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

Die Einzelheiten des Verfahrens zum Betreiben der volumetrischen Handoptoakustischen Sonde wird nachstehend beschrieben. Dieser Vorgang wird nach anerkannten institutionellen Regelungen zur Tier- und Menschenversuche durchgeführt. 1. Systemvorbereitungs <ol><li> Einschalten des Lasers für eine Aufwärmzeit von ca. 15 min vor dem Betrieb zur Stabilisierung der Ausgangslichtstrahl. </li><li> Setzen Sie den Wasser umschließenden Teil mit der Trennmembran, die in Kontakt mit der Haut (Abbildung 1) ist. HINWEIS: Der Abstand zwischen der Trennmembran (in Kontakt mit der Haut) und die Region mit der maximalen Empfindlichkeit des Wandlers (Zentrum der sphärischen Sonde) stellt die effektive Abbildungstiefe. </li><li> Füllen des gesamten Volumens von ca. 100 ml zwischen der Trennmembran und der Oberfläche des Wandlers mit entionisiertem Wasser mittels einer Pumpe. </li><li> Stellen Sie sicher, dass keine Wasserleckage noch Luftblasen Present. Alternativ zu vermeiden Luftblasen durch Rezirkulation des Wassers. </li><li> Führen die Versuche bei RT und gewährleisten die Koppelmedium (Wasser) wird bei dieser Temperatur gehalten. </li></ol> 2. Imaging Vorbereitung <ol><li> Menschliche Bildgebung Vorbereitung. </li><li> Entfernen von Haar des zu bild Teil mit einer Depilation Lotion, um einen unerwünschten Hintergrund in den Bildern zu vermeiden (dieser Schritt ist optional). </li><li> Anwendung Ultraschallgel auf die Haut, um den Bereich zu, um eine effiziente akustische Kopplung bereitzustellen abgebildet werden. Legen Sie die optoakustischen Sonde in der Region von Interesse. Darauf, dass keine Luftblasen in der Ultraschallankopplung Gel vorhanden sind. </li><li> Tierbildgebung Vorbereitung. </li><li> Stellen Sie sicher, dass die Pflege und experimentellen Verfahren mit Tieren sind in Übereinstimmung mit institutionellen und staatlichen Vorschriften. </li><li> Entfernen das Fell des Tieres in dem Bereich mit einer Rasierwasser abgebildet werden. Schützen Sie die Augen des Tieres mitTierarzt Salbe, die Trockenheit und Schäden durch Einwirkung von intensiver gepulster Laserstrahlung verhindert. </li><li> Betäubt das Tier mit Hilfe intraperitoneale Injektion (IP) von Ketamin / Xylazin (100 mg / kg KG Ketamin + 5 mg / kg KG Xylazin) vor dem Experiment oder mit Isofluran-Narkose (2-3% (Volumen) mit 0,9 l / min Gasstrom) während des Experiments. Betäubung Bestätigen durch Überprüfung der Reflex der hinteren Gliedmaßen des Tieres. </li><li> Anwendung Ultraschallgel auf die Haut um den Bereich zu, um eine effiziente akustische Kopplung bereitzustellen und legen die optoakustischen Sonde in dem interessierenden Bereich abgebildet werden. Darauf, dass keine Luftblasen in der Ultraschallankopplung Gel vorhanden sind. </li></ol> 3. Pre-view Operation Mode <ol><li> Stellen die Abbildungswellenlänge (n) zwischen 690 nm und 900 nm und die Pulswiederholungsrate zwischen 10 und 50 Hz. Die Parameter für die akustische Datenerfassungssystem - 1 MEingangsimpedanz. Erwerben 2030 Abtastwerte für jeden Laserpuls mit einer Abtastrate von 40 Megasamples pro Sekunde und 12-Bit-Vertikalauflösung. Auslösen der Erfassung mit Q-Schalter-Ausgang des Lasers. </li><li> Stellen Sie sicher, dass sowohl der Betreiber und der Patient Schutzbrille tragen zur optischen Anregungswellenlänge (n) angepasst. Eingestellt, die Laserleistung so, dass der Lichtfluss auf der Gewebeoberfläche unter 20 mJ / cm 2 während des Experiments zur nahen Infrarotwellenlängen, um die Sicherheit Grenzwerte für Menschenversuche 19 erfüllen und thermische Belastung und Schädigung der Haut bei Tieren verhindern gehalten . </li><li> Starten des pre-view-Software mit einer GPU Umsetzung Verarbeitungsalgorithmen ermöglichen die Visualisierung dreidimensionaler Bilder mit einer Bildrate entsprechend der Impulswiederholungsrate des Lasers. </li><li> Bewegen Sie die Sonde und / oder das Objekt, um Visualisierungsleistung zu optimieren und zu lokalisieren, die Strukturen von Interesse abgebildet werden. </li></ol> 4. Datenerfassung <ol><li> Die Datenerfassung für das Scannen (Handheld) Modus. </li><li> Falls erforderlich, Injizieren eines Kontrastmittels nach der Akquisition der Kontrast in der Region von Interesse zu machen. HINWEIS: In unseren Experimenten haben wir nicht kontrastverstärkten menschlichen Bildgebung durchgeführt. Dennoch können verschiedene Kontrastmittel potentiell für diesen Zweck verwendet werden. Indocyanin-Grün (ICG) ist ein Beispiel einer klinisch zugelassenen optische Kontrastmittel, die für die Kontrastverstärkung bei maximal empfohlenen Dosis von 2 mg / kg Körpergewicht bei Erwachsenen verwendet werden können. </li><li> Starten der Hardware für die Datenerfassung mit den in 3.1 beschriebenen Parameter Aufrechterhaltung der Ausführung des Vorschau Software. Bewegen vorsichtig die Sonde um den abgebildeten Bereich, um die Strukturen von Interesse zu verfolgen. HINWEIS: Wenn Bilder an mehreren Laserwellenlängen gleichzeitig erfasst, hat die Geschwindigkeit der Sondenbewegung im Handbetrieb deutlich verringert werden (vorzugsweise unter 2 mm / secfür eine Laserimpulswiederholungsrate von 50 Hz), um bewegungsbezogenen Artefakten in den spektral entmischten Bilder zu vermeiden. </li><li> Die Datenerfassung für den stationären Betrieb. </li><li> Montieren Sie das abgebildete Objekt (z. B. Tier) und der Handsonde auf den Halter und starten Sie die Übernahme mit den in 3.1 beschriebenen Parameter Aufrechterhaltung der Ausführung des Pre-View Software. </li><li> Aufrechterhaltung der optoakustischen Sonde und das Abbildungsteil in der gleichen Position während des Versuchs, um dynamische biologische Ereignisse in dem Bereich von Interesse darzustellen. </li><li> Injizieren eines Kontrastmittels, um seine dynamische Verteilung in der Region von Interesse zu verfolgen. HINWEIS: In unserem Mausexperimenten, Indocyaningrün (ICG) wurde zur Kontrastverstärkung eingesetzt. Als allgemeine Richtlinie gilt, einer Menge von 10 nmol oder 0,4 mg / kg ICG hat, um nachweisbare Unterschied multispektralen Optoakustik in vivo erzeugen in den Kreislauf eingebracht werden, der Maus. HINWEIS: Das Kontrastmittel mussfür den menschlichen und / oder tierischen Gebrauch durch den jeweiligen Behörde genehmigt. </li></ol> 5. Abschließen der Experiment <ol><li> Stoppen Sie den Laser. </li><li> Entfernen Sie die optoakustischen Sonde von der abgebildeten Region. Für Tierstudie, stoppen Sie die Anästhesieversorgung. </li><li> Positionieren Sie das Tier unter einer Infrarotheizung, um es warm zu halten und den Kontakt mit anderen Tieren zu verhindern, bis er aus der Narkose vollständig erholt. Sie das Tier nicht unbeaufsichtigt während der Erholung von der Narkose. </li></ol> 6. Off-line Datenverarbeitung <ol><li> Laden Sie die Datei (en) enthält, die erworbenen optoakustischen Signale in der Software-Anwendung für die Datenverarbeitung eingesetzt. </li><li> Verwenden eines Rekonstruktionsalgorithmus auf einer dreidimensionalen Matrixanordnung entsprechend einem volumetrischen Bild der optischen Absorption für jeden Rahmen, und jede Wellenlänge zu erhalten. HINWEIS: Für die Rekonstruktion ist es bevorzugt, einen Algorithmus zu verwenden, entfallen Störfaktoren, wie heterogenekeiten und Dämpfung in dem abgebildeten Objekt, Auswirkungen der endgültigen Bandbreite und geometrische Form der Erfassungselemente und Lichtfluenz Variationen, um eine quantitative Darstellung der Verteilung der absorbierten Energie zu erhalten. </li><li> Verwenden einen Algorithmus, um Entmischung zu erhalten, aus jedem Mehrfachwellenlängen-Rahmen, einen neuen Satz von dreidimensionalen Matrixarrays, die die optische Absorption für jeden in der Probe vorhandenen absorbierenden Substanz. </li><li> Bei Bedarf weiterverarbeiten die Matrix-Arrays, die die optischen Absorptionsverteilung bis zur Visualisierung und Lesen von biologisch relevanten Parameter zu erleichtern. </li></ol>

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The authors have nothing to disclose.

Die einzigartigen Vorteile, die durch optoakustischen Bildgebungstechniken in kleinen Tierforschung angeboten haben starke Motivation für die Übersetzung der Technik in der klinischen Praxis mit einer Reihe von Diagnose und Therapieüberwachung Anwendungen vorgesehen, zB erstellt., Brust- und Hautkrebs, Entzündung oder periphere Gefäßkrankheiten. Jedoch, im Gegensatz zu Mäusen oder kleinere Tiere, die mit einer ausreichenden Anzahl von Lichtquellen und Detektorelementen umgeben sein kann, um eine wirksame...

Optoakustische (photoakustische) Bildgebung zieht wachsende Interesse aus den biologischen und medizinischen Forschungsgemeinschaften, wie sie in der ständig wachsenden Zahl von Publikationen umfassenden Vielzahl von neuen Anwendungen, die die einzigartigen Vorteile von der Technik 5.1 zu nutzen manifestiert. Insbesondere eröffnet die Fähigkeit, Bild spektral unverwechselbaren Foto-Absorptionsmittel mit hoher raum-zeitlicher Auflösung in Tiefen weit über die Diffusionsgrenze von Licht beispiellose Fähigkeiten für funktionelle und molekulare Bildgebung 6-10. Tatsächlich Übersetzung der optoakustischen Technologie in die klinische Praxis kommt mit vielversprechenden Perspektiven in der Diagnostik und Therapieüberwachung von vielen Krankheiten. Noch der begrenzte Ausbreitung von Photonen in optisch streuenden und absorbierenden Gewebe und der generell schwachen Reaktionen mit der optoakustischen Phänomen zu begrenzen den anwendbaren Tiefe des Verfahrens. Als Ergebnis Hand optoacoustic Sonden haben, um Bildteile von außerhalb des Körpers 11,12 zugänglich versucht worden, während endoskopischer Systeme werden verwendet, um Bilder aus dem Körperinneren, indem Sie sie über natürliche Körperöffnungen 13 bereitzustellen. Einige Low absorbierenden Teile des menschlichen Körpers, wie der weiblichen Brust sind auch von tomographischen optoakustische Scanner 14,15 zugänglich. Von besonderem Interesse ist das Hand Ansatz, da es ermöglicht große Vielseitigkeit, ähnlich wie Ultraschall. Hier bleibt die Anpassung der gemeinsamen Ultraschall lineare Anordnung Sonden für optoakustischen Bildgebung anspruchsvoll, vor allem auf grundlegende Unterschiede in Bildgebungsanforderungen zwischen Ultraschall und Optoakustik. Während hohe Frameraten in Standard-Sonographie werden durch sequentielle aktiviert Sende-Empfangs-Systeme, die Hochimpulsfolgefrequenzen im kHz-Bereich, wird in Echtzeit dreidimensionale optoakustischen Bildgebung durch gleichzeitige Sammlung von volumetrischen tomographischen Daten aus einer einzigen int erreichterrogating Laserpuls. Somit impliziert hochwertigen optoakustischen Tomographie-Erfassung von dreidimensionalen Daten aus einem möglichst großen Raumwinkel um das abzubildende Objekt. Kürzlich haben wir den ersten akusto-optischen Handsonde zur dreidimensionalen (volumetrisch) Bildgebung in Echtzeit 16. Das System basiert auf einem zweidimensionalen Array 256 auf einer Kugeloberfläche (blaue Punkte in 1A) angeordneten piezoelektrischen Elementen abdeckt einen Winkel von 90 ° bezogen. Die Größe der einzelnen Elemente von ca. 3 x 3 mm 2, sowie deren Orientierung und Frequenzbandbreite (ca. 2-6 MHz) garantieren effektive Signal Sammlung von einer zentimetergroßer Lautstärke rund um das Zentrum der Kugel (schwarz Würfel in Abbildung 1A). Optische Anregung der Abbildungsbereich mit einem Faserbündel durch einen zentralen zylindrischen Hohlraum der Anordnung eingesetzt ist, so dass jede Wellenlänge Susceptible der durch das Faserbündel übertragen kann für die Bildgebung verwendet werden. Eine tatsächliche Bild der Anordnung von Wandlern zusammen mit dem optischen Faserbündel ist in 1B gezeigt. Die effiziente Anregung und gleichzeitige Detektion von Signalen ermöglicht tiefen Gewebe-Bildgebung mit Single-Shot-Anregung (ein Laser-Impuls), so daß eine Echtzeitabbildung mit einer Bildrate von der Pulswiederholungsfrequenz des Lasers bestimmt ist ferner mit einer Grafik- aktiviert Verarbeitungseinheit (GPU) Durchführung des Wiederaufbauverfahren 17. Ein zylindrisches Gehäuse mit einem transparenten Polyethylenmembran (1C) mit der Wandleranordnung befestigt ist, um eine akustisch Übertragungsflüssigkeit (Wasser) zu umschließen. Die Membran wird auf die Gewebe mittels akustischer Gel gekoppelt. Ein Bild von der optoakustischen Sonde in handgehaltenen Betriebsmodus verwendet wird in 1D gezeigt. Das demonstriert three dimensional Handoptoakustischen Bildgebung in Verbindung mit dem Echtzeit-Funktionsabbildungsleistung wichtige Vorteile für die klinische Diagnostik und eine Anzahl von potentiellen Anwendungen sind für verschiedene Indikationen, wie periphere arterielle Verschlusskrankheit, des Lymphsystems, Brustkrebs, Hautläsionen vorgesehen, Entzündung oder Arthritis 18. Weiterhin schnelle Abbildungsleistung ermöglicht die Visualisierung von dynamischen biologischen Ereignisse mit dem in einer stationären Position angeordnet Sonde. Kombiniert mit schneller Wellenlängenabstimmung optischen parametrischen Oszillator (OPO), Lasertechnik, ermöglicht die Echtzeit-Bildgebung von biologischen Verteilung von Photoabsorptionsmittel dieser Ansatz. Dadurch kann neue Möglichkeiten gleichermaßen entstehen in der Kleintierbildgebungsanwendungen, z. B. bei der Untersuchung Gewebe Hämodynamik, in vivo Zellverfolgung, Visualisierung der Pharmakokinetik, Organperfusion, gezielte molekulare Bildgebung von Tumoren und Herz-Kreislauf-System oder Neuroimaging. <p class = &quot;jove_content&quot;&gt; In dieser Arbeit stellen wir eine detaillierte Beschreibung der experimentellen Bildgebung Protokoll, um mit dem sphärischen Array optoakustischen Handsonde und Schaufenster Leistung in mehreren typischen klinischen und Kleintierbildgebung Szenarien arbeiten.

<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.

Repräsentative Ergebnisse, demonstriert die Fähigkeiten des beschriebenen volumetrischen Handoptoakustischen Sonde, werden in diesem Abschnitt vorgestellt. In allen Fällen wurde der Lichtfluss auf der Hautoberfläche unter den Sicherheitsgrenzwert von 20 mJ / cm 2 19 gehalten. Die Leistung der Sonde in Echtzeit-Tracking peripheren menschlichen Gefäß ist in Figur 2 vorgestellt. Im Verlauf dieses Versuches wurde die Sonde langsam entlang der Hand eines gesunden...

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.

Universal-Hand Dreidimensionale Optoakustische Bildgebung Sonde für Deep Tissue Menschen Angiographie und Funktions präklinischen Studien in Echtzeit

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 Aufrufe.  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.

Serie: 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.

Dreidimensionale Optische Auflösung photoakustische Mikroskopie

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

Forschung

Biotechnik

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.

Planar-und Drei-Dimensional Printing von Leitfähige Druckfarben

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 Dreidimensionale-Flow-Kammer-Geräte zu Chemokine gerichtet Extravasation von zirkulierenden Zellen unter physiologischen Bedingungen zu studieren Durchfluss

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.

Menschliches Knorpelgewebe Herstellung unter Verwendung von dreidimensionalen Inkjet-Drucktechnologie

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.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

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.

Entwicklung eines unter Schwerelosigkeits Vielzellige Dreidimensionale Organotypischen Modell des menschlichen Darmschleimhaut Grown

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.

Dreidimensionale Gewebezüchtungen ausgerichtet Astrozyten Netzwerke Nervensystem Regeneration zu rekapitulieren Entwicklungsmechanismen

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.

Elektrospinnen Schreiben von dreidimensionalen Poly(ε-caprolactone) Gerüste mit steuerbaren Morphologien für Tissue Engineering Anwendungen zu schmelzen

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.

Hand-Held klinische photoakustische Imaging-System für Echtzeit-nicht-invasive kleine Tier Imaging

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.

Kultivierung von Säugerzellen in dreidimensionale Peptid Gerüste

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