Name: measurement of tension release during laser induced axon lesion to evaluate axonal adhesion to the substrate at piconewton and millisecond resolution
Uploaded: 2013-05-27T12:00:00
Description: Watch this Scientific Journal Video about Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution at JoVE.

Alberto Guiggiani for at udvikle tidstro kontrolsystem, Evelina Chieregatti og Hanako Tsushima for indsigtsfulde diskussioner, Giacomo Pruzzo og Alessandro Parodi til udvikling af brugerdefinerede elektronik og software, og Claudia Chiabrera og Marina Nanni for deres ekspert rådgivning og bistand i cellekultur forberedelse.

1.. Optisk opsætning Hele det optiske system blev beskrevet tidligere 15.. Kort fortalt er det optiske pincet-system baseret på en ytterbium kontinuert bølge (CW) fiberlaser kører ved 1064 nm (IPG Laser GmbH). En rumlig lysmodulator (SLM) (LCOS-SLM, model X10468-07 - Hamamatsu) varierer fasen af ​​den indkommende IR laserstråle til at styre positionen af ​​fældefangst fokus plet på dyrkningsskålen ved computer genererede hologrammer. De frit tilgængelige Blue-pincet...

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Vi rapporterer i dette arbejde en kvantitativ metode til at sammenligne den neurit vedhæftning til dyrkningssubstrat, ved at udføre samtidig kraft spektroskopi målt i løbet laserinduceret cellelæsion. Den målte frigivelse af spænding er relateret til graden af ​​adhæsion af cellen til substratet: celler med et højere antal fokale adhæsioner bør frigive mindre spænding. Måling frigivelsen af spænding i form af piconewtons giver en fysisk mængde, som at vurdere axon vedhæftning til kulturen støtte i f...

Optisk mikroskopi er en af ​​de mindre invasiv billeddannende system til rådighed til at observere levende celler. Med udnyttelse af effekter såsom stråling tryk (som i optisk pincet 1), eller høj-energi fotonflux (som i laser dissektor 2), blev denne teknologi udvidet til nano-manipulation. Det optiske billedsystem fremlægger en præcis kontrol at visualisere og manipulere sub cellulære mål 3.. Samtidig, at præcis kalibrering af den leverede laser magt tak, optiske værktøjer udrette enten blødt eller invasive prøve manipulation med en hidtil uset reproducerbarhed. Adskillige laboratorier integreret i den samme forsøgsopstilling, optisk pincet og laser dissektoren for at polere organeller 4, at smelte sammen forskellige celler 5, eller at stimulere celler ved optisk drevet laster 6,7. Mens optisk pincet, efter kalibrering af den optiske stivhed, tilladerkontrol af påførte kraft til cellen på en piconewton skala, kan laser dissektion systemer modulere optisk manipulation, som spænder fra membran foto-porering til ablation af enkelte organeller eller dissektion af sub-cellulære strukturer. Men laser dissektion kalibrering afhængig kvalitativ vurdering af den enhed af optisk manipulation med hensyn til energi, der leveres til prøven, hovedsagelig baseret på billedanalyse illustrerer morfologiske ændringer forårsaget til modellen 8.. I det præsenterede metode, viser vi, hvordan du udfører force spektroskopi målinger under laseren axonal dissektion af et udviklingsland neuron, at kvantificere på piconewton skala den kraft der produceres af en ændret balance i cytoskelettet struktur af en sub-cellulært rum 9.. Dyrkede neuroner klæbe til substratet, og polarisere under udvikling. Polariseringen fase opstår i løbet af de første fem dage in vitro. Ved fase to af polarisering, en af ​​extrudING neuritter bliver længere, og det vil differentiere at blive Axon 10. Axonal forlængelse i respons på trækkraft på væksten kegle er tidligere modelleret af Dennerl model 11.. For nylig er denne model blevet udvidet 12 til også at omfatte den rolle neurit vedhæftning til den ekstracellulære matrix substrater. Denne biofysisk model foreslået efter eksperimentelle observationer 13, viste, at trækkræfter på vækst kegle, udbreder sig langs neurit, moduleres ved fokale adhæsioner til underlaget. Ligeledes axonal læsion producerer en lokal frigivelse af spænding formerings mod cellen kroppen. Således har vi foreslået, at måling af sådan frigivet spænding i en placering langs Axon mellem læsionen og den celle soma giver mulighed for at vurdere den afdæmpende resultat af upåvirket fokale adhæsioner. Vi kalibrerer den nødvendige energi foton-flux af laser dissektoren at styre omfanget af den påførte axonal damage, fra fuldstændig transection til delvis læsion. Efter kalibrering, gentog vi delvis læsion til axoner i flere differentierende neuroner og udviklet protokollen at kvantificere spændingen frigivelse, og således opnåede et kvantitativt parameter at estimere vedhæftning Axon til substratet 14.. I det foreliggende arbejde, beskriver vi i detaljer udviklede protokol, som repræsenterer en præcis eksperimentel procedure for at vurdere og sammenligne med piconewton følsomhed den axonal vedhæftning til underlaget i forskellige eksperimentelle tilstande såsom kemisk behandling 14, eller forskellige typer af cellekultur støtte.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Polymer microspheres, &Oslash; 4 &mu;m, COOH coated</td>
			<td>Bangs laboratories</td>
			<td>PC05N/6700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PolyLink Protein Coupling Kit</td>
			<td>Polyscience</td>
			<td>19539</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>IR laser</td>
			<td>IPG Laser GmbH</td>
			<td>YLM-5-SC-LP</td>
			<td>ytterbium continuous wave (CW) fiber laser operating at 1064 nm, with linear polarization</td>
		</tr>
		<tr>
			<td>Spatial light modulator</td>
			<td>Hamamatsu</td>
			<td>LCOS-SLM 10468-07</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue-tweezers software</td>
			<td>Optics group, University of Glasgow</td>
			<td>Free downloadable software</td>
			<td><a href="http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/" target="_blank">http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/</a></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>Free downloadable software</td>
			<td><a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/ </a></td>
		</tr>
		<tr>
			<td>QPD</td>
			<td>Thorlabs</td>
			<td>S5980 with C5460SPL 6041 board</td>
			<td>Four quadrant photo-diode to measure x, y trapped probe displacement</td>
		</tr>
		<tr>
			<td>PD</td>
			<td>Teem Photonics</td>
			<td>PDA100A-EC</td>
			<td>Photodiode to measure z trapped probe displacement</td>
		</tr>
		<tr>
			<td>nano-Pulse UV laser</td>
			<td>AA-optoelctronics</td>
			<td>PNV-001525-040</td>
			<td>Pulsed UVA laser, pulse length 400 ps</td>
		</tr>
		<tr>
			<td>Acoustic Optic Modulator</td>
			<td>Olympus</td>
			<td>MQ110-A3-UV, 355 nm fused silica</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Andor</td>
			<td>BX51</td>
			<td>Equipped with a 60X, 0.9 NA, water dipping objective</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>Warner Instruments</td>
			<td>V887ECSUVB EMCCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peltier device</td>
			<td>Physic Instruments</td>
			<td>QE1 resistive heating with TC-344B dual channel heater controller</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope stage: micro+piezo stage</td>
			<td>National Instruments</td>
			<td>Three linear stages M-126.CG1 carrying a separate 3-axis piezoelectric nano-positioning stage P-733.3DD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Daq</td>
			<td>&nbsp;</td>
			<td>NI PCI-6229</td>
			<td>Acquiring the x, y, z position of the trapped probe, and sending feedback loop signals to microscope stage</td>
		</tr>
		<tr>
			<td>Linux Real Time Application Interface (RTAI) machine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Real time feedback loop system, to control stage position, developed on a dedicated PC desktop</td>
		</tr>
	</tbody>
</table>

measurement of tension release during laser induced axon lesion to evaluate axonal adhesion to the substrate at piconewton and millisecond resolution

Dannelsen af ​​funktionelle forbindelser i et udviklingsland neuronal netværk er påvirket af udefrakommende signaler. Den neurite væksten i udviklingslandenes neuroner er underlagt kemiske og mekaniske signaler, og de mekanismer, som det sanser og reagerer på mekaniske signaler dårligt forstået. Belyse rollen af ​​kræfter i cellemodning vil gøre udformningen af ​​stilladser, der kan fremme celleadhæsion og cytoskelet kobling til underlaget, og dermed forbedre kapaciteten af ​​forskellige neuronale typer at regenerere efter skade. Her beskriver vi en metode til at anvende samtidige kraft spektroskopi målinger under laserinduceret cellelæsion. Vi måler spændingen frigivelse i delvis læderede Axon ved samtidig interferometrisk sporing af en optisk fanget sonde klæbet til membranen af ​​Axon. Vores forsøgsprotokol registrerer spændingen udgivelse med piconewton følsomhed og dynamikken i spændingen frigivelsemillisekund tidsopløsning. Derfor, det giver en høj opløsning metode til at undersøge, hvordan den mekaniske kobling mellem celler og substrater kan moduleres ved farmakologisk behandling og / eller ved forskellige mekaniske egenskaber af substratet.

Cellen genererer trækkræfter på substratet ved dens fokale adhæsioner. Kraft, der genereres af cytoskeletelementer er i ligevægt med den modvirkende kraft dyrkningssubstrat. Efter laser induceret læsion af neurit, er nogle af de spændte cytoskelet kabler forstyrret og deres ækvilibreret spændingen udløses, fordi den modsatrettede kraft af substratet vedhæftning elimineres. Den frigjorte spænding er delvist fordelt på de upåvirkede fokale adhæsioner, og vulsten knyttet til cellemembranen, holdt i en optisk...

Watch this Scientific Journal Video about Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution at JoVE.

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

Vi målte spænding frigivelse i en Axon der blev delvist læderet med en laser dissektor ved samtidig kraft spektroskopi måling udføres på en optisk fanget sonde klæbet til membranen af ​​Axon. Den udviklede eksperimentelle protokol evaluerer Axon vedhæftning til dyrkningssubstrat.

Måling af Tension frigivelse i løbet Laser Induced Axon Læsion at evaluere axonale vedhæftning til underlaget ved Piconewton og millisekund

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Påvisning af Exosomal Biomarkør af elektrisk felt-induceret frigivelse og måling (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

En metode til at anslå dødt lårbenet kortikale stammer under fraktur test ved hjælp af Digital billed korrelation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

Intraductal Delivery to the Rabbit Mammary Gland

Intraduktal Levering til kanin mælkekirtlerne

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

Miljømæssige dynamisk mekanisk analyse til at forudsige den blødgørende opførsel af neurale implantater

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Imaging Integrin spændinger og cellulære kraft på Submicron opløsning med en integrativ spænding Sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...