Name: a protocol for real-time 3d single particle tracking
Uploaded: 2018-01-03T13:00:00
Description: Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

Dette arbeidet ble støttet av National Institute of General Medical Sciences av National Institutes of Health prisen nummer R35GM124868 og Duke University.

1. oppsett oppsett og justering
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	<li>Installasjon og kollimasjon av sporing magnetisering laser<ol>
		<li>Påføre laser optisk tabellen ved hjelp av en hjemme-bygget mount. Fjellet er en enkel aluminiumsplate med monteringshuller laser hodet og optisk tabellen. Laser bør være godt festet til et metall montere for stabilitet og varmespredning. Til dette bruker du en 488 nm solid state laser for belysning (figur 1), men bølgelengden kan velges for å passe en bestemt fluorophore ell...

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	<li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat. Methods. 3 (10), 793-796 (2006).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Super-Resolution+Imaging+by+Stochastic+Optical+Reconstruction+Microscopy.">Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy.</a> Science. 319 (5864), 810-813 (2008).</li><li>Jones, S. A., Shim, S. H., He, J., Fast Zhuang, X. 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Selv om mange varianter av 3D enkelt partikkel oppfølgingsmetoder har dukket opp de siste årene, er robust sanntidssporing av høyhastighets 3D diffusjon til lav Foton teller priser enkelt fortsatt en utfordring, som begrenser sin søknad til viktige biologiske problemer. 3D-DyPLoT metoden beskrevet i denne protokollen løser disse utfordringene på en flere måter. Først er eksitasjon og oppdagelsen veier forenklet sterkt sammenlignet med andre implementeringer gjør justering enkel og robust. Dernest gir er bevegeli...

Fremveksten av avanserte imaging teknikker har åpnet et vindu for å se mer detaljert struktur av mobilnettet fenomener, helt ned til molekylært nivå. Metoder som Stokastisk optisk rekonstruksjon mikroskopi (STORM)1,2,3, foto-aktivert lokalisering mikroskopi (PALM)4,5,6,7 , strukturert belysning mikroskopi (SIM)8,9,10,11og stimulert utslipp uttømming mikroskopi (STED)12,13, 14 har gått langt utover Diffraksjon grensen til levere enestående detaljer i struktur og funksjon av levende celler. Men krever full forståelse av hvordan disse systemene fungerer dynamisk informasjon samt strukturinformasjon. Super-oppløsning metodene ovenfor innebære en avveining mellom romlig oppløsning og midlertidig løsning, begrense timelige presisjonen som dynamisk prosesser kan bli undersøkt. En metode som gir både høy romlig presisjon og midlertidig løsning er RT-3D-SPT15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29. Her vi trekke et skille mellom tradisjonelle 3D-SPT30 og RT-3D-SPT. tradisjonell 3D-SPT bare krever en gang rekke tredimensjonale bildedata (som kan erverves enten med AC confocal mikroskop eller et mikroskop-epifluorescence gitt den riktige konfigurasjonen). I tradisjonelle 3D-SPT bestemmes koordinatene av partikkel etter innsamling av finne partikkelen i hver bildestakk og å sette sammen stedene i påfølgende volumer til å opprette en bane. For disse metodene bestemmes ultimate timelige oppløsningen av volumetriske tenkelig hastigheten. For AC confocal mikroskoper er dette lett på omfanget av sekunder å titalls sekunder. For epifluorescence metoder, hvor den optiske banen endres slik at aksial stedsinformasjonen kan hentes, begrenses timelige oppløsningen kameraet eksponering eller avlesning da. Metodene epifluorescent er begrenset i området som aksial opplysninger kan samles, men framskritt i Fourier flyet fase masker design og adaptive optikk utvider disse områdene 10 µm eller mer31,32 , 33 , 34.
Derimot stole RT-3D-SPT ikke på å anskaffe en 3D bildestakk og finne partikler i ettertid. I stedet, sanntid stedsinformasjon er hentet via enkeltpunkt detektorer og tilbakemeldinger brukes effektivt "låse" partikkel i fokal mengden av linsen ved hjelp av et høyhastighets piezoelectric stadium. Dette tillater kontinuerlig måling av partikkels posisjon begrenset bare av hvor mange fotoner kan samles. Denne metoden kan videre spectral avhør av partikkel når den beveger seg over lange avstander. RT-3D-SPT faktisk fungerer som en kraft-fri optisk felle nanoskala objekter, der partikkelen er kontinuerlig analysert og målt i sanntid uten behov for stor laser krefter eller optisk styrker. Gitt at RT-3D-SPT gir et middel for kontinuerlig avhør raskt diffusive objekter (opptil 20 µm2/s)25,29 i tre dimensjoner for lav Foton teller priser20,29, 35, bør det gi et vindu inn fort eller forbigående biologiske prosesser som intracellulær Last transport, ligand-reseptor bindende og ekstracellulære dynamikken i én virions. Imidlertid til dette punktet er anvendelse av RT-3D-SPT begrenset til håndfull grupper som arbeider for å fremme denne teknologien.
En barriere er kompleksiteten av optisk oppsettet kreves av RT-3D-SPT metoder, som varierer. For de fleste metoder gis det optisk tilbakekobling av en piezoelectric scene. Som partikkel gjør små bevegelser i X, Y eller Z, readouts fra eneste detektorer konverteres til feilfunksjoner og matet på høy hastighet til en piezoelectric nanopositioner, som i sin tur går prøve å motvirke den partikkelen bevegelse, effektivt låse den på plass i forhold til linsen. Å måle små posisjonelle bevegelser i X, Y og Z, flere detektorer (4 eller 5 avhengig av implementeringen)15,18,21 eller flere eksitasjon flekker (2-4, lavere som kan brukes hvis en låsbare forsterker brukes til å hente X og Y posisjon ved hjelp av en roterende laser spot)25,28 brukes. Overlapping av disse flere gjenkjenning og utslipp flekker gjøre systemer vanskelig å justere og vedlikeholde.
Her presenterer vi en høyhastighets mål-låst 3D-SPT metode med en forenklet optisk design kalt 3D-DyPLoT29. 3D-DyPLoT bruker en 2D-EOD og en TAG linsen36,37,38 dynamisk flytte en fokusert laser spot gjennom objektive fokal volumet til en høy rente (50 kHz XY, 70 kHz Z). Laser fokus plasseringen og Foton ankomsttid kan partikkels 3D posisjon innhentes raskt selv på lav Foton teller priser. 2D-EOD kjører laser fokus i en ridders tour mønster39 square størrelse på 1 x 1 µm i X-Y flyet og TAG linsen flytter laser fokus i aksial retning med en rekke 2-4 µm. 3D partikkel plassering oppnås med en optimalisert posisjon estimering algoritmen29,40 i 3D. Kontroll av den 3D dynamisk bevegelse laser spot, Foton teller fra skred photodiode (APD), sanntid partikkel posisjon beregning, piezoelectric scenen tilbakemeldinger og datainnspilling utføres på en feltet programmerbare gate array (FPGA).I denne protokollen, vi beskriver hvordan å bygge en 3D-DyPLoT mikroskop trinnvis, inkludert optisk justering, kalibrering med faste partikler, og endelig gratis partikkel sporing. Som en demonstrasjon, ble 110 nm fluorescerende perler kontinuerlig sporet i vann for minutter om gangen.
Metoden beskrevet heri er et ideelt valg for alle programmer der det er ønskelig å kontinuerlig overvåke en raske fluorescerende sonde tåler lite lys, inkludert virus, nanopartikler og blemmer som endosomes. I motsetning til tidligere metoder er det bare en enkelt eksitasjon og enkelt oppdagelsen pathway, gjør justering og vedlikehold enkelt. Videre gjør området stort oppdagelsen denne mikroskop for å enkelt hente raskt spre partikler, mens muligheten til å spore på lav signal nivåer (ned 10 kHz) gjør denne metoden perfekt for dårlige programmer29.

<table border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">2D Electro-optic Deflector&#160;</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">M310A</td>
			<td style="width:541px;">2 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Power supply for EOD</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">412</td>
			<td>Converts FPGA ouput to high voltage for EOD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TAG&#160; Lens</td>
			<td style="width:145px;">TAG Optics</td>
			<td style="width:160px;">TAG 2.0</td>
			<td>Used to deflect laser along axial direction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">XY piezoelectric nanopositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-PDQ275HS</td>
			<td>Used for moving the sample to lock the particle in the objective focal volume in&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Z piezoelectric nanoposiitoner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-OP65HS</td>
			<td>Used to move the objective lens to follow the diffusing particle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Micropositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">MicroDrive</td>
			<td>Used to coarsely position sample and evaluate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Objective Lens</td>
			<td style="width:145px;">Zeiss</td>
			<td style="width:160px;">PlanApo</td>
			<td>High numerical aperture required for best sensitivity. 100X, 1.49 NA, M27, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">sCMOS camera</td>
			<td style="width:145px;">PCO</td>
			<td style="width:160px;">pco.edge 4.2</td>
			<td>Used to monitor the particle&#39;s position</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">APD</td>
			<td style="width:145px;">Excelitas</td>
			<td style="width:160px;">SPCM-ARQH-15</td>
			<td>Lower dark counts beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Field programmable gate array</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">NI-7852r</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Software</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">LabVIEW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tracking excitation laser</td>
			<td style="width:145px;">JDSU</td>
			<td style="width:160px;">FCD488-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-150-A-ML</td>
			<td>L1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Pinhole</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">P75S</td>
			<td>PH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Glan-Thompson Polarizer</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">GTH5-A</td>
			<td>GT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Half-wave plate</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">WPH05M-488</td>
			<td>WP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-75-A-ML</td>
			<td>L3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-250-A-ML</td>
			<td>L4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dichroic Mirror</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">ZT405/488/561/640rpc</td>
			<td>DC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Fluorescence Emission Filter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">D535/40m</td>
			<td>F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">10/90 beamsplitter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">21012</td>
			<td>BS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">190 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/9942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">110 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/10617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-545A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Powermeter</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">PM100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">CMOS</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">DCC1545M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Iris</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">SM1D12D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:145px;">Mad City Labs</td>
			<td style="width:160px;">RM21</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for real-time 3d single particle tracking

Sanntid tredimensjonale enkelt partikkel sporing (RT-3D-SPT) har potensial til å belyse rask, 3D prosesser i cellular systemer. Selv om ulike RT-3D-SPT metoder har blitt lagt frem de siste årene, fortsatt sporing høyhastighets 3D spre partikler til lav Foton teller priser en utfordring. Videre er RT-3D-SPT oppsett generelt komplisert og vanskelig å gjennomføre, begrense deres omfattende programmet biologiske problemer. Denne protokollen presenterer en RT-3D-SPT system kalt 3D dynamisk Foton lokalisering sporing (3D-DyPLoT), som kan spore partikler med høy diffusive hastighet (opptil 20 µm2/s) på lav Foton teller priser (ned 10 kHz). 3D-DyPLoT benytter en 2D elektro-optisk deflektor (2D-EOD) og en tunable akustisk gradering (TAG) linse å kjøre en enkelt fokusert laser spot dynamisk i 3D. Kombinert med en optimalisert posisjon estimering algoritme, kan 3D-DyPLoT låse på enkelt partikler med høy hastighet og høy lokalisering presisjon. Enkelt eksitasjon og enkelt oppdagelsen bane layout er 3D-DyPLoT robust og lett å konfigurere. Denne protokollen diskuterer hvordan bygge 3D-DyPLoT steg for steg. Først er optisk oppsettet beskrevet. Deretter er systemet kalibrert og optimalisert av raster skanning en 190 nm fluorescerende perle med piezoelectric nanopositioner. Til slutt, for å demonstrere real-time 3D-sporing evne, 110 nm fluorescerende perler spores i vann.

Fast partikkel skanning (Figur 4) og fritt spre 110 nm fluorescerende partikkel sporing (figur 5) ble utført etter protokollen ovenfor. Partikkel skanningen ble utført ved å flytte piezoelectric nanopositioner og bin fotoner mens samtidig beregne partikkels estimerte posisjon på hvert punkt i søket. Skanning bildet viser en firkant med intensitet (figur 4a) og anslått stillingene viser en line?...

Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

A Protocol for Real-time 3D Single Particle Tracking

Denne protokollen detaljer bygging og drift av real-time 3D enkelt partikkel sporing mikroskop kan sporing nanoskala fluorescerende sonder på diffusive høye og lave Foton teller priser.

En protokoll for sanntids 3D enkelt partikkel sporing

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Research

JoVE Journal

Bioengineering

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Langsgående Måling av ekstracellulære matrix Stivhet i 3D tumormodeller Bruke Partikkel-sporing Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

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

3D Orbital Tracking i en modifisert to-foton Microscope: en søknad til sporing av intracellulær Blemmer

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

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Bestilling av enkeltceller og enkelt Embryoet i 3D Confinement: En ny enhet for høyt innhold Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

In Situ Kartlegging av de mekaniske egenskapene til Biofilm av Partikkel-sporing Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

Sanntidsovervåking av Intracellulær gallesyre Dynamics Ved hjelp av en genetisk kodet FRET baserte gallesyre Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting brusk og huden vev Analogs utnytte en roman passiv miksing enhet teknikk for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ Mikroskopi for Real-Time bestemmelse av single-celle morfologi i Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Produksjon av en Nafion-belagt, redusert Grafén oksid/Polyaniline Chemiresistive Sensor til skjermen pH i sanntid under mikrobiell gjæring

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Opptak av nye Lipid-belagt nanopartikler som inneholder Falcarindiol av menneskelige stamceller

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...