Wir danken für die großzügige Unterstützung durch die National Science Foundation (CAREER CBET-1.055.379) und den National Institutes of Health (1RC4EY021357, 1R01EY019951). Wir erkennen auch die Unterstützung aus dem China Scholarship Council Wei Song.

Ein. System Configuration <ol><li> PAOM Subsystem <ol><li> Beleuchtungsquelle: ein Nd: YAG-Laser (SPOT-10-100, Elforlight Ltd, UK: 20 uJ / Puls; 2 nsec Pulsdauer; 30 kHz maximalen Impulswiederholungsrate). </li><li> Der Ausgang Lasers bei 1064 nm wird frequenzverdoppelten bis 532 nm durch ein Beta-Barium-Borat (BBO)-Kristall (Castech, San Jose, CA). Nach weiteren Spaltung durch eine Laserlinie Spiegel, wird 532 nm Licht durch eine Single-Mode-Lichtleitfaser (P1-460A-FC-5, Thorlabs) geliefert und 1064 nm-Laser wird durch eine Photodiode (DET10A, Thorlabs), die aufgezeichnet auslöst PA Signalerfassung. </li><li> Der Laser kommende Licht aus der Monomode-Lichtleitfaser wird auf der Netzhaut durch ein Galvanometer (GM, QS-7, Nutfield Technology) und ein Teleskop Konfiguration geliefert (f1 = 75 mm und f2 = 14 mm, Edmund Optics) 6. </li><li> Ein unkonzentriert Nadel Wandler (40-MHz Mittenfrequenz, 16-MHz-Bandbreite, 0,4 × 0,4 mm 2 aktive Element Größe, NIH Ressource Center für Ultraschall-Transducer Technologies, The University of Southern California) in Kontakt mit dem Augenlid platziert, um die PA-Signale von der Netzhaut erzeugt erkennen. Ultraschallgel (Sonotech) zwischen dem Wandler Spitze Augenlid und tierischer für gute akustische Kopplung angewendet wird. </li><li> Das PA-Signal wird von zwei Verstärkern (ZFL-500LN +, Mini-Schaltungen und 5073PR, Olympus) verstärkt, und wird durch eine Datenerfassungsplatine digitalisiert (CS14200, Gage Applied). </li></ol></li><li> SD-OCT Subsystem <ol><li> Geringer Kohärenz Lichtquelle: eine breitbandige Superlumineszenzdiode (IPSDD0804, InPhenix; Mittenwellenlänge: 840 nm; 6-dB-Bandbreite: 50 nm), die die axiale Auflösung von 6 um bestimmt. </li><li> Die Nah-Infrarot-Licht wird Aufgeteilt Arm und Probenarm durch einen 50 × 50 referenzieren angepassten Einmodenfaser Koppler (OZ Optics). </li><li> Nach Kombination mit PAOM Beleuchtungslicht von einem heißen Spiegel (FM02, Thorlabs), Aktien Oktober Probenarm die gleiche Scan-und Lieferbedingungen Optik with PAOM 6. </li><li> Ein selbstgebauten Spektrometer verwendet, um die SD-OCT Störsignalen, wo eine Zeilenkamera CCD-Kamera (Aviiva SM2, e2v) ermöglicht eine A-Zeilenfrequenz von 24 kHz erfassen. Design von typischen Spektrometer können aus mehreren bereits veröffentlichten Literatur 11 und fasergekoppelten SD-OCT-Spektrometer zu finden sind ab sofort im Handel erhältlich. Die SD-OCT-Empfindlichkeit wird gemessen, um besser als 90 dB. </li></ol></li><li> Abtastschema <ol><li> Fast 2-D Rasterabtastung des Galvanometers wird von einem Analog-Ausgabe-Karte (PCI-6731, National Instruments), die ebenfalls löst sowohl das PAOM Laseransteuerung und die Signalerfassung der OCT-Spektrometer gesteuert. Als Ergebnis werden die Daten Akquisitionen PAOM und OCT Subsysteme synchronisiert. </li><li> Die PAOM Datenerfassung von einer Fotodiode Aufnahme PAOM Laser-Sequenz (siehe 1.1.2) ausgelöst. </li><li> 3-D volumetrischen Bildern oder 2-D Fundusbilder von 256 B-Scan-Bilder konstruiert (256 A-Linien pro BScannen Bild). </li></ol></li></ol> 2. System Alignment <ol><li> Maximieren des frequenzverdoppelnden Wirkungsgrad der BBO-Kristall und die Kopplungseffizienz des Einmoden-Lichtleitfaser ist. Tragen LG3 Schutzbrille (Thorlabs) für Personal Augenschutz bei der Optimierung der PAOM Beleuchtungslichts. </li><li> Kollimieren Faserausgang Laser nach PAOM bis 2,0 mm im Durchmesser. </li><li> Richten Sie die kombinierte Beleuchtung Lichter PAOM und SD-OCT zu sein koaxial. </li><li> Stellen Sie die PAOM Anregungslicht bei ~ 40 nJ / Impuls und SD-OCT Sondieren Licht bei ~ 0,8 mW, welche beide berichtet augensicher 6,12 sein. </li></ol> 3. In vivo Multimodal Retinal Imaging <ol><li> Übertragen der Ratte auf eine transparente Polypropylen-Feld, und das Tier zu betäuben durch eine Mischung von Isofluran (Phoenix pharmazeutische, Inc.) und normaler Luft bei einer Konzentration von 1,5% und mit einer Fließgeschwindigkeit von 2,0 l / min für 10 min. </li><li> Begrenze die narkotisierten Ratte in einem geficktde-Halter mit Fünf-Achs-verstellbare Freiheit (Abbildung 1), und halten ihre Körpertemperatur bei ~ 37 ° C durch ein Heizkissen (Repti therm, Zoomed Labors, Inc.). Pflegen Sie die Narkose durch Einatmen Gas gemischten Isofluran und normale Luft mit 1,0% iger Konzentration und 1,5 l / min Volumenstrom während des gesamten Experiments. </li><li> Schneiden Sie die Wimpern mit einer chirurgischen Schere, erweitern die Schüler mit 1% Tropicamide ophthalmische Lösung, und lähmen die Iris Schließmuskel mit 0,5% Tetracainhydrochlorid ophthalmische Lösung. , Künstliche Tränenflüssigkeit (Systane, Alcon Laboratories, Inc.) mit dem Auge Ratte alle zwei Minuten, um Cornea Dehydratisierung und Kataraktbildung verhindern. Überwachen Sie den tierischen Herzfrequenz, Atmung und Blutoxygenierung von einem Pulsoximeter (8.600 V, Nonin Medical, MN) während der Bildgebung. </li><li> Schalten Sie SD-OCT Beleuchtungslicht und überprüfen Sie die Sondierung Licht ~ 0,8 mW sein. </li><li> Aktivieren Sie das Galvanometer Scannen. Richten Sie die SD-OCT Bestrahlungslichts Lieferungauf die Rattenretina und Identifizierung der retinalen Bereich von Interesse (ROI) durch Einstellen der Fünf-Achsen Tierhalter. Hierbei wird die Papille absichtlich in der Mitte des Sichtfeldes plaziert, während der ROI basierend auf verschiedenen Forschungsarbeiten Anforderungen gewählt werden sollte. </li><li> Weitere einstellen Tierhalter, die SD-OCT-Bildgebung Qualitäten Netzhaut Querschnitt in beiden Scan-Richtung (durch Umschalten der Rasterabtastung Richtung) zu optimieren, bis die besten optischen Schwerpunkt erreicht wird. </li><li> Bereiten Sie die Nadel Wandler auf einer Fünf-Achs-verstellbare Plattform, einen Tropfen von Ultraschall-Gel auf den Schallkopf Spitze und sanft an den Wandler Spitze des Tieres Augenlid. </li><li> Set PAOM Laser an den externen Trigger-Modus, starten Sie den Galvanometer Scannen, und aktivieren Sie Echtzeit-Anzeige der PAOM Querschnitt Bild des Tieres Netzhaut. Sorgfältig einstellen Schallkopforientierung bis die PAOM Bild hat das beste Signal-Rausch-Verhältnis (SNR) und in der Zwischenzeit, zeigt eine gleichmäßig distributed PA Amplitudenmuster in beiden Scan-Richtungen. </li><li> Stellen Sie die Scan-Parameter, und führen die gleichzeitige Netzhaut der SD-OCT und PAOM. Rekonstruieren dreidimensionale Bilder von SD-OCT und PAOM off-line. Unsere Rekonstruktion Codes wurden in Matlab geschrieben und dreidimensionale Visualisierung erreicht uing ein Freeware (Volview, Kitware). Der Algorithmus für die SD-OCT Rekonstruktion kann in Ref gefunden werden. 11 und der Algorithmus für PAOM Rekonstruktion kann in Ref gefunden werden. 6 und Nr. 13. Wenn nötig, wiederholen Verfahren 3.7) -3,9). </li><li> Nach Experiment, schalten Sie die SD-OCT Sondieren Licht, entfernen Sie das Tier aus der Halterung sofort und warm halten, bis er aufwacht natürlich. Halten Sie das Tier in einer dunklen Umgebung für eine zusätzliche Stunde für die Augen zu erholen. Das gesamte Versuchsdauer einschließlich des Tieres Anästhesie und bildgebenden Erfassung, weniger als 30 min für einen erfahrenen Bediener. </li></ol>

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	<li>Kinyoun, J. L., Martin, D. C., Fujimoto, W. Y., Leonetti, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ophthalmoscopy+versus+fundus+photographs+for+detecting+and+grading+diabetic+retinopathy.">Ophthalmoscopy versus fundus photographs for detecting and grading diabetic retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 33 (6), 1888-1893 (1992).</li><li>Schuman, J. S., Wollstein, G., Farra, T., Hertzmark, E., Aydin, A., Fujimoto, J. G., Paunescu, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+optic+nerve+head+measurements+obtained+by+optical+coherence+tomography+and+confocal+scanning+laser+ophthalmoscopy.">Comparison of optic nerve head measurements obtained by optical coherence tomography and confocal scanning laser ophthalmoscopy.</a> Am. J. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+photoacoustic+microscopy+and+computed+tomography.">Multiscale photoacoustic microscopy and computed tomography.</a> Nat. Photonics. 3 (9), 503-509 (2009).</li><li>Jiao, S., Jiang, M., Hu, J., Fawzi, A., Zhou, Q., 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 (4), 3967-3972 (2010).</li><li>Wei, Q., Liu, T., Jiao, S., 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=Image+chorioretinal+vasculature+in+albino+rats+using+photoacoustic+ophthalmoscopy.">Image chorioretinal vasculature in albino rats using photoacoustic ophthalmoscopy.</a> J. Mod. Optic. 58 (21), 1997-2001 (2011).</li><li>Liu, T., Wei, Q., Wang, J., Jiao, S., Zhang, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H.F+Combined+photoacoustic+microscopy+and+optical+coherence+tomography+can+measure+metabolic+rate+of+oxygen.">H.F Combined photoacoustic microscopy and optical coherence tomography can measure metabolic rate of oxygen.</a> Biomed. Opt. Express. 2 (5), 1359-1365 (2011).</li><li>Yu, D., Cringle, S. 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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), 1-3 (2010).</li><li>Zhang, H. F., 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+imaging+of+subcutaneous+structures+using+functional+photoacoustic+microscopy.">In vivo imaging of subcutaneous structures using functional photoacoustic microscopy.</a> Nature protocols. 2, 797-804 (2007).</li><li>Ling, T., Chen, S. L., Guo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+and+wide-directivity+ultrasound+detection+using+high+Q+polymer+microring+resonators.">High-sensitivity and wide-directivity ultrasound detection using high Q polymer microring resonators.</a> Appl. Phys. Lett. 98 (20), 204103 (2011).</li><li>Xie, Z., Jiao, S., 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=Laser-scanning+optical-resolution+photoacoustic+microscopy.">Laser-scanning optical-resolution photoacoustic microscopy.</a> Opt. Lett. 34, 1771-1773 (2009).</li></ol>

Alle tierexperimentellen Verfahren wurden vom Institutional Animal Care und Use Committee der Northwestern University zugelassen.

Hier präsentieren wir eine detaillierte Anleitung bei gleichzeitiger in vivo Netzhaut von Ratten Augen mit PAOM mit SD-OCT kombiniert. Optical-Streuung auf SD-OCT ist vielleicht die klinische &quot;Goldstandard&quot; für die Netzhaut 3, jedoch ist es nicht empfindlich auf die optische Absorption in der Netzhaut zu erkennen. Der neu entwickelte PAOM ist das einzige optische Absorption basierende ophthalmische Bildgebungsmodalität die optischen Absorptionseigenschaften der Netzhaut 6 biet...

integrated photoacoustic ophthalmoscopy and spectral-domain optical coherence tomography

Sowohl die klinische Diagnose und grundlegende Untersuchung der wichtigsten Augenkrankheiten stark von verschiedenen nicht-invasive ophthalmologische Bildgebung Technologien profitieren. Bestehende Netzhaut Modalitäten wie Fundusfotografie 1, konfokale Laser-Scanning-Ophthalmoskopie (cSLO) 2, und die optische Kohärenztomographie (OCT) 3 haben wesentliche Beiträge bei der Überwachung Krankheit Einsätze und Progressionen, und die Entwicklung neuer therapeutischer Strategien. Jedoch verlassen sie überwiegend auf den Back-reflektierten Photonen von der Netzhaut. Als Folge sind die optischen Absorptionseigenschaften der Netzhaut, die üblicherweise stark mit retinalen Pathophysiologie Status verbunden, unzugänglich durch die traditionellen Imaging-Technologien. Photoakustische Ophthalmoskopie (PAOM) ist eine aufstrebende Netzhaut Modalität, die den Nachweis der optischen Absorption Kontraste im Auge ermöglicht mit einer hohen Empfindlichkeit 4-7. In PAOM nanosecond Laserpulse durch die Pupille geliefert und abgetastet über den hinteren Auge photoakustischen (PA) Signalen, die durch einen unfokussierten Ultraschall-Wandler an dem Augenlid erkannt werden induzieren. Wegen der starken optischen Absorption von Hämoglobin und Melanin ist PAOM fähig nichtinvasiv Bildgebung die retinale und choroidale Blutgefäßverteilungen und dem retinalen Pigmentepithel (RPE) Melanin bei hohen Kontrasten 6,7. Noch wichtiger ist, basierend auf dem gut ausgebauten spektroskopische photoakustische Bildgebung 5,8 hat PAOM das Potenzial, die Sauerstoffsättigung des Hämoglobins in retinalen Gefäßen, die entscheidend sein Studium der Physiologie und Pathologie von mehreren blendenden Krankheiten 9 wie diabetische Retinopathie kann abzubilden und neovaskulärem altersbedingter Makula-Degeneration. Darüber hinaus ist das einzig existierende optische Absorption basierenden Augenheilkunde Bildgebungsverfahren können PAOM mit etablierten klinischen ophthalmologische Bildgebung te integriert werdenchniques zu erreichen umfassendere anatomische und funktionelle Auswertungen des Auges auf mehreren optischen Kontrast 6,10 berechnet. In dieser Arbeit, integrieren wir PAOM und Spektral-Domain-OCT (SD-OCT), um gleichzeitig in vivo Netzhaut von Ratten, bei denen sowohl optische Absorption und Streuung Eigenschaften der Netzhaut enthüllt. Die Konfiguration des Systems, System-Ausrichtung und Imaging Acquisition dargestellt.

Abbildung 2 zeigt die 2-D Fundus Bilder von SD-OCT und PAOM erworbenen gleichzeitig in einem Albino-Ratte (A und B) und einer pigmentierten Ratte (C und D), jeweils. In den SD-OCT Fundus Bilder (2A und 2C), haben Netzhautgefäße dunkles Aussehen aufgrund der Hämoglobinabsorption der Sondierung Licht. Neben Netzhautgefäße (RV in 2B), visualisiert PAOM die Aderhaut Blutgefäßverteilungen (CV in 2B) in albino Auge weg...

Watch this Scientific Journal Video about Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography at JoVE.com

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Photoakustischen Ophthalmologie (PAOM), eine optische Absorption-basierenden Bildgebungsmodalität, bietet der ergänzenden Bewertung der Netzhaut zu den derzeit verfügbaren ophthalmische Imaging-Technologien. Wir berichten über die Verwendung von PAOM mit spektraler-Domain Optischen Kohärenztomographie (SD-OCT) zur gleichzeitigen multimodalen Netzhaut bei Ratten integriert.

Integrierte Photoakustische Ophthalmoskopie und Spectral-Domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

integrated-photoacoustic-ophthalmoscopy-spectral-domain-optical

Research

JoVE Journal

Bioengineering

11.4K Aufrufe.  Northwestern University. Photoakustischen Ophthalmologie (PAOM), eine optische Absorption-basierenden Bildgebungsmodalität, bietet der ergänzenden Bewertung der Netzhaut zu den derzeit verfügbaren ophthalmische Imaging-Technologien. Wir berichten über die Verwendung von PAOM mit spektraler-Domain Optischen Kohärenztomographie (SD-OCT) zur gleichzeitigen multimodalen Netzhaut bei Ratten integriert.

Serie: Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

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

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Erkennung und Isolierung von zirkulierenden Melanomzellen mit Photoakustische Flowmetrie

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

In vitro Synthese von Native, Fibröse Lange Abstände und Segmentberichterstattung Lange Spacing Collagen

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

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.

Photoakustische Zystographie

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

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Vorbereitung und photoakustische Analyse von Zell Fahrzeuge, die Gold-Nanostäbchen

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

Computertomographie und Optical Imaging von Osteogenesis-Angiogenese-Kopplung an Integration von Schädelknochen Autografts und Allografts Beurteilen

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

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.

Umschaltbare akustische und optische Auflösung Photoakustische Mikroskopie für<em&gt; In vivo</em&gt; Kleintier-Blut-Vaskulatur-Bildgebung

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

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

Ein leistungsstarkes kompaktes Fotoakustik-Tomographie-System für<em&gt; In vivo</em&gt; Kleintier Gehirn Bildgebung

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodale Volumetrische Retinal Imaging von schrägen Scanning Laser Ophthalmoskopie (oSLO) und optische Kohärenztomografie (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Zerstörungsfreie Überwachung der abbaubaren Gerüst-basierten Tissue-Engineering Blutgefäß Entwicklung mit optischen Kohärenztomographie

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...