The research leading to these results has received funding from the European Research Council under grant agreement ERC-2010-StG-260991.

La procedura dettagliata per il funzionamento con la sonda optoacustica volumetrico portatile è descritto di seguito. Questa procedura viene eseguita secondo le norme istituzionali approvate in materia di sperimentazione animale e umana. 1. Sistema di Preparazione <ol><li> Accendere il laser per un periodo di riscaldamento di circa 15 minuti prima operazione per stabilizzare il fascio luminoso in uscita. </li><li> Posizionare la parte di acqua che racchiude con la membrana di separazione che è in contatto con la pelle (figura 1). NOTA: La distanza tra la membrana isolante (a contatto con la pelle) e la regione di massima sensibilità del trasduttore (centro del tastatore sferico) stabilisce la profondità effettiva di imaging. </li><li> Riempire l&#39;intero volume di circa 100 ml tra la membrana isolante e la superficie del trasduttore con acqua deionizzata per mezzo di una pompa. </li><li> Assicurarsi che siano perdite di acqua né bolle d&#39;aria sono Present. In alternativa, evitare bolle d&#39;aria, fornendo ricircolo dell&#39;acqua. </li><li> Eseguire gli esperimenti a temperatura ambiente e garantire il mezzo di accoppiamento (acqua) viene mantenuta a questa temperatura. </li></ol> 2. Imaging Preparazione <ol><li> Preparazione imaging umano. </li><li> Rimuovere capelli dalla parte essere-ripreso con una lozione depilazione per evitare uno sfondo indesiderato nelle immagini (questa operazione è opzionale). </li><li> Applicare gel per ultrasuoni sulla cute intorno alla regione da acquisire per fornire accoppiamento acustico efficiente. Posizionare la sonda optoacustica nella regione di interesse. Verificare che non vi siano bolle d&#39;aria sono presenti nel gel di accoppiamento ultrasuoni. </li><li> Preparazione di immagini di animali. </li><li> Verificare che le procedure di cura e sperimentali con gli animali sono in accordo con le norme e regolamenti istituzionali e di governo. </li><li> Rimuovere il pelo dell&#39;animale nella regione per essere ripreso con una lozione da barba. Proteggere gli occhi dell&#39;animale conUnguento veterinario, che previene la secchezza e danni da esposizione a intense radiazioni laser pulsato. </li><li> Anestetizzare l&#39;animale mediante iniezione intraperitoneale (IP) di ketamina / xilazina (100 mg / kg KG ketamina + 5 mg / kg KG Xilazina) prima dell&#39;esperimento o utilizzare isoflurano (2-3% (in volume) con 0,9 l / min flusso di gas) durante l&#39;esperimento. Conferma anestesia controllando il riflesso dell&#39;arto posteriore dell&#39;animale. </li><li> Applicare gel per ultrasuoni sulla cute intorno alla regione da acquisire per fornire accoppiamento acustico efficiente e posizionare la sonda optoacustica nella regione di interesse. Verificare che non vi siano bolle d&#39;aria sono presenti nel gel di accoppiamento ultrasuoni. </li></ol> Modalità operativa 3. Pre-view <ol><li> Stabilire la lunghezza d&#39;onda di imaging (s) tra 690 nm e 900 nm e la frequenza di ripetizione degli impulsi tra 10 e 50 Hz. Selezionare i parametri per il sistema di acquisizione dati acustici - 1 MWimpedenza di ingresso. Acquisire 2030 campioni per ogni impulso laser ad una frequenza di campionamento di 40 megacampioni al secondo e 12 bit risoluzione verticale. Attivare l&#39;acquisizione con uscita Q-switch del laser. </li><li> Assicurarsi che sia l&#39;operatore che il paziente usa occhiali protettivi adatti alla lunghezza d&#39;onda di eccitazione ottica (s). Impostare la potenza del laser in modo che la fluenza luce alla superficie del tessuto viene mantenuta al di sotto di 20 mJ / cm 2 durante l&#39;esperimento per lunghezze d&#39;onda nel vicino infrarosso per soddisfare i limiti di esposizione di sicurezza per esperimenti umani 19 e prevenire sollecitazioni termiche e danni alla pelle negli animali . </li><li> Avviare il software pre-vista con una implementazione di algoritmi di elaborazione delle GPU per consentire la visualizzazione di immagini tridimensionali ad un frame rate corrispondente alla frequenza di ripetizione degli impulsi del laser. </li><li> Spostare la sonda e / o l&#39;oggetto da esporre al fine di ottimizzare le prestazioni di visualizzazione e localizzare le strutture di interesse. </li></ol> 4. Acquisizione Dati <ol><li> L&#39;acquisizione dei dati per la modalità di scansione (a mano). </li><li> Se necessario, iniettare un agente di contrasto prima dell&#39;acquisizione di arricchire il contrasto nella regione di interesse. NOTA: Nei nostri esperimenti non abbiamo eseguito l&#39;imaging umano con mdc. Tuttavia, vari mezzi di contrasto possono essere potenzialmente utilizzati per questo scopo. Indocianina verde (ICG) è un esempio di mezzo di contrasto ottico clinicamente approvato che può essere utilizzato per migliorare il contrasto a somministrazione della dose massima raccomandata di 2 mg / kg di peso corporeo negli adulti. </li><li> Avviare l&#39;hardware di acquisizione dati con i parametri descritti in 3.1 mantenendo l&#39;esecuzione del software di anteprima. Spostare delicatamente la sonda intorno alla regione ripreso per monitorare le strutture di interesse. NOTA: Quando le immagini a più lunghezze d&#39;onda laser sono acquisiti contemporaneamente, la velocità di movimento della sonda in modalità manuale deve essere diminuito significativamente (preferibilmente inferiore a 2 mm / secper un laser a impulsi frequenza di ripetizione di 50 Hz) per evitare artefatti da movimento legati nelle immagini spettralmente miscelati. </li><li> L&#39;acquisizione dei dati per la modalità fermo. </li><li> Montare l&#39;oggetto ripreso (ad es., Animali) e la sonda portatile sul supporto e avviare l&#39;acquisizione dei parametri descritti in 3.1 mantenendo l&#39;esecuzione del software pre-view. </li><li> Mantenere la sonda optoacustica e la parte di imaging nella stessa posizione durante l&#39;esperimento di visualizzare eventi biologici dinamici della regione di interesse. </li><li> Iniettare un agente di contrasto per monitorare la distribuzione dinamica nella regione di interesse. NOTA: Nei nostri esperimenti del mouse, verde indocianina (ICG) è stato utilizzato per migliorare il contrasto. Come regola generale, una quantità di 10 nmol o 0,4 mg / kg di ICG deve essere introdotto nella circolazione del mouse per creare contrasto rilevabile con optoacoustics multi-spettrali in vivo. NOTA: Il mezzo di contrasto deve essereapprovato per uso umano e / o animale dal rispettivo ente. </li></ol> 5. Finalizzazione l&#39;esperimento <ol><li> Arrestare il laser. </li><li> Rimuovere la sonda optoacustica dalla regione ripreso. Per lo studio degli animali, interrompere l&#39;erogazione dell&#39;anestesia. </li><li> Posizionare l&#39;animale sotto un riscaldatore a raggi infrarossi per tenerlo al caldo ed evitare il contatto con altri animali fino a quando non pienamente recuperato dalla anestesia. Non lasciare l&#39;animale incustodito durante il recupero dall&#39;anestesia. </li></ol> 6. Off-line Data Processing <ol><li> Caricare il file (s) che contiene i segnali optoacustica acquisite nell&#39;applicazione software utilizzati per l&#39;elaborazione dei dati. </li><li> Utilizzare un algoritmo di ricostruzione per ottenere una matrice tridimensionale corrispondente ad una immagine volumetrica dell&#39;assorbimento ottico per ogni fotogramma e ogni lunghezza d&#39;onda. NOTA: Per la ricostruzione è preferibile usare un algoritmo che rappresentano fattori di distorsione, come hétérogèneimpianti e delle attenuazione nell&#39;oggetto fotografato, effetti di banda finale e la geometria degli elementi di rilevazione e varianti luce fluenza, al fine di ottenere una rappresentazione più quantitativa della distribuzione dell&#39;energia assorbita. </li><li> Utilizzare un algoritmo unmixing di ottenere, a ogni frame multi-lunghezza d&#39;onda, una nuova serie di matrici matrice tridimensionale che rappresenta l&#39;assorbimento ottico per ciascuna sostanza assorbente presente nel campione. </li><li> Se necessario, trattare ulteriormente gli array a matrice che rappresenta la distribuzione di assorbimento ottico per facilitare la visualizzazione e la lettura dei parametri biologicamente rilevanti. </li></ol>

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The authors have nothing to disclose.

I vantaggi unici offerti dalle tecniche di imaging optoacustica in piccola ricerca su animali hanno creato una forte motivazione per tradurre la tecnologia nella pratica clinica, con un numero di diagnostica e applicazioni di monitoraggio di trattamento immaginati ad es., In seno e il cancro della pelle, infiammazioni o malattie vascolare periferico. Tuttavia, al contrario di topi o animali più piccoli, che possono essere circondati da un numero sufficiente di fonti di illuminazione e di elementi di rilevament...

Optoacustica (fotoacustico) di imaging attrae un crescente interesse da parte delle comunità di ricerca biologica e medica, come manifestato dal numero sempre crescente di pubblicazioni che comprendono varietà di nuove applicazioni che sfruttano i vantaggi unici offerti dalla tecnologia 1-5. In particolare, la capacità di immagine agenti foto-assorbimento spettrale distintivi ad alta risoluzione spazio-temporale a profondità ben oltre il limite diffusiva della luce apre capacità senza precedenti per l&#39;imaging funzionale e molecolare 6-10. Infatti, la traduzione della tecnologia optoacustica nella pratica clinica viene con prospettive promettenti in diagnostica e monitoraggio trattamento di molte malattie. Tuttavia, la propagazione limitata di fotoni in dispersione otticamente e tessuti assorbenti e le risposte generalmente deboli associati al fenomeno optoacustica limitare la profondità applicabile del metodo. Come risultato, optoa portatilesonde Coustic sono tentato di parti di immagine accessibili dall&#39;esterno del corpo 11,12 mentre i sistemi endoscopici vengono utilizzati per fornire immagini all&#39;interno del corpo inserendoli tramite orifizi naturali 13. Alcune parti a basso assorbimento del corpo umano, come il seno femminile, sono accessibili anche da scanner tomografici optoacustica 14,15. Di particolare interesse è l&#39;approccio a mano in quanto consente grande versatilità, in modo simile a ecografia. Qui, l&#39;adattamento dei comuni sonde ecografiche di array lineari per l&#39;imaging optoacustica resta difficile, soprattutto a causa delle differenze fondamentali nei requisiti di imaging tomografico tra ultrasuoni e optoacoustics. Mentre frame rate elevati in ecografia di serie sono attivati ​​per sequenziale sistemi che impiegano frequenze di ripetizione degli impulsi ad alta nel range kHz di trasmissione-ricezione, in tempo reale, tridimensionale delle immagini optoacustica si ottiene con la raccolta simultanea di dati tomografici volumetrico da un singolo interrogating impulso laser. Così, immagini optoacustica alta qualità implica acquisizione di dati tridimensionali dal più grande angolo solido possibile intorno all&#39;oggetto fotografato. Recentemente, abbiamo introdotto la prima sonda optoacustica tenuto in mano per tridimensionale (volumetrico) di imaging in tempo reale 16. Il sistema è basato su una matrice bidimensionale di elementi piezoelettrici 256 disposti su una superficie sferica (punti blu in Figura 1A) che coprono un angolo di 90 °. La dimensione dei singoli elementi di circa 3 x 3 mm 2, così come il loro orientamento e la larghezza di banda di frequenza (circa 2-6 MHz) garantiscono l&#39;incasso efficace segnale da un volume centimetri scala circonda il centro della sfera (cubo nero nella Figura 1A). Eccitazione ottica della regione di imaging è fornito di un fascio di fibre inserito attraverso una cavità cilindrica centrale della matrice, in modo che qualsiasi susc lunghezza d&#39;ondaeptible di essere trasmesso attraverso il fascio di fibre può essere utilizzato per l&#39;imaging. Una immagine reale della matrice di trasduttori con il fascio di fibre ottiche è mostrato nella Figura 1B. L&#39;eccitazione efficiente e rilevazione simultanea di segnali permette l&#39;imaging dei tessuti profondi con eccitazione a colpo singolo (un impulso laser), in modo che le immagini in tempo reale ad una frequenza determinata dalla frequenza di ripetizione degli impulsi del laser è ulteriormente attivata con un Grafica- trasformazione-unità (GPU) l&#39;attuazione della procedura di ricostruzione 17. Un involucro cilindrico con una membrana in polietilene trasparente (Figura 1C) è collegato alla trasduttori per racchiudere un mezzo liquido acusticamente trasmissione (acqua). La membrana è ulteriormente accoppiato al tessuto mediante gel acustica. Un&#39;immagine della sonda optoacustica come utilizzati nella modalità di funzionamento manuale è mostrato nella Figura 1D. Il thr dimostratoee dimensionale immagini optoacustica a mano in combinazione con il tempo reale capacità di imaging funzionale sono dotati di importanti vantaggi per la diagnostica clinica e una serie di potenziali applicazioni sono immaginato per diverse patologie, come la malattia vascolare periferica, disturbi del sistema linfatico, cancro al seno, lesioni cutanee, infiammazione o artrite 18. Inoltre, la capacità di imaging veloce permette la visualizzazione di eventi biologici dinamici con la sonda disposti in posizione stazionaria. In combinazione con lunghezza d&#39;onda veloce a punto parametrico ottico oscillatore (OPO) tecnologia laser, questo approccio consente in tempo reale di immagini di biodistribuzione di agenti foto-assorbimento. In tal modo, nuove possibilità possono altresì emergere in piccole applicazioni di imaging animale, ad esempio., Nello studiare l&#39;emodinamica del tessuto, in vivo il monitoraggio delle cellule, la visualizzazione di farmacocinetica, la perfusione degli organi, mirato imaging molecolare dei tumori e sistema cardiovascolare, o neuroimaging. <p class = &quot;jove_content&quot;&gt; In questo lavoro forniamo una descrizione dettagliata del protocollo sperimentale di imaging ad operare con la matrice sferica optoacustica sonda e vetrina prestazioni a mano in diversi scenari clinici e di imaging animale di piccole dimensioni tipiche.

<table border="0" cellpadding="0" cellspacing="0" width="1663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Name of Material/ Equipment</td>
			<td style="width:389px;">Company</td>
			<td style="width:171px;">Catalog Number</td>
			<td style="width:777px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Optical parametric oscillator (OPO)-based laser</td>
			<td style="width:389px;">Innolas Laser GmbH, Krailling (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The laser provides laser pulses with a duration around 10ns and an energy up to 80mJ. 
			The wavelength is tunable between 680-950nm.</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:327px;">Spherical array of piezocomposite detectors</td>
			<td style="width:389px;">Imasonic SaS, Voray (France)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The array consists of 256 piezoelectric sensors distributed on a spherical surface. 
			Each element has dimensions 3x3mm2, a central frequency of 4MHz and a bandwidth of 100%.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data acquisition system (DAQ)</td>
			<td style="width:389px;">Falkenstein Mikrosysteme GmbH, Taufkirchen (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The DAQ simultaneously acquires 256 signals at 40 megasamples per second and 2030 samples. 
			The input impedance is 1MW.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Fiber bundle</td>
			<td style="width:389px;">CeramOptec GmbH, Bonn (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The bundle consists of 480 individual fibers randomly distributed in the input and output. 
			The numerical aperture of each individual fiber is 0.22.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Athymic Nude mouse</td>
			<td style="width:389px;">Harlan Laboratories (The Netherlands)</td>
			<td style="width:171px;">Athymic nude - Foxn1nu</td>
			<td style="width:777px;">The mouse was 8 weeks old (adult) at the time of the experiment. 
			The ethical protocol was approved by the Bavarian goverment (number 55.2.1.54-2632-102-11)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bepanthen cream</td>
			<td style="width:389px;">Bayer AG (Germany)</td>
			<td style="width:171px;">N/A</td>
			<td>Vet ointment to protect the eyes during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data processing software</td>
			<td style="width:389px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The data processing software was devoped at our institute. 
			It allows reconstruction at each wavelength and multi-wavelength unmixing, as well as further data processing.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:327px;">Water-enclosing part</td>
			<td style="width:389px;">N/A</td>
			<td style="width:171px;">custom-made</td>
			<td>This part contains the water that acts as an acoustic coupling medium between skin and transducer elements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Indocyanine green (ICG)</td>
			<td style="width:389px;">PULSION Medical Systems SE</td>
			<td style="width:171px;">N/A</td>
			<td style="width:777px;">ICG-PULSION (active ingredient: indocyanine green dye) is a drug used in cardiac, circulatory and micro-circulatory diagnostics, liver function diagnostics and ophthalmic angiography diagnostics.</td>
		</tr>
	</tbody>
</table>

universal hand-held three-dimensional optoacoustic imaging probe for deep tissue human angiography and functional preclinical studies in real time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

Rappresentante dei risultati, che dimostrano le capacità del sensore portatile volumetrico descritto optoacustica, sono in mostra in questa sezione. In tutti i casi, la fluenza della luce sulla superficie della pelle è stata mantenuta al di sotto del limite di esposizione di sicurezza del 20 mJ / cm 2 19. Le prestazioni della sonda in tempo reale inseguimento vascolare periferico umano è presentato nella figura 2. Nel corso di questo esperimento, la sonda è st...

Watch this Scientific Journal Video about Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time at JoVE.com

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Universale Mano libera tridimensionale optoacustica Imaging Probe per Deep Tissue angiografia umana e funzionali studi preclinici in tempo reale

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

universal-hand-held-three-dimensional-optoacoustic-imaging-probe-for

Research

JoVE Journal

Bioengineering

10.6K Views.  Helmholtz Zentrum München. We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Video: Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

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 ...

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 ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

A Novel tridimensionale del flusso Camera dispositivo per studiare Chemokine-diretto stravaso di cellule circolanti in condizioni di flusso fisiologici

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Sviluppo di un multicellulare tridimensionale Organotipica Modello della mucosa intestinale umana coltivate in microgravità

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Tessutale tridimensionale allineato Astrocyte reti per ricapitolare i meccanismi dello sviluppo e facilitare la rigenerazione del sistema nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Sciogliere elettrofilatura scrittura di scaffold tridimensionale Poly(ε-caprolactone) con morfologie controllabile per applicazioni di ingegneria del tessuto

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Mano clinico fotoacustico sistema di Imaging per Imaging animale piccolo Non-invasivo in tempo reale

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Coltura di cellule di mammifero in scaffold tridimensionale Peptide

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...