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

Preparazione del campione 1 <ol><li> Mantenere le cellule CHOK1 in tessuto fiasche di coltura utilizzando DMEM supplementato con 10% di siero fetale bovino e 100 UI / ml di penicillina 50 mg / ml di streptomicina. Incubare le cellule in un 5% di CO 2 incubatore umidificato a 37 ° C. </li><li> Raccolta e poi plate cellule CHOK1 su un diametro di micro-pozzetto 14 mm e uno spessore superficie di 0,16 mm. Cellule seme per densità ottimale per l&#39;imaging, intorno al 60-70% di confluenza. </li><li> Incubare le cellule per una notte a 37 ° C, 5% di CO 2. Lavare le cellule tre volte in HBSS (Hank Buffered Saline Solution), e incubare le cellule in una soluzione contenente 50 nM di Lysotracker DND26 verde e 150 Nm di tubulina inseguitore verde. Incubare le cellule per 1 ora a 37 ° C. Passaggio facoltativo: Prima di incubazione, eseguire una macchia supplementare con un colorante colorazione matrice mitocondriale (25 Nm). </li><li> Lavare le cellule tre volte in HBSS per rimuovere il colorante non legato. Aggiungere fresco terreno di coltura DMEM prima imagzione le cellule. </li></ol> 2. Configurazioni Microscopio Il microscopio per il tracciamento di particelle descritto nel protocollo video viene montato sul telaio di un microscopio invertito disponibile in commercio (Figura 4). Tuttavia moduli commerciali per 3D tracciamento di particelle orbitali sono ora disponibili. <ol><li> Utilizzare un coerente Chameleon-Ultra II Ti: Sa a femtosecondi sorgente di luce di eccitazione laser, con una gamma di lunghezze d&#39;onda di uscita regolabile tra 690 nm-1.040 nm. </li><li> Assicurarsi che il raggio laser è allineato nella porta posteriore del microscopio utilizzando specchi IR-rivestito. Il raggio laser viene attenuato mediante una piastra semionda rotante seguito da un polarizzatore lineare calcite. Attenuazione del fascio in modo che la potenza media al campione è compreso tra 0,5 e 2 mW. NOTA: Il raggio laser viene riflesso da una coppia di specchi a comando galvanometro motori che consentono il controllo della posizione e traiettoria del fascio focalizzato nel piano campione. Inconfigurazione tipica del fascio laser collimato uscire degli specchi galvanometro viene espanso (10X), che passa attraverso un espansore del fascio, prima di entrare nel retro dell&#39;obiettivo microscopio dopo la riflessione nel passo corto specchio dicroico. </li><li> Raccogliere la luce di fluorescenza dal campione ponendo un obiettivo numerico ad elevata dell&#39;acqua di apertura (60X, NA 1.2) nel percorso ottico. Scegli un cubo filtro di fluorescenza in funzione delle lunghezze d&#39;onda di emissione desiderati in configurazione sia uno o due canali. Impiegare filtri passa-banda di emissione per selezionare ulteriormente la gamma spettrale della fluorescenza emessa. </li><li> Porre il campione su un palco motorizzato, e regolare la multa movimento dell&#39;obiettivo del microscopio con un piezo-controllore obiettivo. </li><li> Dirigere la luce dal cubo del filtro in tubi fotomoltiplicatori dove il segnale viene discriminati e inviato a una scheda di acquisizione dati I / O digitale. Generato dal computer forme d&#39;onda sono l&#39;uscita analogica della scheda I / O e sono provid: NOTAed agli scanner di controllo elettronica. L&#39;ingresso di conteggio di fotoni dai fotomoltiplicatori e il segnale di uscita per gli scanner sono misurati e controllati tramite la scheda I / O dal Laboratorio per il software Fluorescence Dynamics SimFCS. </li></ol> 3. Imaging <ol><li> Posizionare le cellule sul palco e mettere a fuoco utilizzando l&#39;illuminazione a luce trasmessa. </li><li> Passa alla scansione di immagini raster per identificare le cellule che hanno incorporato il colorante e di visualizzare i microtubuli sottostanti. Identificare l&#39;area iniziale in cui il movimento delle vescicole è presente. </li><li> Una volta che una vescicola isolato è identificato, impostare i seguenti parametri per l&#39;inseguimento orbitale: <ol><li> Selezionare il raggio dell&#39;orbita per definire le dimensioni della scansione circolare secondo la dimensione della particella monitorata. Per un emettitore punto, impostare il raggio dell&#39;orbita pari alla vita della Point Spread Function (PSF) del fascio di eccitazione al massimo la sensibilità e la risposta. </li><li> Impostare il assialedistanza per definire la distanza tra le due orbite che vengono eseguite per localizzare la posizione della particella lungo la direzione assiale. Impostare la distanza assiale a 1,5-3 volte la vita PSF. </li><li> Definire il tempo di sosta in base alla luminosità della particella per impostare il tempo speso su ogni punto dell&#39;orbita, che determinerà anche il tasso-photo sbiancamento. Utilizzare un tempo di sosta tra 10-100 msec. </li><li> Impostare il numero di punti in ciascuna orbita di 64 o 128 per produrre periodi orbita nell&#39;ordine di 4-32 msec e fornire un&#39;elevata risoluzione temporale per la determinazione della posizione della particella. </li></ol></li><li> Modificare il segnale di offset DC inviata agli specchi in modo da centrare il fascio sulla particella attraverso l&#39;interfaccia utente grafica mediante un cursore di selezione nell&#39;immagine raster-digitalizzata. </li><li> Inizia il monitoraggio commutando la modalità microscopio da raster-imaging per la scansione orbitale. </li><li> Attivare la raccolta di feedback e dati. </li></ol> 4. TrajectAnalisi ory <ol><li> Utilizzare il software per visualizzare la fluorescenza raccolte in ogni punto lungo l&#39;orbita e ad ogni tempo sotto forma di un &#39;tappeto intensità&#39;. L&#39;intensità raccolto lungo il tappeto fornisce informazioni sulle interazioni della particella con altri oggetti luminosi. Utilizzare il software per visualizzare sia le informazioni traiettoria (cioè lo spostamento CC nel tempo di x, y scanner e del -piezo z) e l&#39;intensità di fluorescenza raccolti dal tubo fotomoltiplicatore nel tempo sotto forma di serie temporali. </li><li> Utilizzare la rappresentazione delle serie storiche e le informazioni &#39;tappeto&#39; intensità &#39;di scegliere solo una regione di interesse nella traiettoria. </li><li> Utilizzare il software per visualizzare una proiezione 2D della porzione selezionata della traiettoria della particella. Selezionare i controlli appropriati per codice colore traiettoria secondo l&#39;intensità di fluorescenza delle particelle. </li><li> Selezionare l&#39;opzione per visualizzare la particolo traiettoria in 3D, e codice-colore che secondo l&#39;intensità di fluorescenza. Ruotare la traiettoria in 3D utilizzando i comandi per visualizzare le caratteristiche del moto lisosomi lungo il microtubulo. </li></ol>

<ol>
	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+photon+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. C., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+nanometer+images+of+biological+fibers+by+directed+motion+of+gold+nanoparticles.">3D nanometer images of biological fibers by directed motion of gold nanoparticles.</a> Nano Lett. 11, 4656-4660 (2011).</li><li>Kis-Petikova, K., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distance+measurement+by+circular+scanning+of+the+excitation+beam+in+the+two+photon+microscope.">Distance measurement by circular scanning of the excitation beam in the two photon microscope.</a> Microscopy Research and Technique. 63, 34-49 (2004).</li><li>Levi, V., Ruan, Q., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+particle+tracking+in+a+two+photon+microscope+application+to+the+study+of+molecular+dynamics+in+cells.">3 D particle tracking in a two photon microscope application to the study of molecular dynamics in cells.</a> Biophys J. 88, 2919-2928 (2005).</li><li>Lund, F. W., Wustner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+single+particle+tracking+and+temporal+image+correlation+spectroscopy+for+quantitative+analysis+of+endosome+motility.">A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility.</a> Journal of Microscopy. 252, 169-188 (2013).</li><li>Nath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+Neurodegenerative+Pathology+via+Neuron+to+Neuron+Transmission+of+beta+Amyloid.">Spreading of Neurodegenerative Pathology via Neuron to Neuron Transmission of beta Amyloid.</a> Journal of Neuroscience. 32, 8767-8777 (2012).</li><li>Balint, S., Vilanova, I. V., Alvarez, A. S., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+live+cell+and+superresolution+microscopy+reveals+cargo+transport+dynamics+at+microtubule+intersections.">Correlative live cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 3375-3380 (2013).</li><li>Huotari, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+maturation.">Endosome maturation.</a> Embo Journal. 30, 3481-3500 (2011).</li><li>Ragan, T., So, P. T., Kwon, H. S., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+particle+tracking+on+the+two+photon+microscope.">3D particle tracking on the two photon microscope.</a> Multiphoton Microscopy in the Biomedical Sciences. 2, 247-258 (2001).</li><li>Konig, K., So, P. T. C., Mantulin, W. W., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+to+near-infrared+femtosecond+laser+pulses+in+two+photon+microscopes.">Cellular response to near-infrared femtosecond laser pulses in two photon microscopes.</a> Optics Letters. 22, 135-136 (1997).</li><li>Caviston, J. P., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+motors+at+the+intersection+of+trafficking+and+transport.">Microtubule motors at the intersection of trafficking and transport.</a> Trends Cell Biol. 16, 530-537 (2006).</li><li>Cardarelli, F., Lanzano, L., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+directed+molecular+motion+in+the+nuclear+pore+complex+of+live+cells.">Capturing directed molecular motion in the nuclear pore complex of live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 9863-9868 (2012).</li></ol>

The authors have nothing to disclose.

Nonostante gli enormi progressi delle tecniche di microscopia a fluorescenza e strumentazioni nel corso degli ultimi anni, raggiungendo traiettorie delle particelle fluorescenti in tre dimensioni con una elevata risoluzione temporale è rimasta una sfida nel campo. Se alta risoluzione temporale è stato raggiunto particelle di inseguimento in due dimensioni, estensione alla direzione assiale comporta tipicamente una drastica riduzione della risposta in frequenza del sistema 10. In...

Un gran numero di approcci sono stati sviluppati fino ad oggi per monitorare le particelle fluorescenti in tre dimensioni utilizzando un microscopio. La maggior parte approcci si basano sull&#39;impiego di telecamere veloci, ideale per monitorare in due dimensioni, di solito in combinazione con modifiche personalizzate dell&#39;ottica emissione del microscopio per ottenere inseguimento in direzione assiale. Microscopi a scansione laser confocale (uno o due fotoni) convenzionalmente in grado di monitorare una particella fluorescente eseguendo una sequenza temporale di z-stack, anche se questo processo è in genere richiede tempo, e produce la risoluzione di tempo ragionevole (10 Hz) solo se la particella monitorata è conservato nel centro di una piccola regione di imaging raster da un meccanismo di feedback attivo 2. L&#39;idea di bloccaggio-in per la particella è alla base del metodo di monitoraggio orbitale. Invece di un raster scan un&#39;orbita circolare viene eseguita intorno alla particella fluorescente. L&#39;intensità della fluorescenza lungol&#39;orbita localizza con precisione la posizione delle particelle 4. La posizione localizzato della particella può quindi essere utilizzato per azionare gli scanner microscopio e ri-centro orbita sulla posizione delle particelle. Gli scanner galvanometro del microscopio sono guidate da una tensione analogica. La componente alternata della tensione permette di eseguire un&#39;orbita con il fascio laser focalizzato, cioè un sinusoidale e un&#39;onda coseno applicata agli specchi X e Y scansione consentirà di eseguire un&#39;orbita circolare. Il DC offset del segnale facilita la modifica della posizione del centro orbita. Dopo un periodo di orbita è determinata e la forma d&#39;onda per il segnale AC è configurato, il sistema di retroazione deve aggiornare solo la componente continua del segnale. Un software in grado di leggere il segnale fluorescente raccolti dai rivelatori è necessario per controllare gli scanner e rivelatori, per calcolare la posizione della particella e aggiornare il DC offset. Per una valutazione di successo di imaging un&#39;accurata scelta dei parametri orbitali (dimensioni e della tempistica) è necessario e questi parametri devono essere regolati in base alle caratteristiche delle particelle fluorescenti che è necessario tenere traccia. I fondamenti fisici e matematici della tecnica sono stati descritti in passato 4,5 per entrambe le applicazioni 2D e 3D. In questo protocollo si illustrano brevemente le principali componenti hardware del setup, e più in dettaglio la scelta dei parametri e la preparazione del campione richiesto per un tipico esperimento che ci permette in seguito lo spostamento di un lisosoma con una risoluzione temporale elevata all&#39;interno di una cellula vivente.

<table border="0" cellpadding="0" cellspacing="0" width="525">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</td>
		</tr>
	</tbody>
</table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

L&#39;obiettivo di questo protocollo video è quello di discutere come eseguire e analizzare un esperimento tridimensionale fluorescente orbitale particle tracking utilizzando una versione modificata del microscopio a due fotoni 1. A differenza di approcci convenzionali (scansione raster o largo campo basato su una pila di frame), il tracciamento orbitale 3D permette di localizzare e seguire con un spaziale elevata (10 precisione nm) e risoluzione temporale (risposta in frequenza 50 Hz) lo spostamento 3D di una particella fluorescente passare lunghezza-scale di centinaia di micron 2. Il metodo si basa su un algoritmo di feedback che controlla l&#39;hardware di un microscopio a scansione laser a due fotoni per eseguire un&#39;orbita circolare intorno all&#39;oggetto da tracciare: il meccanismo di feedback manterrà dell&#39;oggetto fluorescente al centro da comandare lo spostamento dei il raggio di scansione 3-5. Per dimostrare i vantaggi di questa tecnica, abbiamo seguito un organello in rapido movimento, il lisosoma, dentro alcellule iving 6,7. Le cellule sono state piastrate secondo protocolli standard, e colorate con un colorante commercialmente lisosoma. Discutiamo brevemente la configurazione hardware e più in dettaglio il software di controllo, effettuare un esperimento di tracciamento orbitale 3D all&#39;interno delle cellule viventi. Si parlerà in dettaglio i parametri richiesti per controllare il microscopio a scansione e consentire il movimento del fascio in un&#39;orbita chiusa attorno alla particella. Concludiamo dimostrando come questo metodo può essere efficacemente utilizzato per tracciare il movimento veloce di un lisosoma marcato lungo i microtubuli in 3D all&#39;interno di una cella dal vivo. Lisosomi possono muoversi con velocità nell&#39;intervallo 0,4-0,5 micron / sec, tipicamente visualizzando un moto diretto lungo la rete microtubuli 8.

Secondo questo protocollo veloce inseguimento singola particella 3D può essere eseguita all&#39;interno di cellule viventi utilizzando un microscopio a due fotoni modificato per monitorare lo spostamento dei lisosomi fluorescente. L&#39;esperimento eseguito consiste di tracciare un lisosoma isolato muoversi all&#39;interno della cella dopo il processo di maturazione endosome 9. I lisosomi sono stati colorati con un colorante verde fluorescente ed eccitato a 930 nm che sfruttano 2-fotone di eccitazione. I nos...

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

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

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 ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Research

JoVE Journal

Bioengineering

10.1K Views.  University of California, Irvine. 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. 

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

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

Tracciamento di segnalazione all&#39;interno di aggregati di cellule ipossiche Encapsulated

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

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 ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

A lungo termine intravitale immunofluorescenza Imaging di tessuto componenti della matrice con Epifluorescenza e due fotoni Microscopia

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Piattaforma microfluidica con multiplex di rilevamento elettronico per spaziale monitoraggio di particelle

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Assemblea e monitoraggio dello sviluppo microbico della comunità all&#39;interno di una piattaforma di array Microwell

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Protocol for Real-time 3D Single Particle 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 various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

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 ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Un orbitale scuotendo la coltura di cellule di mammifero in vasi O a forma di produrre aggregati uniformi

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentazione della crescita delle cellule endoteliali in piastre a 6 pozzi su uno shaker orbitale per studi meccanobiologici

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.