Name: afm-based mapping of the elastic properties of cell walls: at tissue, cellular, and subcellular resolutions
Uploaded: 2014-07-24T14:00:00
Description: Watch this Scientific Journal Video about AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions at JoVE.com

Vi giver en særlig tak til Yves Couder til mange nyttige diskussioner. Vi takker Atef Asnacios til kalibrering af bjælker og diskussion. Vi takker Lisa Willis, Elliot Meyerowitz, og Oliver Hamant til kritisk læsning. Dette arbejde blev finansieret delvist af Human Frontier Science Program tilskud RGP0062/2005-C; Agence Nationale de la Recherche projekter&#39;&#39; Growpec,&#39;&#39; og&#39;&#39; Mechastem&#39;&#39;.

1.. Forbered glas Slides til montering Sample <ol><li> Forbered imbedding agarose medier: 0,7% lavtsmeltende agarose i 10% mannitol (i vand). </li><li> Anvendelse af en stærk metal-instrument (f.eks borespids, lime), etch en 0,5 x 0,5 cm område i centrum af et mikroskopi objektglas. Eller i stedet, lim et lille stykke glas lameller (ca. 20 x 200 um) til objektglas ved hjælp af Araldite lim. BEMÆRK: Denne roughens overfladen for at lette vedhæftning af agarose at sikre, at agarose medier pinde eller rette...

<ol>
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I planter spiller skiftende mekaniske egenskaber en stor rolle i at lede vækst og morfogenese. Til dato har der været store fremskridt i at udrede de genetiske og kemiske netværk, der styrer planternes vækst, men vores viden om, hvordan disse netværk bidrager til og er påvirket af ændringer i mekaniske egenskaber er rudimentære. Denne metode skal gøre det muligt for os at udfylde dette hul, og så bør det være af stor interesse for forskere studerer ethvert aspekt af plantevækst eller morfogenese. Vi har nu ...

Væksten i planter opnås ved koordineret udvidelse af de stive cellevægge, der omgiver hver celle af organismen. Akkumulerende tyder på, at det er gennem en ændring af cellevæggen kemi, planter lokalt styre denne ekspansion. Udvidelsen menes primært at være drevet af belastning af cellevæggene, forårsaget af cellen høje saftspændingen; denne stamme respons på saftspændingen er underlagt de mekaniske egenskaber af cellevæggene 1. Lidt er kendt af disse mekaniske egenskaber, og hvordan de ændrer sig under udvikling. Endvidere lidt er kendt, hvordan disse mekaniske egenskaber er kontrolleret og om feedback bidrage til at ændre cellevæg kemi på en måde, at der åbenbart er koordineret på en væv. Hvis vi skal forstå sammenhængen mellem kemiske og mekaniske ændringer i planternes cellevægge under udviklingen, og i sidste ende, hvordan disse mikroskopiske interaktioner styrer en plante&#39;S makroskopisk vækst, en metode, der kan overvåge mekaniske egenskaber af cellevægge i udviklingslandene organer på celler eller væv skala er påkrævet. Atomic force mikroskopi (AFM), der er beskrevet her, som er baseret på mikrometer eller nanometer væv kompressioner eller fordybninger, blev udviklet præcist at måle de mekaniske egenskaber af cellevægge i udviklingslandene organer samtidigt på subcellulære opløsninger og på tværs af hele regioner i væv. Andre metoder har enten en opløsning, der er for lav eller for høj: extensometerets er kun i stand til at måle de gennemsnitlige mekaniske egenskaber af en hel væv ved millimeter skala 2-4, en skala, der er for eksempel for stor til at måle tidlige hændelser i organogenesis; den microindenter kan tage målinger på subcellulære opløsning på nanometer skala, men den er begrænset til måling af isolerede celler og ikke grupper af celler eller organer 5-7. Med AFM, den kræverd væv, cellulære og subcellulære resolutioner kan opnås 8-10. For nylig flere protokoller er blevet udviklet specielt til at måle mekanik af planter væv, der også kan benyttes 11, 12. Vi præsenterer her, hvordan man vurderer elasticitet af væv gennem måling af den tilsyneladende Youngs modul 13. The Young modul er almindeligt anvendt til at beskrive stivheden af ​​et materiale. Under lille deformation den nødvendige kraft til at deformere et materiale er proportional med arealet af indrykning. The Young modul er denne koefficient. I tilfælde af en kontinuerlig homogent materiale vil blive målt samme koefficient uanset indrykningen type (størrelse og form), men vil ændre sig med hastigheden af ​​målingen. I tilfælde af den komplekse struktur af planter væv, har vi observeret, så langt, at kraften er proportional med deformation tillader bestemmelse afen koefficient på proportionalitet, som vi kalder &quot;tilsyneladende ung modul&quot;. I kontrast fra kontinuerlige medier i anlæggene, denne tilsyneladende unge modul er følsom over for størrelsen af ​​indrykning. Det svarer ikke til den unge moduli af en ren cellevæg. Det bedst beskriver elasticiteten af ​​stilladser af cellevæggen af ​​vævet.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">NanoWizard</td>
			<td style="width:167px;">All the 3-generation are abele to do the work withe the same preferment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM stage</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">CellHesion</td>
			<td>Required for sample withe low topography (les then 11&#181;m between the lowest and the highest point in the aria of force scanning).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM optics</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">Top View Optics&#160;</td>
			<td>Very important in order to position the sample. Cold be replaces by long range a binocular or microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo Microscopes&#160;</td>
			<td style="width:348px;">Leica</td>
			<td style="width:119px;">M125</td>
			<td>Any type of stereo microscopes could do.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">150nm mounted cantilever&#160;</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td style="width:119px;">R150-NCL-10</td>
			<td>To measure only the cell wall at the surface of the epidermis use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1&#181;m mounted cantilever&#160;</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td>SD-Sphere-NCH-S-10&#160;</td>
			<td>to measure the mechanics of the cell wall orthogonal to the surface of the epidermis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tipless cantiliver</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td>TL-NCH-20&#160;</td>
			<td>to measure the local mechanics of the tissue (2-3 cell wide) use a 5&#181;m mounted cantilever. We attached a 5&#181;m borasilicate bead to a tipless cantiliver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5&#181;m silicon microspheres</td>
			<td style="width:348px;">Corpuscular</td>
			<td style="width:119px;">C-SIO-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aradilte&#160;</td>
			<td style="width:348px;">Bartik S.A. 77170 Coubet France</td>
			<td style="width:119px;"></td>
			<td>Aradilte for fixing the bead to the tip les cantiliver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;low melting Agarows</td>
			<td style="width:348px;">Fishersci Fair Lawn , new jersey 07410</td>
			<td style="width:119px;">BP160-100</td>
			<td>34-45 Gelation Temperature</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">D-Mannitol</td>
			<td style="width:348px;">Sigma-Aldrich, 3050 Spruce Street, St Louis Mo 63103 USA)</td>
			<td style="width:119px;">M4125-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2&#160; Stainless Steel No. 5 Tweezers</td>
			<td style="width:348px;">Ideal-Tek 6828 Balerna Switzerland&#160;</td>
			<td align="right">951199</td>
			<td></td>
		</tr>
	</tbody>
</table>

afm-based mapping of the elastic properties of cell walls: at tissue, cellular, and subcellular resolutions

Vi beskriver en nyligt udviklet fremgangsmåde til at måle mekaniske egenskaber af overfladen af ​​plantevæv ved hjælp af atomic force mikroskopi (AFM) mikro / nano-indsnit, til JPK AFM. Konkret i denne protokol vi måler den tilsyneladende Youngs modul af cellevægge ved subcellulære beslutninger på tværs af regioner på op til 100 um x 100 um i blomstret meristems, hypokotyler, og rødder. Det kræver omhyggelig forberedelse af prøven, det korrekte valg af mikro-Indenters og indrykning dybder. At redegøre for cellevægs egenskaber kun er målinger udført i stærkt koncentrerede opløsninger af mannitol for at plasmolyze cellerne og dermed fjerne bidrag celle saftspændingen. I modsætning til andre bevarede teknikker, ved hjælp af forskellige Indenters og indrykning dybder, denne metode giver mulighed for samtidig multiscale målinger <em&gt; Dvs ved subcellulære resolutioner og på tværs af hundredvis af celler, der omfatter et væv. Dette betyder, at det nu er muligt at rumligt tidsmæssigt karakterisere de ændringer, der finder sted i de mekaniske egenskaber af cellevægge under udviklingen, således at disse ændringer korreleres med vækst og differentiering. Dette er et afgørende skridt for at forstå, hvordan koordinerede mikroskopiske cellulære ændringer medføre makroskopiske morfogenetiske begivenheder. Men flere begrænsninger er fortsat: metoden kan kun bruges på forholdsvis små prøver (ca. 100 um i diameter), og kun på eksterne væv; fremgangsmåden er følsom over for væv topografi; den måler kun visse aspekter af væv komplekse mekaniske egenskaber. Teknikken udvikles hurtigt, og det er sandsynligt, at de fleste af disse begrænsninger vil blive løst i den nærmeste fremtid.

I figur 1 præsenterer vi typiske Unge moduli kort over blomstermotiver meristems (figur 1A og 1B), unge og gamle hypokotyler (figur 1C-F), og rod meristem (figur 1G og 1H). I alle eksperimenter indrykning er halvkugleformet, men dens radius er forskellig, således at forskellige geografiske opløsninger kan opnås Figurerne 1C og 1D viser typiske resultater for meso-nanoskala Indenters...

Watch this Scientific Journal Video about AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions at JoVE.com

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

AFM-baserede Kortlægning af de elastiske egenskaber af cellevægge: ved Tissue, Cellular, og Subcellulære resolutioner

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

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Isolering af murint Ventil endotelceller

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Spænding og Calcium Dual Channel Optical Kortlægning af dyrkede HL-1 Atriel myocyt Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

Dissektion af Auditory Bulla i postnatale mus: Isolering af mellemøret Knogler og Histologisk analyse

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Analyse af Beta-celle funktion ved hjælp af encellede opløsning Calcium Imaging i zebrafisk Holme

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

Dynamisk cellebilledbehandling i superopløsning af undercellulære strukturer

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...