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 arbejde blev støttet af National Institute of General Medical Sciences af National Institutes of Health under award nummer R35GM124868 og ved Duke University.

1. opsætning af Layout og justering
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	<li>Installation og collimation af tracking excitation laser<ol>
		<li>Anbringe laser den optiske tabel ved hjælp af et hjem-bygget mount. Mount er en simpel aluminiumplade med monteringshuller for laser hovedet og tabellen optisk. Laser skal være solidt fastgjort til en metal mount for stabilitet og varmeafledning. For dette arbejde, bruge en 488 nm solid state laser til belysning (figur 1), selvom bølgelængden kan vælges der passer til en b...

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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 sorter af 3D enkelt partikel sporingsmetoder er dukket op i de seneste år, er robust real-time tracking af høj hastighed 3D diffusion til lav foton count priser med en simpel opsætning stadig en udfordring, hvilket begrænser dets anvendelse til vigtige biologiske problemer. 3D-DyPLoT metode beskrevet i denne protokoladresser disse udfordringer på flere måder. Først, excitation og påvisning veje forenkles betydeligt i forhold til andre implementeringer gør justering, enkel og robust. For det andet, ...

Fremkomsten af avanceret billedbehandling teknikker har åbnet et vindue for at se mere detaljerede struktur af cellulære fænomener, hele vejen ned til det molekylære niveau. Metoder såsom stokastiske optisk genopbygning mikroskopi (STORM)1,2,3, foto-aktiveret lokalisering mikroskopi (PALM)4,5,6,7 , struktureret belysning mikroskopi (SIM)8,9,10,11, og stimuleret emission udtynding mikroskopi (STED)12,13, 14 er gået langt ud over diffraktion grænsen til at levere hidtil usete detaljer i struktur og funktion af levende celler. Dog kræver fuld forståelse af hvordan systemerne opføre sig dynamisk information samt strukturelle oplysninger. Super-opløsning metoder ovenfor indebærer en afvejning mellem rumlige og tidsmæssige opløsning, begrænse den tidsmæssige præcision som dynamiske processer kan blive aftestede. En metode, der giver både højt rumlige præcision og tidsmæssige opløsning er RT-3D-SPT15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29. Her, vi skelne mellem traditionelle 3D-SPT30 og RT-3D-sømløse rør traditionelt 3D-SPT blot kræver en tidsserie af tre-dimensionelle billeddata (som kan erhverves enten ved hjælp af en Konfokal mikroskop eller et epifluorescensmikroskop givet de rigtige konfiguration). I traditionel 3D-SPT fastsættes koordinaterne af partiklen efter indsamling af data ved at finde partiklen i hver billedstak og sammenskriver steder i successive diskenheder til at oprette en bane. For disse metoder bestemmes den ultimative tidsmæssige opløsning af den volumetriske imaging sats. For Konfokal mikroskoper er dette uden sammenligning på skala fra sekunder til snesevis af sekunder. For epifluorescensmikroskop metoder, hvori den optiske transmissionslængde manipuleres således at den aksiale placeringsoplysninger kan udtrækkes, er den tidsmæssige opløsning begrænset af kamera eksponering eller udlæsning tid. Disse epifluorescerende metoder er begrænset i rækken over som aksial oplysninger kan indsamles, selv om de seneste fremskridt i Fourier flyet fase masker design og adaptiv optik udvidelse af disse intervaller til 10 µm eller flere31,32 , 33 , 34.
Derimod stole RT-3D-sømløse rør ikke på erhverve en 3D billedstak og finde partikler efter kendsgerning. I stedet, real-time placeringsoplysninger udvindes via singlepoint detektorer og feedback er anvendt til effektivt "låse" partiklen af fokale omfanget af formålet objektivet ved hjælp af en highspeed piezoelektriske fase. Dette giver mulighed for kontinuerlig måling af den partikel holdning begrænset kun af hvor mange fotoner kan afhentes. Desuden, denne metode gør det spektral forhør af partiklen, som den bevæger sig over lange afstande. RT-3D-sømløse rør i realiteten fungerer som en kraft-fri optisk fælde for nanoskala objekter, hvori partiklen er løbende aftestede og målt i realtid uden behovet for stor laser beføjelser eller optisk styrker. Eftersom at RT-3D-sømløse rør giver mulighed for kontinuerlig forhør af hurtigt diffuserende objekter (op til 20 µm2/s)25,29 i tre dimensioner på lav foton count satser20,29, 35, bør det give et vindue ind hurtigt eller forbigående biologiske processer såsom intracellulære fragt transport, ligand-receptor binding og ekstracellulære dynamikken i enkelt virioner. Men til dette punkt, anvendelsen af RT-3D-SPT har været begrænset til håndfuld af grupper, der arbejder for at fremme denne teknologi.
En hindring er kompleksiteten af den optiske layout kræves af RT-3D-SPT metoder, der er varieret. For de fleste metoder, er den optiske feedback fra en piezoelektriske fase. Som partikel gør små bevægelser i X, Y eller Z, udlæsninger fra enkelt punkt detektorer er konverteret til fejl funktioner og fodret på højhastighedstog til en piezoelektriske nanopositioner, som i slå flytter prøve at modvirke den partikel bevægelse, effektivt låse det på plads i forhold til de objektive linse. Til at måle små positionelle bevægelser i X, Y og Z, enten flere detektorer (4 eller 5 afhængigt af gennemførelsen)15,18,21 eller flere excitation steder (2-4, nederst som kan anvendes, hvis en lock-in forstærker bruges til at udtrække X og Y position ved hjælp af en roterende laser spot)25,28 anvendes. Overlapningen af disse flere påvisning og emission steder besværliggør systemerne til at justere og vedligeholde.
Heri, præsenterer vi en højhastigheds target-låst 3D-SPT metode med en forenklet optisk design kaldes 3D-DyPLoT29. 3D-DyPLoT bruger en 2D-EOD og et TAG linse36,37,38 dynamisk flytte en fokuseret laser spot gennem den objektive fokale volumen ved en høj (50 kHz XY, 70 kHz Z). Kombinere laser fokus position og photon ankomsttid gør det muligt for den partikel 3D position opnås hurtigt selv ved lav foton count priser. 2D-EOD drev laser fokus i en ridder tour mønster39 med en kvadratisk størrelse på 1 x 1 µm i X-Y plane og TAG objektivet flytter laser fokus i aksial retning med et udvalg af 2-4 µm. 3D partikel position er opnået med en optimeret position estimering algoritme29,40 i 3D. Kontrol af den 3D dynamisk flytte laser spot, foton optælling fra lavine fotodiode (APD), real-time partikel holdning beregning, piezoelektriske fase feedback og dataregistrering udføres på et felt programmerbare gate array (FPGA).I denne protokol, vi beskriver, hvordan man opbygger en 3D-DyPLoT mikroskop trinvise, herunder optiske justering, kalibrering med faste partikler, og endelig gratis partikel tracking. Som en demonstration, blev 110 nm fluorescerende perler sporet kontinuerligt i vand i minutter ad gangen.
Den metode beskrevet heri er et ideelt valg for enhver anvendelse, hvor det ønskes at kontinuerligt overvåge en holdbar-omflytning fluorescerende sonden ved lave lysniveauer, herunder vira, nanopartikler og blærer som endosomes. I modsætning til tidligere metoder er der kun en enkelt excitation og enkelt påvisning pathway, hvilket gør justering og vedligeholdelse ligetil. Desuden muliggør store bevægelsesdetektering område denne mikroskop til nemt at afhente hurtigt spreder partikler, mens mulighed for at spore ved lav signal niveauer (ned til 10 kHz) gør denne metode ideel til lav-lys 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

Real-time tredimensionale enkelt partikel tracking (RT-3D-SPT) har potentiale til at kaste lys over hurtig, 3D processer i cellulære systemer. Selvom forskellige RT-3D-SPT metoder har været fremlagt i de seneste år, stadig sporing af høj hastighed 3D spreder partikler til lav foton count priser en udfordring. Derudover er RT-3D-SPT opsætninger generelt komplekse og vanskelige at gennemføre, begrænse deres udbredte anvendelse til biologiske problemer. Denne protokol præsenterer en RT-3D-SPT system opkaldt 3D dynamisk Photon lokalisering Tracking (3D-DyPLoT), som kan spore partikler med høj diffuserende hastighed (op til 20 µm2/s) til lav foton count priser (ned til 10 kHz). 3D-DyPLoT beskæftiger en 2D elektro-optisk deflektor (2D-EOD) og en afstemmelige akustiske gradient (TAG) linse til at drive en enkelt fokuseret laser spot dynamisk i 3D. Kombineret med en optimeret position estimering algoritme, kan 3D-DyPLoT låse på én partikler med høj sporing hastighed og høj lokalisering præcision. Enkelt excitation og enkelt registrering sti layout er 3D-DyPLoT robust og let at sætte op. Denne protokol beskriver hvor hen til opbygge 3D-DyPLoT trin for trin. Først, den optiske layout er beskrevet. Næste, systemet er kalibreret og optimeret af raster scanning en 190 nm fluorescerende perle med den piezoelektriske nanopositioner. Endelig, for at vise real-time 3D inddeling evne, 110 nm fluorescerende perler er sporet i vand.

Fast partikel scanning (figur 4) og frit sprede 110 nm fluorescerende partikel tracking (figur 5) blev udført efter den ovennævnte protokol. Partikel scanning blev udført ved at flytte de piezoelektriske nanopositioner og bin fotoner, mens samtidig beregner partiklen anslået position på hvert punkt i scanningen. Scanning billedet viser en square selv intensitet (figur 4a) og de anslåede positio...

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 protokol detaljer opførelse og drift af en real-time 3D enkelt partikel tracking mikroskop i stand til at spore nanoskala fluorescerende sonder ved høj diffuserende hastighed og lav foton count priser.

En protokol til Real-time 3D enkelt partikel Tracking

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

The overall goal of this protocol is to implement a real-time, 3D, single particle tracking microscope. The main advantage of this technique is that a single diffusing nano scale object can be monitored continuously with photon limited temporal resolution. This technique can help answer key questions in the field of virology, such as following a single virion through the entire infection process.Prepare the apparatus on an optical table. This setup is ready for calibration. Key components include a 488 nanometer solid state laser, two electro-optic deflectors that are along the beam's path, a tunable acoustic gradient lens, and a sample stage with micro and nano positioners.This schematic of the setup provides additional details. Here is the laser beam path to the sample stage. The electro-optic deflectors and the tunable acoustic lens are along the beam's path.These devices dynamically drive the laser spot at the sample stage. Photons from the sample are counted by an avalanche photo diode. A camera monitors the position of the particle.The position of the sample is set using micro and nano controllers. A field programmable gate array controls the laser spot, real-time particle position calculation, stage feedback, and photon counting. Two samples have to be prepared.The first, requires 190 nanometer fluorescent beads diluted in phosphate buffered saline. Use a cover slip to prepare a fixed particle sample. Place 400 microliters of the bead solution onto the cover slip.Now, produce the free moving particles sample. It requires 110 nanometer fluorescent beads diluted in water. Create a free moving particle sample by placing 400 microliters of the solution onto a cover slip.Take the fixed particle sample to the microscope to mount. The sample is on the XY nano positioner. Turn on the laser, the photo detector, the controllers for the beam line elements, and for the positioners.Run the Piezo nano positioner in a closed loop. Next, remove the fluorescence emission filter from in front of the photo detector. Then, vary the sample's z-position with the micro positioner.As the position varies, use the photo detector to monitor the intensity of the laser's reflection. Maximize the intensity to identify the focal plane of the objective lens. With the sample in the focal plane, replace the fluorescence emission filter.Now, work with the computer control software. Use custom software to raster scan the sample. First, set a large scanning range, the difference between start and finish.Here, the range is 10 micrometers in Y and 10 micrometers in X.Also, set a large step size in each direction. Here, 200 nanometers. Set both Z fields to the previously identified position of the focal plane.Pushing scan will start the Raster scan. Start the scan. The software drives the electro-optic deflectors in a night's tour, collects counts from the photo detector, drives the nano positioner, and performs position calculations.The scan will find a particle that is identifiable in the software through a plata fluorescence. For each site in the scan, the software will also determine estimates of the particle position in X and Y, relative to the center of the electro-object deflector scan. After finding the particle, shrink the scanning range around it to two micrometers in X and Y.Also, decrease the step size for both to 100 nanometers.Add a scan range in the Z direction of two micrometers to bracket the fluorescent focus. Set the Z step size to 200 nanometers. Start the 3D raster scan to find the fluorescent focus.The data produced from the scan show the focal plane before the tunable acoustic gradient lens is powered on. Move on to work with the tunable acoustic lens setting within its control software. First, click connect, then, power on.Go to the pulse and gating control section, change the output trigger mode from RGB to multi-plane. Then, change it back to RGB to allow changing the output phase. Use the sliders to set the output phase between zero degrees, 90 degrees, and 270 degrees.Next, go to the frequency control section. There, select a resonance frequency of 68, 500 hertz. Move to the top of the screen and click advanced.Then, choose the settings tab. On the new screen, find the maximum frequency of the zero column. Adjust it to change the frequency search range.Click, save calibration, then, exit calibration. Return to frequency controls and change the resonance frequency. Immediately change it back to 68, 500 hertz to have the calibration take effect.In the amplitude control section, use the slider to slowly raise the amplitude to 35%After this, click lock resonance. When the frequency is locked, click unlock resonance. Return to the raster scan software.Input two micrometer scanning ranges centered on the particle in X and Y.Use a step size of 100 nanometers. Center the particle in a four micrometer range in Z, with a step size of 200 nanometers. In the scanning image that results the region of illumination representing the particle is about one micrometer wide.Based on this, adjust the XY scale parameter so the estimated positions in X and Y range from negative 0.5 to 0.5 micrometers. After a similar procedure with the Z scale set the nano positioner to the zero position. Turn off the power to the nano positioner before continuing.Replace the fixed particle sample with the freely moving particle sample. Then, make sure all of the equipment is on. Run the Piezo nano positioner in open loop.At the computer, set the tunable acoustic gradient lens software as before. Remove the fluorescence emission filter from the path to the photo detector. Find the focal plane by varying the sample Z position and maximizing the intensity of laser reflection, from the cover slip.After finding the focal plane, increase the focus position 15 micrometers into the solution. Replace the fluorescence emission filter. Open the custom tracking software.Set the position estimation parameters to the values found during optimization. Now, set the integral control constants, which dictate the response speed of the nano positioner. Start with the X constant.Enter a low value and increase it slowly. When oscillations begin in the particle position read-out, note the value of the control constant. Reduce the integral control constant to about 70%of its value at the onset of oscillation.Repeat the steps with the Y and Z control constants. Set he trigger to start tracking the track threshold to three kilohertz. Set track minimum, the trigger to end tracking, at 1.5 kilohertz.Start the experiment by clicking search and then clicking auto track. This movie shows the result of using the three dimensional, dynamic photon localization tracking microscope to track the diffusive motion of a 110 nanometer fluorescent nano particle in water. The particle's position, measured using the position read-out of the nano positioner is shown on the 3D axes.The inset is the read-out of the scientific sea moss camera. It demonstrates that the particle is held, locked in the focal volume, by the tracking system. The particle was tracked and its position recorded for more than two minutes.Once mastered, this technique can be done in under 50 minutes, if the system is properly aligned, and the calibration parameters set. After its development, this technique will help researchers in the field of drug delivery explore the pathways of internalization and transport of cellular cargo. After watching this video, you should have a good understanding of how to operate a zero-time, 3D single particle tracking microscope.

a-protocol-for-real-time-3d-single-particle-tracking

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 within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

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

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Longitudinal Måling af ekstracellulær matrix Stivhed i 3D tumormodeller med Partikel-tracking 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

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

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.

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

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

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Bestilling enkelte celler og Single Embryoner i 3D Indespærring: En ny enhed for High Content 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

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

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

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

In Situ Kortlægning af de mekaniske egenskaber af biofilm fra Particle-tracking 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

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

Real Time Overvågning af intracellulære Bile Acid Dynamics Ved hjælp af en genetisk indkodede FRET-baserede Bile Acid Sensor

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

In Situ Microscopy for Real-time Determination of Single-cell Morphology in 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 oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

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

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

In situ Mikroskopi for realtids bestemmelse af enkelt 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

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Fremstilling af en Nafion-belagt, reduceret Graphene oxid/Polyaniline Chemiresistive Sensor til skærm pH i Real-time under mikrobiel gæring

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

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Optagelsen af nye Lipid-belagt nanopartikler som indeholder Falcarindiol af humane mesenkymale stamceller

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

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Generering af heterogene stof gradienter på tværs af Cancer populationer på en Microfluidic Evolution Accelerator for real-time observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...