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

Questo lavoro è stato supportato dal National Institute of General Medical Sciences del National Institutes of Health, sotto numero premio R35GM124868 e dalla Duke University.

1. impostare il Layout e l'allineamento
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	<li>Installazione e collimazione dell'eccitazione rilevamento laser<ol>
		<li>Apporre il laser alla tabella ottico usando un montaggio autocostruite. Il Monte è un piatto semplice in alluminio con fori per la testa del laser e la tabella ottica di montaggio. Il laser deve essere ben fissato a un supporto di metallo per stabilità e dissipazione del calore. Per questo lavoro, è necessario utilizzare un laser a stato solido 488 nm per l'illuminazione (F...

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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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A., Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+photoactivated+localization+microscopy+of+nanoscale+adhesion+dynamics.">Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.</a> Nat. Methods. 5 (5), 417 (2008).</li><li>Shao, L., Kner, P., Rego, E. H., Gustafsson, M. G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+3D+microscopy+of+live+whole+cells+using+structured+illumination.">Super-resolution 3D microscopy of live whole cells using structured illumination.</a> Nat. Methods. 8 (12), 1044 (2011).</li><li>Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L., Gustafsson, M. G. 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H., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+three+dimensional+tracking+of+a+single+nanoparticle+with+concurrent+spectroscopic+readouts.">Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts.</a> Appl. Phys. Lett. 88 (22), 223901 (2006).</li><li>Han, J. J., Kiss, C., Bradbury, A. R. M., Werner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Resolved,+Confocal+Single-Molecule+Tracking+of+Individual+Organic+Dyes+and+Fluorescent+Proteins+in+Three+Dimensions.">Time-Resolved, Confocal Single-Molecule Tracking of Individual Organic Dyes and Fluorescent Proteins in Three Dimensions.</a> ACS Nano. 6 (10), 8922-8932 (2012).</li><li>Wells, N. P., Lessard, G. A., Goodwin, P. M., Phipps, M. E., Cutler, P. J., Lidke, D. 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Anche se molte varietà di 3D singola particella metodi di monitoraggio sono emerse negli ultimi anni, robusto tracking in tempo reale di diffusione 3D ad alta velocità al prezzo di conteggio basso del fotone con un semplice programma di installazione è ancora una sfida, che limita la sua applicazione al biologico importante problemi. Il metodo 3D-DyPLoT descritto in questo protocollo indirizzi queste sfide in vari modi. In primo luogo, le vie di eccitazione e di rilevamento vengono semplificate notevolmente rispetto a...

L'emergere di tecniche avanzate di imaging ha aperto una finestra per vedere la struttura sempre più dettagliata dei fenomeni cellulari, tutta la strada fino al livello molecolare. Metodi quali stocastici ricostruzione ottica microscopia (tempesta)1,2,3, foto-attivata localizzazione microscopia (PALM)4,5,6,7 , strutturato illuminazione microscopia (SIM)8,9,10,11e stimolato emissione svuotamento microscopia (STED)12,13, 14 vanno ben oltre il limite di diffrazione per consegnare un dettaglio senza precedenti nella struttura e nella funzione delle cellule vive. Tuttavia, la comprensione completa di come questi sistemi si comportano richiede informazioni dinamiche, nonché informazioni strutturali. I metodi di Super-risoluzione sopra elencati implicano un compromesso tra la risoluzione spaziale e temporale ad alta risoluzione, limitando la precisione temporale con cui possono essere sondati processi dinamici. Un metodo che fornisce sia alta precisione spaziale e temporale ad alta risoluzione è RT-3D-SPT15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29. Qui, abbiamo una distinzione tra tradizionale 3D-SPT30 e RT-3D-TSS. tradizionale 3D-SPT richiede semplicemente una serie temporale di dati di immagine tridimensionale (che possono essere acquistati o utilizzando un microscopio confocale o un microscopio a epifluorescenza Data la configurazione giusta). In tradizionale 3D-SPT, le coordinate della particella sono determinate dopo la raccolta dei dati di individuazione della particella in ogni serie di immagini e concatenando le posizioni in volumi successivi a creare una traiettoria. Per questi metodi, l'ultima risoluzione temporale è determinata dal tasso di imaging volumetrico. Per microscopi confocali, questo è facilmente sulla scala dei secondi per decine di secondi. Per i metodi epifluorescenza, in cui il percorso ottico viene manipolato in modo che le informazioni di posizione assiale possono essere estratte, la risoluzione temporale è limitata con il tempo di esposizione o della lettura di fotocamera. Questi metodi di epifluorescente sono limitati nella gamma sopra cui assiale informazioni possono essere raccolte, anche se recenti progressi nella fase di Fourier aereo maschere design e ottica adattiva è allungabile questi intervalli di 10 µm o più31,32 , 33 , 34.
Al contrario, RT-3D-SPT non si basa su l'acquisizione di una serie di immagini 3D e trovare particelle dopo il fatto. Invece, informazioni sulla posizione in tempo reale viene estratta tramite rivelatori di singolo punto e feedback viene applicato in modo efficace "bloccare" la particella nel volume focale della lente dell'obiettivo attraverso l'uso di una fase piezoelettrico highspeed. Questo consente la misura continua della posizione della particella limitata solo da quanti fotoni possono essere raccolti. Inoltre, questo metodo consente spettrale interrogatorio della particella che si muove su lunghi tragitti. RT-3D-SPT in effetti funziona simile a una forza-libero ottica trappola per oggetti su scala nanometrica, in cui la particella è continuamente sondata e misurata in tempo reale senza la necessità di alimentazioni grandi laser o ottico le forze. Dato che RT-3D-SPT fornisce un mezzo per l'interrogatorio continuo di oggetti veloce diffusivo (fino a 20 µm2/s)25,29 in tre dimensioni a basso fotone conteggio tariffe20,29, 35, dovrebbe fornire una finestra in processi biologici veloci o transitori come trasporto cargo intracellulare, legame ligando-recettore e la dinamica extracellulare dei virions singolo. Tuttavia, a questo punto, l'applicazione di RT-3D-SPT è stato limitato per la manciata di gruppi di lavoro per far progredire questa tecnologia.
Una barriera è la complessità del layout ottico richieste dai metodi RT-3D-SPT, che sono di vario tipo. Per la maggior parte dei metodi, il feedback ottico è fornito da una fase piezoelettrica. Come il particella fa piccoli movimenti in X, Y, o Z, letture da singolo punto rilevatori vengono convertite in funzioni di errore e alimentato a ad alta velocità per un nanopositioner piezoelettrico, che a sua volta muove il campione per contrastare efficacemente il movimento della particella, bloccandola rispetto alla lente dell'obiettivo. Per misurare piccoli movimenti posizionali in X, Y e Z, o più rivelatori (4 o 5 a seconda dell'implementazione)15,18,21 o punti multipli dell'eccitazione (2-4, il più basso dei quali può essere applicato se una amplificatore di lock-in è utilizzato per estrarre X e posizione Y utilizzando un rotante laser spot)25,28 vengono applicate. La sovrapposizione di questi punti di rilevamento ed emissione multipli rendono difficile allineare e mantenere i sistemi.
Qui, presentiamo un metodo ad alta velocità 3D destinazione-bloccato-SPT con un design ottico semplificato denominato 3D-DyPLoT29. 3D-DyPLoT utilizza un 2D-EOD e un TAG lente36,37,38 per spostare dinamicamente un punto laser focalizzato attraverso il volume focale obiettivo ad un ritmo elevato (50 kHz XY, 70 kHz Z). La posizione di messa a fuoco del laser e l'orario di arrivo del fotone la combinazione consente la posizione della particella 3D di ottenere rapidamente anche a tassi di conteggio basso del fotone. 2D-EOD spinge il fuoco del laser di un cavaliere tour modello39 con un quadrato di 1 x 1 µm nel piano X-Y e la lente di TAG si sposta il fuoco del laser in direzione assiale con una gamma di 2-4 µm. La posizione di particelle 3D si ottiene con una posizione ottimizzata stima algoritmo29,40 in 3D. Il controllo del 3D in modo dinamico movimento laser spot, fotone contando dal fotodiodo a valanga (APD), calcolo della posizione in tempo reale delle particelle, fase piezoelettrico feedback e registrazione dei dati vengono eseguite su un allineamento di cancello programmabile del campo (FPGA).In questo protocollo, descriviamo come costruire un microscopio 3D-DyPLoT passo dopo passo, compreso l'allineamento ottico, calibrazione con particelle fisse e finalmente libero il rilevamento di particelle. Come dimostrazione, perline fluorescenti di 110 nm sono stati monitorati continuamente in acqua per minuti alla volta.
Il metodo qui descritto è la scelta ideale per qualsiasi applicazione dove si desidera monitorare continuamente una sonda fluorescente veloci a bassi livelli di luce, tra cui virus, nanoparticelle e vescicole come gli endosomi. Contrariamente ai metodi precedenti, c'è solo una singola eccitazione e via sola rilevazione, rendendo semplice l'allineamento e la manutenzione. Inoltre, l'area di rilevamento grande consente questo microscopio raccogliere facilmente diffondere rapidamente le particelle, mentre la possibilità di monitorare a livello di segnale basso (fino a 10 kHz) rende questo metodo ideale per applicazioni di basso-luce29.

<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

Particelle singole tridimensionale in tempo reale (RT-3D-SPT) di rilevamento ha il potenziale per far luce sui processi veloci, 3D in sistemi cellulari. Anche se vari metodi di RT-3D-SPT sono state presentate negli ultimi anni, alta velocità inseguimento 3D diffondente particelle al prezzo di conteggio basso fotone rimane una sfida. Inoltre, configurazioni di RT-3D-SPT sono generalmente complessi e difficili da implementare, limitando la loro diffusa applicazione a problemi biologici. Questo protocollo presenta un sistema di RT-3D-SPT denominato 3D dinamico del fotone localizzazione Tracking (3D-DyPLoT), che permette di monitorare le particelle con diffusiva ad alta velocità (fino a 20 µm2/s) al prezzo di conteggio basso fotone (fino a 10 kHz). 3D-DyPLoT si avvale di un deflettore di elettro-ottica 2D (2D-EOD) e una lente sintonizzabile sfumatura acustica (TAG) all'unità un singolo focalizzato punto dinamicamente in 3D laser. In combinazione con un algoritmo di stima della posizione ottimizzata, 3D-DyPLoT può agganciare di singole particelle con inseguimento ad alta velocità e precisione di localizzazione alta. Grazie alla singola eccitazione e la struttura dei percorsi sola rilevazione, 3D-DyPLoT è robusto e facile da configurare. Questo protocollo viene descritto come costruire 3D-DyPLoT passo per passo. In primo luogo, è descritto il layout ottico. Successivamente, il sistema è calibrato e ottimizzato da raster di scansione una perlina fluorescente di 190 nm con la nanopositioner piezoelettrico. Infine, per dimostrare la capacità di tracciatura in tempo reale 3D, perline fluorescenti di 110 nm sono tracciati in acqua.

Particella fissa scansione (Figura 4) e liberamente diffondente 110 nm fluorescente particella di rilevamento (Figura 5) sono stati effettuati seguendo il protocollo di cui sopra. La particella di scansione è stata eseguita spostando i fotoni piezoelettrici nanopositioner e bin mentre simultaneamente calcolo della particella stimato posizione ad ogni punto nella scansione. L'immagine di scansione mostra un quadrato di intensità...

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

Questo dettagli del protocollo la costruzione e il funzionamento di un microscopio di rilevamento in tempo reale 3D singola particella capace di rilevamento su scala nanometrica fluorescente sonde ad alta velocità diffusiva e tassi di conteggio basso del fotone.

Un protocollo per il monitoraggio in tempo reale 3D singola particella

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.

Un semplice Hanging goccia Cultura protocollo cellulare per la generazione di sferoidi 3D

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

Ricerca

Bioingegneria

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.

Misurazione longitudinale di matrice extracellulare rigidità in 3D Tumore modelli utilizzando-particelle inseguimento 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 Inseguimento in una Modified microscopio a due fotoni: un&#39;applicazione per il monitoraggio di intracellulare vescicole

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.

Ordinazione di singole cellule e singola embrioni in 3D confinamento: Un nuovo dispositivo per l&#39;alta Screening contenuti

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 mappatura delle proprietà meccaniche del biofilm da-Particle tracciamento 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.

Monitoraggio in tempo reale di intracellulare Bile Acid Dynamics Utilizzando una sede a FRET geneticamente codificato Bile Acid sensore

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 Microscopia per la determinazione in tempo reale della morfologia a singola cellula nei bioprocessi

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.

Produzione di Nafion rivestite, ridotto grafene ossido/Polyaniline Chemiresistive sensore al pH Monitor in Real-Time durante la fermentazione microbica

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.

Assorbimento di nuove nanoparticelle rivestite con lipidi contenenti Falcarindiol di cellule staminali mesenchimali umane

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.

Generazione di gradienti di farmaci eterogenei in tutte le popolazioni di cancro su un acceleratore evoluzione microfluidico per l'osservazione in tempo reale

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