte Hemi-Unterkiefer geladen gesammelt werden. Die main Ziel der Ansatz in dieser Studie präsentiert wird, um die Wirkung von dreidimensionalen Morphologie der Zahn-Knochen durch den Vergleich Bedingungen bei betonen: 1) Leerlauf und unter Last, und wenn 2) konzentrisch und exzentrisch geladen. Wodurch die Notwendigkeit für Schnitt Proben und Experimente auf ganze Organe intakt unter feuchten Bedingungen durchführen wird für maximale Erhaltung der 3D-Spannungszustand zu ermöglichen. Es öffnet sich ein neues Gebiet der Untersuchung dynamischer Prozesse im Verständnis des Komplexes unter verschiedenen Belastungsszenarien. In dieser Studie wurden die Methoden zur Prüfung PDL Biomechanik innerhalb einer intakten Fasergelenk einer Sprague Dawley-Ratte, wird ein Joint als optimale Biotechnik als Modellsystem beschrieben werden. Die Experimente werden in der Reihenfolge auf drei wichtige Merkmale des gemeinsamen markieren, wie sie Organebene beziehen, umfassen Biomechanik-Simulation des Kauens Lasten unter hydratisierten Bedingungen. Die drei Punkte sind: 1) reaktionäre Kraft-Verschiebung:Zahnbewegung innerhalb der Alveole und reaktionären Reaktion auf Belastung, 2) dreidimensionale (3D-) räumlichen Konfiguration und Morphometrie: geometrische Beziehung des Zahns mit der Alveole und 3) Veränderungen der Auslesungen 1 und 2 aufgrund einer Änderung in Ladeachse, also von konzentrischen exzentrischen Lasten. Die drei grundlegenden Auslesungen der vorgeschlagenen Technik kann angewendet werden, um die adaptive Natur der Gelenke bei Wirbeltieren entweder aufgrund von Veränderungen in der funktionellen Anforderungen und / oder Krankheiten zu untersuchen. Veränderung der zuvor genannten Anzeigen, insbesondere die Korrelation zwischen reaktionären Lasten mit Verschiebung, und die daraus resultierenden reaktionären Last-Zeit-und Kraft-Verschiebungskurven bei verschiedenen Belastungsraten können angewendet werden, um Veränderungen in der Gesamt Joint Biomechanik markieren. Die Wirksamkeit der vorgeschlagenen Protokoll wird durch die Kopplung der mechanischen Prüfung Anzeige in die 3D-Morphometrie und die allgemeine Biomechanik des Gelenks ausgewertet werden.

<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

Diese Studie zeigt eine neuartige Biomechanik Testprotokoll. Der Vorteil dieses Protokolls umfaßt die Verwendung eines in situ-Ladevorrichtung, um eine hochauflösende Röntgenmikroskops gekoppelt ist, wodurch die Visualisierung interner Strukturelemente unter simulierten physiologischen Belastungen und Nässe. Experimentelle Proben gehören intakte Knochen Desmodont (PDL)-Zahn-Faser Gelenke. 1) reaktionäre Kraft-Verschiebung: Zahnverschiebung innerhalb der Alveole und ihre reaktionäre Antwort auf Laden, 2) drei-dimensionale (3D) räumliche Konfiguration Ergebnisse werden drei wichtige Merkmale des Protokolls, wie sie können, um Organebene Biomechanik angewendet werden, veranschaulichen und Morphometrie: geometrische Beziehung des Zahns mit der Alveole und 3) Veränderungen der Auslesungen 1 und 2 aufgrund einer Änderung der Ladeachse, also von konzentrisch zur exzentrischen Belastungen. Die Wirksamkeit der vorgeschlagenen Protokoll wird durch die Kopplung mechanischer te ausgewertet werdenStachel Anzeige in die 3D-Morphometrie und die allgemeine Biomechanik des Gelenks. Darüber hinaus wird diese Technik auf die Notwendigkeit der Versuchsbedingungen äquilibrieren speziell reaktionären Lasten vor Erwerb Tomogramme von Faserverbindungen hervorzuheben. Es sei darauf hingewiesen, dass die vorgeschlagene Protokoll beschränkt sich auf die Prüfung Proben unter ex-vivo-Bedingungen, und dass die Verwendung von Kontrastmitteln Weichgewebe mechanische Antwort visualisieren könnte zu falschen Schlüssen über die Gewebe-und Organebene Biomechanik führen werden.

Die Einschätzung der Ladevorrichtung &quot;backlash&quot;, &quot;Pushback&quot;, Steifigkeit und Drift-System unter einer konstanten Last Backlash: Zwischen Be-und Entladen des Zyklus gibt es eine Pause von 3 Sekunden, während der umgekehrte Räder innerhalb des Motors, bevor wahr Entladen beginnt, dh als die Probe zieht weg von der Aufsatzbacke (Abbildung 3). Dieser Zeitraum wird als ein Spiel in dem System, das einen Zeitabschni...

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

In dieser Studie wird die Verwendung eines in situ-Ladevorrichtung in Verbindung mit Mikro-Röntgenstrahl-Computertomographie zur gemeinsamen Faser Biomechanik diskutiert. Experimentelle Anzeigen erkennbar mit einer Gesamtänderung der Gelenkbiomechanik gehören: 1) reaktionäre Kraft-Verschiebung, dh Zahnbewegung innerhalb der Alveole und ihre reaktionäre Antwort auf Laden, 2) drei-dimensionale (3D) räumliche Konfiguration und Morphometrie, dh geometrische Verhältnis der Zahn mit der Alveole und 3) Veränderungen der Auslesungen 1 und 2 aufgrund einer Änderung in Lastachse, dh konzentrische oder exzentrische Belastung.

In-situ-Druckbelastung und Correlative Nichtinvasive Bildgebung des Knochen parodontalen Ligament-Zahn-Faser-Joint

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 Aufrufe.  University of California San Francisco.  In dieser Studie wird die Verwendung eines in situ-Ladevorrichtung in Verbindung mit Mikro-Röntgenstrahl-Computertomographie zur gemeinsamen Faser Biomechanik diskutiert. Experimentelle Anzeigen erkennbar mit einer Gesamtänderung der Gelenkbiomechanik gehören: 1) reaktionäre Kraft-Verschiebung, dh Zahnbewegung innerhalb der Alveole und ihre reaktionäre Antwort auf Laden, 2) drei-dimensionale (3D) räumliche Konfiguration und Morphometrie, dh

Serie: 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.

Hochauflösende LWL-Mikroendoskopiesysteme für In situ Cellular Imaging

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

Forschung

Biotechnik

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.

Demonstration der Verwendung des neuartigen Gravitationskraft Spectrometer auf Stretch and Measure Faserproteinen

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.

In vitro Synthese von Native, Fibröse Lange Abstände und Segmentberichterstattung Lange Spacing Collagen

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.

Entwurf eines Biaxiale Mechanische Belastung Bioreaktor für Tissue Engineering

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.

FIBS-fähigen Nichtinvasive 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 ...

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.

Elektro Growth Factor Releasing Mikrosphären in Fasergerüste

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.

Studieren der Stöchiometrie der Epidermal Growth Factor Receptor in intakten Zellen mit korrelative Mikroskopie

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.

Optimierte Setup und ein Protokoll für magnetische Domäne Bildgebung mit In Situ Hysterese-Messung

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.

Nanosensoren Protease-Aktivität In Vivo für die nicht-invasive Diagnostik zu erkennen

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.

Korrelative Licht Elektronenmikroskopie (CLEM) für die Verfolgung und Imaging virales Protein verbundenen Strukturen in den Cryo-immobilisierten Zellen

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