Ringraziamo il Biological Imaging Development Center (BIDC) dell&#39;Università della California, a San Francisco per l&#39;uso del Imaris, Imaris XT e Matlab. Ringraziamo V. Kharazia per l&#39;assistenza tecnica e AT Henry, LK Floren, L. Daitch per il loro contributo alla redazione del manoscritto. Questo lavoro è stato sostenuto da un finanziamento da parte dello Stato della California Medical Research su abuso di alcool e di sostanze attraverso la UCSF di SEB, il National Institutes of Health: 1R21DA029966-01 e di aggiudicazione NIH pista veloce per schermare la raccolta MLSMR di SEB, UCSF Facoltà di Farmacia ( Ufficio di Presidenza e Farmacia Clinica) e la Scuola di Medicina (Clinical Pharmacology &amp; Therapeutics sperimentali) a CLHK.

1. Tridimensionale analisi morfometrica delle variazioni fenotipiche cella singola <ol><li> Umane embrionali renali (HEK293) cellule sono state trasfettate con emoagglutinina (HA)-tagged fattore di rilascio della corticotropina recettore-2 (CRF-R2), una proteina recettore accoppiato G (GPCR) come precedentemente descritto 4, 5. </li><li> Le cellule sono state lasciate non trattate (nessun trattamento, NT), stimolate con il CRF-R2 ligando endogeno, fattore di rilascio della corticotropina, CRF (1 pM, 30 min), o pretrattato con un selettivo CRF-R2 antagonista, anti-sauvagine 30 (AS -30, 1 pM, 30 min) prima del trattamento agonista. </li><li> Le cellule sono state poi fissate, permeabilizzate e trattati con anti-HA. CRF-R2 è stata visualizzata con Alexa 594 nm coniugato anti-topo (IgG 1) anticorpo. DAPI è stato usato per visualizzare la fase mitotica nuclei. </li><li> Per limitare la soggettività sperimentatore, le condizioni sperimentali non erano noti solo dopo le immagini sono state acquisite ed analizzate. </li><li> Abbiamo acquisito immagines da fisso HEK293 cellule usando un piano-apocromatico 63x/1.4 obiettivo olio DIC e Zeiss LSM 510 META microscopio confocale collegato ad un coerente integrato a due fotoni sistema laser composto da un Verdi-V5 laser e un Mira 900-F sistema laser. </li><li> Durante il processo di acquisizione dei dati, le cellule sono state compartimenti sia dal sezionamento multispettrale, 488 nm e 790 nm (~ 350 nm Ex 2PH.) E z-partizionamento (incrementi di 0,5 micron) per includere dati dalla membrana nucleare del recettore esterno extracellulare estremità. </li><li> I dati di fluorescenza sono stati elaborati utilizzando Imaris, che permette la visualizzazione e segmentazione di dataset microscopia 3D, e un modello 3D composto voxel cubici è stato creato per l&#39;analisi morfometrica. Poi, Imaris XT modulo è stato utilizzato per Imaris interfaccia con il linguaggio MATLAB programma per computer per determinare il punto coordinate delle estensioni GPCR. Per tener conto della variabilità cellulare, abbiamo ottenuto e analizzato le immagini di fluorescenza taken da 22 celle: nessun trattamento (NT) (n = 7), agonista (CRF) trattamento (n = 8) e di pre-trattamento con antagonista (AS-30) prima del trattamento con agonisti (n = 7) (Figura 2) . <ol><li> La regione di interesse (ROI) dovrebbe includere una cella che non è in fase di mitosi attiva e non vicino ad altre cellule. In questo modo, l&#39;analisi comprenderà cellule con un solo nucleo e le estensioni recettore non sono disturbati da vicinanza di altre cellule. </li><li> La struttura 3D cellulare è stato ricostruito dai dati multispettrali di fluorescenza utilizzando Imaris (v.7.1.1). </li><li> Dopo l&#39;algoritmo progettato da Imaris, prima il rendering di superficie è stato utilizzato per rappresentare la membrana nucleare. Imaris determinerà se vi è più di un nucleo del ROI. </li><li> Poi la creazione di punti di algoritmo è stato utilizzato per individuare il CRF-R2 estensione. Rilevamento Spot è stato utilizzato in quanto compensa il rumore di fondo e l&#39;intensità irregolare delcomplessa rete di cellule a forma amorfa. </li><li> Per massimizzare l&#39;inclusione di ciascuna unità di rivelazione di fluorescenza del CRF-R2, diametro dei punti &#39;stato impostato a 0,2 micron, che è la più piccola unità all&#39;interno dell&#39;immagine per estrapolare informazioni distinte nella forma di una intensità misurata utilizzando un filtro gaussiano. Posto per il filtraggio è stata costituita nel processo di creazione automatica punti. Il software, tuttavia, offre all&#39;utente la possibilità di utilizzare i filtri per definire i parametri. </li><li> Per evitare il troncamento dei dati, il set di dati è stato convertito da 8 bit (unsigned) punto fisso, a 32-bit decimale. </li><li> I dati sono stati scambiati voxel intensità di individuare dati di coordinate utilizzando Imaris modulo XT interfacciato con MATLAB e l&#39;esatta posizione spaziale di ogni spot è stato determinato eseguendo una trasformazione distanza utilizzando la membrana nucleare come punto di riferimento (figura 3). </li></ol></li><li> I dati ottenuti possono essere quantificati e presentati in formato grafico per la sanalisi tatistical. I confronti tra i gruppi sono state eseguite utilizzando ANOVA a due vie e post-test di Bonferroni. I dati sono presentati come media ± DS. Le differenze sono considerati significativi a * p &lt;0,05. Calcoli sono stati fatti con GraphPad Prism 5.02 (Figura 4). </li></ol> 2. Risultati rappresentativi Per dimostrare la potenza del nostro approccio, abbiamo quantificato i cambiamenti cellulari che derivano dalla interazione di G recettori accoppiati a proteine ​​(GPCR) e del fattore di rilascio della corticotropina recettore-2 (CRF-R2) con il suo ligando endogeno CRF in cellule HEK293 trasfettate. Mostriamo che CRF-R2 recettori si trovano nella membrana plasmatica e sporgono da regioni finiti della membrana delle cellule (Figura 2A e Movie 1). Utilizzando l&#39;analisi convenzionale 2D, è possibile rilevare questo sottoinsieme di extracellulari CRF-R2 recettori solo se si analizza il recettore di adesione pointeri sul vetro copre. Di conseguenza, si perde qualsiasi altra informazione derivata da z-impilati dati multispettrali (Figura 5). Quando le cellule vengono trattate con CRF, i recettori extracellulari sono notevolmente ridotti, come mostrato dalla diminuzione della distanza dei punti della membrana plasmatica. Sono anche ridistribuiti da postazioni principalmente finite in un numero di posizioni discrete (Figura 2B e Movie 2). L&#39;effetto della CRF sulla distribuzione recettore di membrana è impedito dal pretrattamento con il CRF-R2 antagonista specifico, antisavagine 30 (AS-30) e troviamo che i CRF-R2 estensioni non cambiano (Figura 2C e Movie 3). La distribuzione distale di macchie, i tracciati in 5 micron spettro di colori intervalli codificati, è utilizzata per visualizzare la distanza dei voxel della membrana nucleare. Nessun trattamento e antagonista pretreatment (AS-30, 1 mM, 30 min) prima del trattamento con agonisti (CRF, 1 pM, 30 min) non mostrano alcun significativo (ns) differenza di contrazione GPCR. Trattamento delle cellule con l&#39;agonista (CRF, 1 pM, 30 min) riduce progressivamente il numero di CRF-R2 contenenti voxel rispetto a nessun trattamento, 0-5 micron (ns), 6-15 micron (** p &lt; 0,01) e&gt; 15 micron (*** p &lt;0,005), o rispetto a AS-30 trattamento, 0-10 micron (ns), 11-15 micron (** p &lt;0.01) e&gt; 15 micron (*** p &lt;0,005) (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/4233/4233fig1.jpg" /> Figura 1. Schema delle tecniche attualmente disponibili e la loro limitazione per analizzare le immagini di fluorescenza. <a href="http://www.jove.com/files/ftp_upload/4233/4233fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 2" src="/files/ftp_upload/4233/4233fig2.jpg" /> <strong&gt; Figura 2. 3D immagini multispettrali di fluorescenza di cellule HEK293 trasfettate con HA-CRF-R2 prima e dopo il trattamento con agonisti e antagonisti. Immagini che mostrano uniti HA-CRF-R2-sondati con anticorpi anti-HA e visualizzati con Alexa 488 coniugato anticorpo anti-topo (IgG 1) secondaria; DAPI è stato utilizzato per visualizzare i nuclei. Le immagini sono state acquisite con scansione laser confocale (CLS) microscopio. Scala bar 5 micron. <img alt="Figura 3" src="/files/ftp_upload/4233/4233fig3.jpg" /> Figura 3. Modello 3D di cellule HEK293 trasfettate con HA-CRF-R2 ricostruita da immagini utilizzando il software Imaris CLS. Il rendering della superficie del nucleo e la creazione di punti di descrizione delle estensioni GPCR convertiti in piccole vescicole. I dati di fluorescenza sono stati elaborati utilizzando Imaris che permette la visualizzazione e la segmentazione del set di dati 3D microscopia. Poi, Imaris XT è stato utilizzato per Imaris interfaccia con MATLAB. Le intensità voxel sono stati scambiati inposto le coordinate. Le macchie spettro codifica a colori (blu, verde 0-5 micron, 6-10 micron micron, giallo, rosso, 11-15 e&gt; 15 micron) rappresentano il modulo di distanza la membrana nucleare. Scala 5 micron. <img alt="Figura 4" src="/files/ftp_upload/4233/4233fig4.jpg" /> Figura 4. Rappresentazione grafica della distribuzione dei punti tracciati distale nello spettro dei colori codificati a intervalli di 5 micron è utilizzata per visualizzare la distanza dei voxel della membrana nucleare. Nessun trattamento e pretrattamento con un antagonista (AS-30, 1 mM, 30 min) prima del trattamento con agonisti (CRF, 1 pM, 30 min) non mostrano alcun significativo (ns) differenza di contrazione GPCR. Trattamento delle cellule con agonisti (CRF, 1 pM, 30 min) riduce progressivamente la distanza del numero di CRF-R2 contenenti voxel rispetto a nessun trattamento 0-5 micron (ns), 6-15 micron (** p &lt;0.01) e&gt; 15 micron (*** p &lt;0,005), o AS-30 di trattamento 0-10 micron (ns), 11-15<html> micron (** p &lt;0.01) e&gt; 15 micron (*** p &lt;0,005). <img alt="Figura 5" src="/files/ftp_upload/4233/4233fig5.jpg" /> Figura 5. Limitazione dell&#39;analisi 2D morfometrica di cellule HEK 293 trasfettate con HA-CRF-R2. Il piano intermedio sezione di celle (3-4 _m sopra il vetrino coprioggetto) che mostra il centro dei nuclei visualizzata con DAPI e HA-CRF-R2 sondati con anticorpi anti-HA e visualizzati utilizzando Alexa 488nm coniugato anti-topo (IgG 1) secondaria anticorpo e acquisite con CLS non mostra alcuna differenza tra (A) nessun trattamento (NT) e (B) agonista (CRF, 1 pM, 30 min), mentre i punti recettori di adesione sono drammaticamente diversi. Movie 1. Rotazione libera modello 3D in modalità &quot;Surpass&quot; di cellule HEK 293 trasfettate con HA-CRF-R2, nessun trattamento, per valutare le differenze tra fenotipo cellulare proteine ​​recettoriali. Bar Scala 5-20 micron./ &quot;Target =&quot; _blank files/ftp_upload/4233/4233movie1.avi &quot;&gt; Clicca qui per visualizzare filmati. Movie 2. Rotazione libera modello 3D in modalità &quot;Surpass&quot; di cellule HEK 293 trasfettate con HA-CRF-R2, trattamento con l&#39;agonista, CRF (Centro Ricerche Fiat, 1 pM, 30 min), per valutare le differenze tra fenotipo cellulare proteine ​​recettoriali. Bar Scala 5-20 micron. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie2.avi" target="_blank">Clicca qui per visualizzare filmati.</a> Movie 3. Rotazione libera 3D in modalità &quot;Surpass&quot; di cellule HEK 293 trasfettate con HA-CRF-R2, il pretrattamento con un antagonista (AS-30, 1 mM, 30 min) prima del trattamento con agonisti (CRF, 1 pM, 30 min ) per valutare le differenze fenotipo cellulare tra proteine ​​recettoriali. Bar Scala 5-20 micron. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie3.avi" target="_blank">Clicca qui per visualizzare filmati.</a>

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	<li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereological+methods+for+counting+neurons.">Design-based stereological methods for counting neurons.</a> Prog, Brain Res. 135, 43-51 (2002).</li><li>Burke, M., Zangenehpour, S., Mouton, P. R., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knowing+what+counts:+unbiased+stereology+in+the+non-human+primate+brain.">Knowing what counts: unbiased stereology in the non-human primate brain.</a> J. Vis. Exp. (27), e1262 (2009).</li><li>Sugawara, Y., Ando, R., Kamioka, H., Ishihara, Y., Honjo, T., Kawanabe, N., Kurosaka, H., Takano-Yamamoto, T., Yamashiro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+three-dimensional+morphometry+and+cell-cell+communication+of+the+osteocyte+network+in+chick+and+mouse+embryonic+calvaria.">The three-dimensional morphometry and cell-cell communication of the osteocyte network in chick and mouse embryonic calvaria.</a> Calcif. Tissue Int. 88, 416-424 (2011).</li><li>Vickery, R. G., von Zastrow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dynamin-dependent+and+-independent+mechanisms+target+structurally+homologous+dopamine+receptors+to+different+endocytic+membranes.">Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes.</a> J. Cell Biol. 144, 31-43 (1999).</li><li>Bartlett, S. E., Enquist, J., Hopf, F. W., Lee, J. H., Gladher, F., Kharazia, V., Waldhoer, M., Mailliard, W. S., Armstrong, R., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+responsiveness+is+regulated+by+targeted+sorting+of+D2+receptors.">Dopamine responsiveness is regulated by targeted sorting of D2 receptors.</a> Pro.c Natl. Acad. Sci. U.S.A. 102, 11521-11526 (2005).</li><li>Gordon, A., Colman-Lerner, A., Chin, T. E., Benjamin, K. R., Yu, R. C., Brent, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+quantification+of+molecules+and+rates+using+open-source+microscope-based+cytometry.">Single-cell quantification of molecules and rates using open-source microscope-based cytometry.</a> Nat. Methods. 4, 175-181 (2007).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial+morphogenesis.">Molecular mechanisms of epithelial morphogenesis.</a> Annu. Rev. Cell Dev. Biol. 18, 463-493 (2002).</li><li>Pincus, Z., Theriot, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+quantitative+methods+for+cell-shape+analysis.">Comparison of quantitative methods for cell-shape analysis.</a> J. Microsc. 227, 140-156 (2007).</li><li>Spiller, D. G., Wood, C. D., Rand, D. A., White, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+single-cell+dynamics.">Measurement of single-cell dynamics.</a> Nature. 465, 736-745 (2010).</li><li>Loo, L. H., Wu, L. F., Altschuler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+multivariate+profiling+of+drug+responses+from+single+cells.">Image-based multivariate profiling of drug responses from single cells.</a> Nat Methods. 4, 445-453 (2007).</li><li>Yarrow, J. C., Totsukawa, G., Charras, G. T., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+cell+migration+inhibitors+via+automated+microscopy+reveals+a+Rho-kinase+inhibitor.">Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor.</a> Chem. Biol. 12, 385-395 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo mostrato che il trattamento CRF indotto un significativo cambiamento nella morfologia e localizzazione del CRF-R2. La variazione CRF-R2 è stata inibita mediante trattamento antagonista selettivo. Abbiamo dimostrato che modifiche recettore non sono stati rilevati e non può essere misurata utilizzando le tecniche standard 2D multispettrali. La capacità di studiare complesse immagini 3D è fondamentale per incorporare la complessità dei parametri biologici per l&#39;analisi morfometrica. Siamo stati in grado di...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Di rene embrionale umano (HEK293) </td><td> Tipo americano Culture Collection </td><td> CRL-1573 </td><td></td></tr><tr><td> Eagle modificato da Dulbecco (DMEM) </td><td> Invitrogen </td><td> 11965118 </td><td></td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Invitrogen </td><td> SH30070.03 </td><td></td></tr><tr><td> AlexaFluor-488 (IgG2b) </td><td> Invitrogen </td><td> A-11001 </td><td></td></tr><tr><td> monoclonale anti-HA.11 (IgG 1) </td><td> Covance </td><td> 16B12 </td><td></td></tr><tr><td> DAPI </td><td> Vector Laboratories </td><td> H-1200 </td><td>; </td></tr><tr><td> CRF </td><td> Sigma </td><td> C2917 </td><td></td></tr><tr><td> Antisauvagine-30 (AS-30) </td><td> Sigma </td><td> A4727 </td><td></td></tr></tbody></table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

I più comuni strumenti di analisi software disponibili per la misurazione della fluorescenza sono le immagini per i dati bidimensionali (2D) che si basano su impostazioni manuali per l&#39;inclusione e l&#39;esclusione dei punti di dati e computer-aided pattern recognition per rafforzare l&#39;interpretazione ei risultati delle analisi. È diventato sempre più importante essere in grado di misurare immagini di fluorescenza costruiti dataset tridimensionali (3D) per essere in grado di catturare la complessità delle dinamiche cellulari e capire la base della plasticità cellulare all&#39;interno di sistemi biologici. Strumenti sofisticati di microscopia hanno permesso la visualizzazione di immagini 3D di fluorescenza attraverso l&#39;acquisizione di immagini multispettrali di fluorescenza e potente software di analisi che ricostruisce le immagini da pile confocale che poi forniscono una rappresentazione 3D delle immagini raccolte 2D. Advanced Design metodi basati Stereology sono progrediti dal ravvicinamento e le ipotesi di original model-based stereology 1, anche in sezioni di tessuto complesse 2. Nonostante questi progressi scientifici in microscopia, rimane una necessità per un metodo analitico automatizzato che sfrutta appieno i dati intrinseci 3D per permettere l&#39;analisi e la quantificazione delle variazioni complesse della morfologia cellulare, la localizzazione delle proteine ​​e traffico recettore. Le attuali tecniche disponibili per quantificare immagini di fluorescenza sono Meta-Morph (Molecular Devices, Sunnyvale, CA) e Image J (NIH) che forniscono l&#39;analisi manuale. Imaris (Andor Technology, Belfast, Irlanda del Nord), il software fornisce la funzione MeasurementPro, che consente la creazione manuale di punti di misura che possono essere inseriti in un volume o un&#39;immagine disegnata su una serie di sezioni 2D per creare un oggetto 3D. Questo metodo è utile per singolo clic misurazioni di punti per misurare una distanza di linea tra due oggetti o per creare un poligono che racchiude una regione di interesse, ma è difficile da applicare a complex strutture di rete cellulare. Filament Tracer (Andor) consente il rilevamento automatico del neuronale 3D filamento simile tuttavia, questo modulo è stato sviluppato per misurare strutture definite come i neuroni, che sono costituite da assoni, dendriti e spine (struttura ad albero). Questo modulo è stato ingegnosamente utilizzato per effettuare misurazioni morfologiche cellule non neuronali 3, tuttavia, i dati di uscita di fornire informazioni di un&#39;estesa rete cellulare utilizzando un software che dipende da una forma cella definita piuttosto che essere una forma amorfa modello cellulare. Per superare il problema di analizzare amorfo a forma di cellule e rendere il software più adatto a una domanda di biologico, Imaris sviluppato cella Imaris. Questo è stato un progetto scientifico con la Eidgenössische Technische Hochschule, che è stato sviluppato per calcolare il rapporto tra cellule e organelli. Mentre il software consente l&#39;individuazione di vincoli biologici, forzando un nucleo per cellamediante membrane cellulari di cellule segmento, esso non può essere utilizzato per analizzare dati di fluorescenza non sono continuativi perché idealmente costruisce superficie cellulare senza spazi vuoti. A nostra conoscenza, per il momento non modificabile dall&#39;utente approccio automatizzato che fornisce informazioni morfometrica 3D da immagini di fluorescenza è stato sviluppato che consente di ottenere informazioni spaziali cellulare di forma indefinita (Figura 1). Abbiamo sviluppato una piattaforma di analisi utilizzando il Imaris modulo software di base e Imaris XT interfacciato con MATLAB (Mat Works, Inc.). Questi strumenti consentono la misurazione 3D di cellule senza una predefinita forma e incoerenti con componenti di rete fluorescenza. Inoltre, questo metodo consentirà ai ricercatori che hanno esteso le competenze nei sistemi biologici, ma non familiarità per applicazioni informatiche, effettuare quantificazione dei cambiamenti morfologici nelle dinamiche cellulari.

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Abbiamo sviluppato una piattaforma software che utilizza Imaris Neuroscienze, ImarisXT e MATLAB per misurare i cambiamenti nella morfologia di una forma indefinita preso da tridimensionale fluorescenza confocale di singole cellule. Questo nuovo approccio può essere usato per quantificare i cambiamenti nella forma cellulare dopo l&#39;attivazione del recettore e rappresenta quindi un possibile strumento aggiuntivo per la scoperta di farmaci.

Uno strumento di analisi che quantifica Modifiche morfologia cellulare dalle immagini a fluorescenza tridimensionali

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

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Research

JoVE Journal

Biology

14.5K Views.  University of California, San Francisco. Abbiamo sviluppato una piattaforma software che utilizza Imaris Neuroscienze, ImarisXT e MATLAB per misurare i cambiamenti nella morfologia di una forma indefinita preso da tridimensionale fluorescenza confocale di singole cellule. Questo nuovo approccio può essere usato per quantificare i cambiamenti nella forma cellulare dopo l'attivazione del recettore e rappresenta quindi un possibile strumento aggiuntivo per la scoperta di farmaci.

Video: An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Basato su chip tridimensionale coltura cellulare in perfuso Micro-bioreattori

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Ricerca

Biologia

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabbricazione di chip di dimensioni Ponteggi per tridimensionale coltura cellulare

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblaggio, carico, allineamento e di una cella analitica del campione Ultracentrifuga

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Cambiamenti nella morfologia della ghiandola mammaria e rischio di cancro al seno in topi

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Analisi rapida ed esplorazione di microscopia a fluorescenza Immagini

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging in idrogel tridimensionali

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Un tessuto metodo di compensazione per l'Imaging tridimensionale del tessuto adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Approcci quantitativi per studiare le strutture cellulari e la morfologia delle orgine in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...