Name: 3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles
Uploaded: 2014-10-01T13:00:00
Description: Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1. Prøvefremstilling <ol><li> Oprethold CHOK1-celler i vævskulturkolber ved hjælp DMEM suppleret med 10% føtalt bovint serum og 100 IE / ml penicillin 50 ug / ml streptomycin. Inkubér cellerne i 5% CO2 befugtet inkubator ved 37 ° C. </li><li> Høst og derefter pladen CHOK1 celler på en 14 mm i diameter mikro-godt med en overflade tykkelse på 0,16 mm. Seed celler for optimal tæthed til billeddannelse, omkring 60-70% sammenflydning. </li><li> Cellerne inkuberes natten over ved 37 ° C, 5% CO2.</...

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Trods de enorme fremskridtene i fluorescensmikroskopi teknikker og besætninger i de seneste år, opnåede baner fluorescerende partikler i tre dimensioner med en høj tidsmæssig opløsning er forblevet en udfordring på området. Hvis høj tidsmæssig opløsning er opnået sporing partikler i to dimensioner, udvidelse til den aksiale retning bringer typisk en drastisk reduktion i frekvensgangen af systemet 10. I denne video-protokol fokuserede vi på den demonstrative anvendelse...

Et stort antal af fremgangsmåder er blevet udviklet til dato til at spore fluorescerende partikler i tre dimensioner ved hjælp af et mikroskop. De fleste metoder hviler på anvendelse af hurtige kameraer, velegnet til at spore i to dimensioner, typisk kombineret med tilpassede modifikationer af emission optik mikroskop for at opnå tracking i den aksiale retning. Laserscanningmikroskoper (enten konfokal eller to fotoner) konventionelt kan spore en fluorescerende partikel ved at udføre en sekvens af z-stakke, selv om denne proces er typisk tidskrævende, og giver rimelig tid opløsning (10 Hz), hvis partikel, der spores, er holdes i centrum af en lille raster billeddannelse region af en aktiv feedback-mekanisme 2. Ideen om at binde sig til partiklen er grundlaget for orbital sporing metode. I stedet for en raster scanne en cirkulær bane udføres omkring fluorescerende partikel. Intensiteten af ​​fluorescensen langskredsløb nøjagtigt lokaliserer partiklen stilling 4. Den lokaliserede stilling af partiklen kan derefter anvendes til at aktivere mikroskop scannere og recentrere orbit på partiklen stilling. Galvanometret scannere mikroskopet som drives af en analog spænding. AC komponent af denne spænding gør det muligt at udføre en bane med den fokuserede laserstråle, dvs en sinus og en cosinus bølge påføres X og Y scanning spejle vil tillade udførelse af en cirkulær bane. DC offset signalet lettere at ændre positionen af ​​kredsløbet centrum. Når en bane, bestemmes og bølgeform til AC signalet er konfigureret, feedback-systemet behov for at opdatere kun DC-komponenten af ​​signalet. En software i stand til at læse det fluorescerende signal opsamlet fra detektorerne er nødvendig for at styre scannere og detektorerne, at beregne positionen af ​​partiklen og opdatere DC fraindstillet. For en succesfuld tilbagemelding imaging et omhyggeligt valg af Orbit parametre (størrelse og timing) er påkrævet, og disse parametre skal justeres på grundlag af de særlige kendetegn ved de fluorescerende partikler, at det er nødvendigt at spore. De fysiske og matematiske grundlag for teknikken blev beskrevet tidligere 4,5 for både 2D og 3D-applikationer. I denne protokol vi kort beskrive de vigtigste hardwarekomponenter opsætningen, og mere detaljeret valget af parametre og af prøveforberedelse kræves for en typisk eksperiment tillader os efter forskydningen af ​​en lysosom ved en høj tidslig opløsning i en levende celle.

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			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
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			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
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			<td></td>
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			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
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			<td></td>
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			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
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			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
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			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
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			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
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			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
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3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

Formålet med denne video protokol er at diskutere, hvordan man kan udføre og analysere en tredimensionel fluorescerende orbital partikel sporing eksperiment ved hjælp af en modificeret to-foton mikroskop 1. I modsætning til konventionelle metoder (rasterskandering eller bredt felt baseret på en stak af rammer), 3D orbital sporing giver mulighed for at lokalisere og følge med en høj rumlig (10 nm nøjagtighed) og tidsmæssig opløsning (50 Hz frekvens respons) 3D forskydning af en bevægende fluorescerende partikel på længde-skalaer hundrede mikrometer 2. Metoden er baseret på en feedback-algoritme, der styrer hardwaren på et to-foton-laserscanningmikroskop for at udføre en cirkulær bane omkring genstanden, der skal spores: feedback mekanisme vil opretholde det fluorescerende objekt i midten ved at styre forskydningen af scanningsstrålen 3-5. For at vise fordelene ved denne teknik, vi fulgte en hurtig bevægelse organel, lysosomet inden aliving celle 6,7. Celler blev udpladet i overensstemmelse med standardprotokoller og farvet under anvendelse af en kommercielt lysosom farvestof. Vi diskuterer kort hardwarekonfiguration og mere detaljeret kontrol software, til at udføre en 3D orbital sporing eksperiment inde levende celler. Vi diskutere i detaljer de parametre, der kræves for at styre scanning mikroskop og muliggøre bevægelse af strålen i et lukket kredsløb omkring partiklen. Vi konkluderer ved at demonstrere, hvordan denne metode kan bruges effektivt til at spore den hurtige bevægelse af en mærket lysosom langs mikrotubuli i 3D i en levende celle. Lysosomer kan bevæge sig med hastigheder i området 0,4-0,5 um / s, der typisk viser en rettet bevægelse langs mikrotubulære netværk 8.

Ifølge denne protokol hurtigt 3D enkelt partikel sporing kan udføres inde i levende celler ved hjælp af en modificeret to-foton mikroskop for at spore forskydning af fluorescensmærkede lysosomer. Forsøget udføres består spore en isoleret lysosom bevæger inde i cellen efter endosomet modningsprocessen 9. De lysosomer blev farvet ved anvendelse af et fluorescerende grønt farvestof og exciteret ved 930 nm, der udnytter to-foton excitation. Vore data viser, at det er muligt at opnå x, y, z fors...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

3D Orbital Tracking i en modificeret to-foton mikroskop: en ansøgning til sporing af intracellulære vesikler

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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