Gli autori apprezzare lettura attenta Dr. Lynnea Brumbaugh del manoscritto. Questo lavoro è stato sponsorizzato dal National Institutes of Borse Salute EB000712 R01, R01 EB008085, R01 CA134539, U54 CA136398 e 5P60 DK02057933. Prof. V. Wang Lihong ha un interesse finanziario in Microphotoacoustics, Inc. e Endra, Inc., che, tuttavia, non hanno sostenuto questo lavoro.

1. Configurazione del sistema <ol><li> Radiazione ottica <ol><li> Sorgente di radiazione ottica: un diodo-pompato a stato solido laser pulsato (INNOSLAB, Edgewave) e un laser a colorante (CBR-D, Sirah). </li><li> Il raggio laser di uscita (ampiezza di impulso: 7 ns) è focalizzata da una lente condensatore (LA1131, Thorlabs) per passare attraverso un foro stenopeico di 50 micron (P50C, Thorlabs). </li><li> Il foro stenopeico è posizionato un po &#39;lontano dal fuoco della lente condensatore che corrisponda al diametro del foro stenopeico con la fondamentale modalità diametro del raggio per un efficace filtraggio spaziale. </li><li> Il raggio filtrato è attenuato da un filtro a densità neutra (NDC-50C-2M, Thorlabs) e poi accoppiati in una modalità singola fibra ottica (P1-460A-FC-2, Thorlabs). </li><li> L&#39;uscita in fibra riempie l&#39;apertura posteriore di un obiettivo microscopio (RMS4X, Thorlabs) per ottenere una diffrazione limitata nel centro ottico di circa 2,6 micron alla lunghezza d&#39;onda di 570 nm. </li></ol></li><li> Rilevamento ultrasonico <ol><li> Trasduttore ad ultrasuoni: 50-MHz di frequenza centrale (V214-BB-RM, Olympus-NDT). </li><li> Il trasduttore ad ultrasuoni è collegato ad un fatto in casa acustico-ottico a fascio combinatore 11 per il rilevamento ultrasonico, che è allineato coassialmente con la diffrazione limitata radiazione ottica. </li><li> Una cavità sferica è terra dal fondo del combinatore per la produzione di una lente acustica. Questa lente acustica ha una apertura numerica di 0,5 in acqua e dà un diametro acustico focale di 43 micron a 50-MHz di frequenza centrale. </li><li> I fuochi ottici e acustici sono allineati confocally per massimizzare la sensibilità di rilevazione. </li></ol></li><li> Accoppiamento acustico <ol><li> Secco accoppiamento ad ultrasuoni è impiegato per evitare sommergendo animali da esperimento in acqua, che è stato utilizzato nei primi mesi del sistemi di imaging fotoacustico 12. </li><li> Una finestra di imaging si apre sul fondo di una piastra di Petri (9 cm di diametro) ed è sigillato con una membrana ad ultrasuoni e otticamente polietilene trasparente. </li><li> Gel ultrasuoni (Clear Image, SonoTech) tra la membrana in polietilene e l&#39;oggetto da coppie ripreso l&#39;onda generata fotoacustico dall&#39;oggetto alla piastra di Petri, e l&#39;acqua deionizzata nella scatola di Petri coppie ulteriormente l&#39;onda al sommerso OR-PAM testa di imaging . </li></ol></li><li> Elettronica <ol><li> Il segnale fotoacustico rilevato dal trasduttore ad ultrasuoni è amplificata da due amplificatori in cascata (ZFL 500LN, Mini-Circuits) </li><li> Il segnale amplificato viene digitalizzato da un 14-bit di acquisizione dati (DAQ) (CompuScope 14200, Gage Scienze Applicate) ad una frequenza di campionamento di 200 MS / s. </li></ol></li><li> Scansione schema <ol><li> Bidimensionale (2-D) raster di scansione del OR-PAM testa di immagini lungo l&#39;orizzontale (xy) piano è controllato da un personal computer, che attiva sia la scheda DAQ e il laser pompa. Il segnale di trigger è sincronizzato con l&#39;orologio-out del segnale dalla scheda DAQ. </li><li> L&#39;asse veloce della 2-D scanner è definita come la direzione della sezione di scansione (B-scan). </li><li> Una sequenza di B-scan le immagini possono essere acquisite traducendo la testa di immagini lungo l&#39;asse lento per formare una immagine volumetrica, che possono essere visualizzati sia in diretta 3-D di rendering o in un 2-D sporgenza massima ampiezza (MAP) immagine . </li></ol></li></ol> 2. Sistema di allineamento <ol><li> Usare il pulse-echo ultrasuoni e un riflettore ad ultrasuoni per determinare la posizione del piano acustico focale (ovvero, il tempo di ritardo dal segnale di trigger al massimo impulso-eco segnale ultrasonico). Questo passaggio è necessario per essere eseguita una sola volta durante la costruzione del sistema O-PAM. </li><li> Massimizzare l&#39;efficienza di accoppiamento della fibra monomodale. </li><li> Applicare il gel ultrasuoni sulla parte superiore di un oggetto otticamente assorbente (ad esempio, un pezzo di nastro adesivo nero) e delicatamente allegare sotto la finestra di imaging nella scatola di Petri riempite con acqua deionizzata. </li><li> Abbassare la testa di esposizione in acqua, ed eliminare le bolle d&#39;aria intrappolate sotto la lente acustica. </li><li> Regolare la testa di esposizione fino a quando il segnale fotoacustico dell&#39;oggetto assorbente acustico dal piano focale, che può essere giudicata dal ritardo acustico. </li><li> Regolare la posizione verticale (cioè, la posizione z) del microscopio obiettivo di massimizzare l&#39;ampiezza del segnale generato dal fotoacustico oggetto piatto. L&#39;ampiezza del segnale massimizzato suggerisce che il focus ottico sia allineato con il centro acustico in direzione verticale. </li><li> Regolare la posizione orizzontale (cioè posizioni, x e y) l&#39;obiettivo microscopio fotoacustico finché il segnale generato dal bersaglio mostra uno schema simmetrico. La simmetria suggerisce che il focus ottico sia allineato con il centro acustico in direzione orizzontale. </li><li> Ripetere i passi 2.6 e 2.7 fino a quando il segnale fotoacustico è ottimizzato sia in simmetria e ampiezza. </li></ol> 3. A sa<html> mple procedura sperimentale in vivo OR-PAM del mouse orecchio vascolarizzazione <ol><li> Questo passaggio non è richiesto per topi nudi. Anestetizzare l&#39;animale con un&#39;iniezione intraperitoneale di un cocktail [ricetta: 1 ml di ketamina (100 mg / ml), 0,1 ml xylazina (100 mg / ml) e 8,9 ml di soluzione salina; dosaggio: 0,1 ml/10 g]. Radersi i capelli nell&#39;orecchio, e ulteriormente depilare i capelli residua con Crema personalizzate e sicure (Categoria #: 82565, American International Industries) prima di pulirlo con acqua deionizzata. Si noti che tale depilazione potrebbe leggermente irritare la vascolarizzazione della pelle e quindi è meglio effettuata 24 ore prima l&#39;esperimento previsto. </li><li> Accendere il sistema laser fotoacustica, e controllare l&#39;allineamento del sistema. </li><li> Anestetizzare il mouse con il 3% isoflurano vaporizzato dal gas inalazione (la portata tipica è 1,0-1,5 l / min, a seconda del peso del corpo dell&#39;animale), e mantenere l&#39;anestesia con l&#39;1% isoflurano tutto l&#39;esperimento. Uso medico aria è raccomandata come l&#39;inalazione di gas per mantenere il mouse in normale stato fisiologico. </li><li> Trasferire il mouse su un palco stereotassica e controllare la sua temperatura corporea a 37 ° C con una piastra elettrica. </li><li> Appiattire l&#39;orecchio del mouse su un piatto di plastica e applicare uno strato di gel per ultrasuoni sulla parte superiore dell&#39;orecchio. Evitare di inglobare bolle d&#39;aria all&#39;interno del gel. Poi, posto l&#39;orecchio sotto la finestra di imaging e sollevare lentamente la fase animale fino contatti gel l&#39;ecografia il fondo della membrana in polietilene. A contatto morbide è necessario perché premendo l&#39;orecchio contro la membrana può influenzare il flusso di sangue nelle orecchie. </li><li> Morsetto un pulsossimetro alla gamba del mouse o la coda di monitorare il suo stato fisiologico, e applicare una pomata per gli occhi per prevenire la secchezza e danni accidentali laser agli occhi del mouse. </li><li> Abbassare la testa di esposizione fino a quando la lente acustica è immerso in acqua deionizzata e rimuovere le bolle d&#39;aria intrappolate sotto la lente acustica. </li><li> Controllare la fluenza laser per assicurarsi che sia nel rispetto degli standard di sicurezza del laser della American National Standards Institute 13. La fluenza laser non deve superare 20 mJ / cm 2, che si traduce in 80 nj di energia laser a impulsi quando il raggio laser viene focalizzato a 150 micron sotto la superficie della pelle. </li><li> Impostare il laser per l&#39;esterno-trigger mode e avviare la scansione di prova. Regolare la posizione z del capo di imaging fino a quando il segnale più forte è fotoacustico dal piano acustico focale. </li><li> Impostare correttamente i parametri di scansione e avviare l&#39;acquisizione formale delle immagini. </li><li> Dopo l&#39;esperimento, spegnere il laser, sollevare la testa immagini fuori dall&#39;acqua deionizzata, abbassare la fase animale a rilasciare il mouse, pulire l&#39;orecchio del mouse con acqua deionizzata, spegnere il sistema di anestesia e il regolatore di temperatura, e scaricare il mouse dal palco stereotassica. </li><li> Se l&#39;imaging ripetitivi è necessario, mettere il mouse in una incubatrice con la temperatura ambiente a 37 ° C. Ritorna il mouse per l&#39;impianto animale dopo che si sveglia in modo naturale. Altrimenti, seguire i protocolli animale eutanasizzare e smaltirlo. </li></ol> 4. Funzionale OR-PAM di concentrazione totale e la saturazione di ossigeno dell&#39;emoglobina <ol><li> Ossiemoglobina (HBO 2) e deossiemoglobina (HBR) sono le due principali forme di emoglobina, la prevalente fonte endogena fotoacustico nel campo del visibile dello spettro. HbO 2 e HBR sono distinti spettri ottici di assorbimento e quindi può essere spettralmente differenziati per quantificare sia la concentrazione totale (HBT) e la saturazione di ossigeno (SO 2) di emoglobina 5. Qui ci sono due linee guida per aiutare a selezionare corretta lunghezze d&#39;onda ottiche per SO 2 misure: </li><li> Lunghezze d&#39;onda dovrebbe essere selezionata all&#39;interno del Q-banda dello spettro di assorbimento dell&#39;emoglobina (ad esempio, 550-600 nm) per garantire un sufficiente rapporto segnale-rumore e la penetrazione adeguata. </li><li> Lunghezze d&#39;onda in cui i coefficienti di assorbimento di HBO 2 e HBR ha una pronunciata differenza (ad esempio, HBR dominante 561 nm e HBO 2-dominante 578 nm) sono raccomandati. </li></ol> Oltre SO 2, HBT può essere calcolato sommando [HBR] e [HbO 2] insieme, o può essere misurata direttamente a lunghezze d&#39;onda dello spettro isosbestic coefficiente di estinzione molare di emoglobina (ad esempio, 530 nm, 545 nm, 570 nm, e 584 nm) 14, dove HBR e HBO 2 hanno uguali coefficienti di estinzione molare. 5. Rappresentante dei risultati: Mostrato nella Figura 1 è l&#39;anatomia vascolare e SO 2 in un orecchio vivente topo nudo ripreso da doppia lunghezza d&#39;onda (561 e 570 nm) O-PAM. Il tipico tempo di acquisizione immagini per doppia lunghezza d&#39;onda sO 2 misurazioni di tale regione di interesse (dimensione immagine: 10 mm x 10 mm, passo: 2,5 micron x 5 micron) è ~ 80 min. p_upload/2729/2729fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Ottico in vivo risoluzione microscopia fotoacustico. MAP di (A) la concentrazione totale di emoglobina che mostra l&#39;anatomia vascolare (acquisita a 570 nm) e (B), la saturazione di ossigeno dell&#39;emoglobina (acquisita a 561 nm e 570 nm) in un orecchio topo nudo. (C) primo piano della regione in scatola a pannello (A). La barra di scala nel pannello (A) vale sia per (A) e (B).

<ol>
	<li>Maslov, K., Zhang, H. F., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-resolution+photoacoustic+microscopy+for+in+vivo+imaging+of+single+capillaries.">Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries.</a> Opt. Lett. 33, 929-931 (2008).</li><li>Zhang, C., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subwavelength-resolution+label-free+photoacoustic+microscopy+of+optical+absorption+in+vivo.">Subwavelength-resolution label-free photoacoustic microscopy of optical absorption in vivo.</a> Opt. Lett. 35, 3195-3197 (2010).</li><li>Wang, Y., Maslov, K., Kim, C., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+photoacoustic+and+fluorescence+confocal+microscopy.">Integrated photoacoustic and fluorescence confocal microscopy.</a> IEEE. Trans. Biomed. Eng. 57, 2576-2578 (2010).</li><li>Jiao, S., Xie, Z., Zhang, H. F., Puliafito, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+multimodal+imaging+with+integrated+photoacoustic+microscopy+and+optical+coherence+tomography.">Simultaneous multimodal imaging with integrated photoacoustic microscopy and optical coherence tomography.</a> Opt. Lett. 34, 2961-2963 (2009).</li><li>Hu, S., Maslov, K., Tsytsarev, V., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transcranial+brain+imaging+by+optical-resolution+photoacoustic+microscopy.">Functional transcranial brain imaging by optical-resolution photoacoustic microscopy.</a> J. Biomed. Opt. 14, 040503-040503 (2009).</li><li>Hu, S., Yan, P., Maslov, K., Lee, J. M., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+amyloid+plaques+in+a+transgenic+mouse+model+using+optical-resolution+photoacoustic+microscopy.">Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy.</a> Opt. Lett. 34, 3899-3901 (2009).</li><li>Hu, S., Rao, B., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+Photoacoustic+Ophthalmic+Angiography.">Label-free Photoacoustic Ophthalmic Angiography.</a> Opt. Lett. 35, 1-3 (2010).</li><li>Jiao, S. L., Jiang, M. S., Hu, J. M., Fawzi, A., Zhou, Q. F., Shung, K. K., Puliafito, C. A., Zhang, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+ophthalmoscopy+for+in+vivo+retinal+imaging.">Photoacoustic ophthalmoscopy for in vivo retinal imaging.</a> Opt. Express. 18, 3967-3972 (2010).</li><li>Oladipupo, S., Hu, S., Santeford, A., Yao, J., Kovalski, J. R., Shohet, R., Maslov, K., Wang, L. V., Arbeit, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+HIF-1+induction+produces+multistage+neovascularization+with+stage-specific+sensitivity+to+VEGFR+inhibitors+and+myeloid+cell+independence.">Conditional HIF-1 induction produces multistage neovascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell independence.</a> Blood. , .</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+functional+chronic+imaging+of+a+small+animal+model+using+optical-resolution+photoacoustic+microscopy.">In vivo functional chronic imaging of a small animal model using optical-resolution photoacoustic microscopy.</a> Med. Phys. 36, 2320-2323 (2009).</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+optical-resolution+photoacoustic+microscopy+with+improved+sensitivity+and+speed.">Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed.</a> Opt. Lett. 36, 1134-1136 (2011).</li><li>Wang, X., Pang, Y., Ku, G., Xie, X., Stoica, G., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+laser-induced+photoacoustic+tomography+for+structural+and+functional+in+vivo+imaging+of+the+brain.">Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain.</a> Nat. Biotechnol. 21, 803-806 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Institute of America, American National Standard for Safe Use of Lasers ANSI Z136. , (2007).</li><li>Jacques, S. L., Prahl, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Absorption+of+Hemoglobin+.">Optical Absorption of Hemoglobin .</a> Oregon Medical Laser Center [Internet]. , (1999).</li></ol>

Tutte le procedure sperimentali sono state effettuate in conformità con il protocollo animale laboratorio riconosciuto dalla Scuola di Medicina degli animali Studi Comitato della Washington University a St. Louis.

In questo video, forniamo istruzioni dettagliate sui protocolli sperimentali di O-PAM, tra cui la configurazione del sistema, l&#39;allineamento del sistema, e la tipica procedure sperimentali. Senza etichetta, non invasivo O-PAM ha permesso studi di funzionamento microvascolare e il metabolismo su una sola base capillare e possiede quindi la possibilità di espandere la nostra comprensione del microcircolo legati fisiologia e patologia. Microphotoacoustics è attualmente in produzione questo O-PAM sistema.

Home-made acustico-ottico fascio combinatore: <ul><li> prisma ad angolo retto (NT32-545, Edmund Optics) </li><li> prisma romboidale (NT49-419, Edmund Optics) </li><li> olio di silicone (1000cSt, Prodotti Clearco) </li><li> O-PAM sistema (Microphotoacoustics) </li></ul>

three-dimensional optical-resolution photoacoustic microscopy

Microscopia ottica, fornendo preziose informazioni a livello cellulare e degli organelli, è stata ampiamente riconosciuta come una tecnologia abilitante biomedica. Come i pilastri in vivo tridimensionale (3-D) microscopia ottica, single-/multi-photon microscopia a fluorescenza e la tomografia a coerenza ottica (OCT) hanno dimostrato la loro sensibilità straordinaria per i contrasti di scattering e fluorescenza ottica, rispettivamente. Tuttavia, il contrasto di assorbimento ottico di tessuti biologici, che codifica le informazioni essenziali fisiologico / patologico, non è stato ancora valutabili. L&#39;emergere di photoacoustics biomediche ha portato ad una nuova branca della microscopia ottica ottici risoluzione microscopia fotoacustico (OR-PAM) 1, dove si concentra la radiazione ottica al limite di diffrazione di raggiungere cellulare 1 o addirittura subcellulare risoluzione di 2 livello laterale. Come un prezioso complemento agli attuali tecnologie di microscopia ottica, O-PAM porta in almeno due novità. Il primo e più importante, O-PAM rileva contrasti l&#39;assorbimento ottico con una sensibilità straordinaria (cioè 100%). La combinazione di O-PAM con microscopia a fluorescenza 3 o con ottiche di diffusione basati su 4 Ottobre (o con entrambi) offre una completa proprietà ottiche dei tessuti biologici. In secondo luogo, O-PAM codifica assorbimento ottico in onde acustiche, in contrasto con i processi di pura ottica in microscopia a fluorescenza e ottobre, e fornisce sfondo senza rilevamento. La rilevazione acustica in O-PAM mitiga gli effetti della dispersione ottica degradazione del segnale ed elimina naturalmente possibili interferenze (cioè crosstalks) tra eccitazione e di rilevamento, che è un problema comune in microscopia a fluorescenza a causa della sovrapposizione tra l&#39;eccitazione e spettri di fluorescenza. Unico per l&#39;imaging assorbimento ottico, O-PAM ha dimostrato ampia applicazioni biomediche dalla sua invenzione, tra cui, ma non limitati a, neurologia 5, 6, oftalmologia 7, 8, 9 biologia vascolare, dermatologia e 10. In questo video, ci insegnano la configurazione del sistema e l&#39;allineamento di O-PAM e le procedure sperimentali per l&#39;imaging in vivo microvascolari funzionale.

Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

Three-dimensional Optical-resolution Photoacoustic Microscopy

Microscopia ottica con risoluzione fotoacustico (OR-PAM) è una tecnologia emergente in grado di assorbimento di imaging ottico contrasti<em&gt; In vivo</em&gt; Cellulari con risoluzione e sensibilità. Ecco, mettiamo a disposizione una istruzione visualizzate sui protocolli sperimentali di O-PAM, inclusa la configurazione del sistema di allineamento del sistema, tipico<em&gt; In vivo</em&gt; Procedure sperimentali, e sistemi di imaging funzionale.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

three-dimensional-optical-resolution-photoacoustic-microscopy

Research

JoVE Journal

Bioengineering

17.7K Views.  Washington University in St. Louis. Microscopia ottica con risoluzione fotoacustico (OR-PAM) è una tecnologia emergente in grado di assorbimento di imaging ottico contrasti In vivo Cellulari con risoluzione e sensibilità. Ecco, mettiamo a disposizione una istruzione visualizzate sui protocolli sperimentali di O-PAM, inclusa la configurazione del sistema di allineamento del sistema, tipico In vivo Procedure sperimentali, e sistemi di imaging funzionale.

Video: Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Ricerca

Bioingegneria

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Stampa planare e tridimensionale di inchiostri conduttivi

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Cistografia fotoacustico

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Umana Cartilagine Tessuto Fabrication Uso tridimensionale Tecnologia Inkjet Printing

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analisi della migrazione delle cellule all&#39;interno di una tridimensionale di collagene Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Preparazione e analisi di<em&gt; In Vitro</em&gt; Tridimensionale carcinoma mammario mondo dei replicanti

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabbricazione di dispositivi microfluidici basati su carta tridimensionale per gli immunodosaggi

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Microscopio fotoacustico acustico e ottico<em&gt; In Vivo</em&gt; Piccolo animale Blood Imaging di Vasculature

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.