Ringraziamo il generoso sostegno della National Science Foundation (CARRIERA CBET-1055379) e il National Institutes of Health (1RC4EY021357, 1R01EY019951). Riconosciamo inoltre il sostegno della Cina Borsa di studio del Consiglio a Song Wei.

1. Configurazione di sistema <ol><li> PAOM sottosistema <ol><li> Illuminazione fonte: un laser Nd: YAG (SPOT-10-100, Elforlight Ltd, Regno Unito: 20 μJ / impulso, 2 nsec durata dell&#39;impulso, 30 kHz frequenza cardiaca massima di ripetizione). </li><li> L&#39;uscita laser a 1064 nm è a frequenza doppia di 532 nm da una beta-bario-borato (BBO) cristallo (CasTech, San Jose, CA). Dopo un&#39;ulteriore suddivisione da uno specchio linea laser, luce 532 nm viene erogata attraverso una modalità singola fibra ottica (P1-460A-FC-5, Thorlabs), e 1064 nm laser viene registrato da un fotodiodo (DET10A, Thorlabs), che innesca PA acquisizione del segnale. </li><li> La luce laser proveniente dalla modalità singola fibra ottica viene erogata sulla retina da un galvanometro (GM, QS-7, Nutfield Technology) ed una configurazione telescopio (f1 = 75 mm e f2 = 14 mm, Edmund Optics) 6. </li><li> Un trasduttore ago sfocata (40 MHz di frequenza centrale, larghezza di banda di 16 MHz, 0,4 × 0,4 mm 2 dimensioni elemento attivo, NIH risorse Center per le tecnologie del trasduttore ad ultrasuoni, la University of Southern California), viene posto a contatto con la palpebra di rilevare i segnali PA generati dalla retina. Gel ultrasuoni (Sonotech) è applicato tra la punta del trasduttore e della palpebra animale per accoppiamento acustico bene. </li><li> Il segnale viene amplificato PA da due amplificatori (ZFL-500LN +, Mini-Circuits, e 5073PR, Olympus), e viene digitalizzato da una scheda di acquisizione dati (CS14200, Gage Applicata). </li></ol></li><li> SD-OCT sottosistema <ol><li> Bassa sorgente luminosa coerenza: una banda larga super-luminescente diodo (IPSDD0804, InPhenix; centro lunghezza d&#39;onda: 840 nm, 6-dB larghezza di banda: 50 nm), che determina la risoluzione assiale di 6 micron. </li><li> La luce nel vicino infrarosso è diviso per fare riferimento a braccio e braccio del campione da un 50 × 50 su misura single-mode in fibra accoppiatore (Ottica OZ). </li><li> Dopo aver combinato con la luce PAOM illuminante da uno specchio caldo (FM02, Thorlabs), braccio del campione ottobre condivide la scansione stessa e la consegna ottica wiPAOM ° 6. </li><li> Una casa costruita spettrometro viene utilizzata per registrare le SD-ottobre segnali di interferenza, in cui una linea di scansione CCD (Aviiva SM2, E2V) permette un tasso di una linea di 24 kHz. Progettazione di spettrometri tipici possono essere trovati da diverse letterature già pubblicati 11 e accoppiato in fibra SD-ottobre spettrometri sono ora disponibili in commercio. L&#39;SD-OTT sensibilità viene misurata per essere migliore di 90 dB. </li></ol></li><li> Schema di scansione <ol><li> Fast 2-D raster scansione del galvanometro è controllato da una scheda di uscita analogica (PCI-6731, National Instruments), che attiva anche la cottura sia PAOM laser e l&#39;acquisizione del segnale di ottobre dello spettrometro. Come risultato, le acquisizioni dati in PAOM e sottosistemi ottobre sono sincronizzati. </li><li> L&#39;acquisizione PAOM dati viene attivato da una sequenza di registrazione fotodiodo PAOM laser (vedi 1.1.2). </li><li> 3-D immagini volumetriche o 2-D immagini del fondo oculare sono costruiti da 256 B-Scan (256 linee A per BLa scansione immagine). </li></ol></li></ol> 2. Sistema di allineamento <ol><li> Massimizzare l&#39;efficienza doppia frequenza del cristallo BBO e l&#39;efficienza di accoppiamento della modalità singola fibra ottica. Indossare occhiali LG3 (Thorlabs) per la protezione degli occhi del personale durante l&#39;ottimizzazione della luce PAOM illuminante. </li><li> Collimare l&#39;uscita laser fibra di PAOM a 2,0 mm di diametro. </li><li> Allineare le luci di illuminazione combinati di PAOM e SD-OCT per essere coassiale. </li><li> Impostare la luce PAOM eccitazione a ~ 40 nJ / impulsi e SD-OCT sondaggio luce ~ 0,8 mW, entrambi i quali sono segnalati per essere sicuro per gli occhi 6,12. </li></ol> 3. Imaging in vivo Multimodale della retina <ol><li> Trasferire il ratto ad una scatola di polipropilene trasparente, e anestetizzare l&#39;animale da una miscela di aria isoflurano (Phoenix farmaceutica, Inc.) e normale ad una concentrazione di 1,5% e una portata di 2,0 litri / min per 10 min. </li><li> Frena il ratto anestetizzato in un homemade supporto con cinque assi libertà regolabile (Figura 1), e mantenere la sua temperatura corporea a ~ 37 ° C da un cuscinetto riscaldante (Repti Therm, laboratori Zoomed, Inc.). Mantenere l&#39;anestesia per inalazione di gas misto aria isoflurano e normale con 1,0% di concentrazione e 1,5 litri / min portata tutta la durata dell&#39;esperimento. </li><li> Tagliare la ciglia con un paio di forbici chirurgiche, dilatare le pupille con la soluzione oftalmica Tropicamide 1%, e paralizzare il muscolo sfintere dell&#39;iride utilizzando 0,5% Tetracainum cloridrato soluzione oftalmica. Applicare artificiali gocce strappo (Systane, Alcon Laboratories, Inc.) ad occhio ratto ogni minuto per evitare la disidratazione cornea e formazione di cataratta. Monitorare la frequenza cardiaca degli animali, la respirazione, e l&#39;ossigenazione del sangue da un pulsossimetro (8.600 V, Nonin Medical, MN) durante l&#39;esposizione. </li><li> Accendere SD-OCT luce che illumina e controllare la luce probe sia accurata al ~ 0,8 mW. </li><li> Attivare la scansione galvanometro. Allineare la SD-OCT consegna di irradiazione di lucesulla retina di ratto e di identificare la regione retinica di interesse (ROI) regolando cinque assi detentore dell&#39;animale. Qui, il disco ottico viene intenzionalmente collocato nel centro del campo di vista, mentre il ROI deve essere selezionato in base a differenti esigenze di ricerca. </li><li> Regolare ulteriormente il titolare animale per ottimizzare le SD-ottobre qualità di imaging retinico sezione trasversale in entrambe le direzioni di scansione (per commutare la direzione di scansione raster) finché il miglior fuoco ottico viene raggiunto. </li><li> Preparare il trasduttore ago su un cinque assi piattaforma regolabile, applicare una goccia di gel ultrasuoni alla punta del trasduttore, e delicatamente contattare la punta del trasduttore al palpebra animale. </li><li> Impostare laser PAOM al-trigger esterno modalità, avviare la scansione galvanometro, e attivare visualizzazione in tempo reale delle PAOM trasversale immagine della retina animale. Regolare accuratamente l&#39;orientamento del trasduttore finché l&#39;immagine PAOM ha il miglior rapporto segnale-rumore (SNR) e, nel frattempo, mostra un uniformemente distributed modelli ampiezza PA in entrambe le direzioni di scansione. </li><li> Impostare i parametri di scansione, e condurre l&#39;imaging simultaneo della retina di SD-OCT e PAOM. Ricostruire le immagini tridimensionali di SD-OCT e PAOM off-line. I nostri codici di ricostruzione sono stati scritti in Matlab e la visualizzazione tridimensionale è stato raggiunto uing un freeware (Volview, Kitware). L&#39;algoritmo per SD-OCT ricostruzione può essere trovato in rif. 11 e l&#39;algoritmo per PAOM ricostruzione può essere trovato in rif. 6 e Ref.. 13. Se necessario, ripetere le procedure di 3,7) -3,9). </li><li> Dopo l&#39;esperimento, spegnere l&#39;SD-OCT luce sondaggio, togliere l&#39;animale dal supporto immediatamente, e tenere in caldo fino a quando non si sveglia in modo naturale. Tenere l&#39;animale in un ambiente buio per un&#39;ora in più per gli occhi per recuperare. L&#39;intera durata sperimentale, compresa l&#39;anestesia animale e di acquisizione di immagini, è inferiore a 30 minuti per un operatore esperto. </li></ol>

<ol>
	<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. Ophthalmol. 135 (4), 504-512 (2003).</li><li>Str&#248;m, C., Sander, B., Larsen, N., Larsen, M., Lund-Andersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+macular+edema+assessed+with+optical+coherence+tomography+and+stereo+fundus+photography.">Diabetic macular edema assessed with optical coherence tomography and stereo fundus photography.</a> Invest. Ophthalmol. Vis. Sci. 43 (1), 241-245 (2002).</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Optical-resolution+Photoacoustic+Microscopy.">Three-dimensional Optical-resolution Photoacoustic Microscopy.</a> J. Vis. Exp. (51), e2729 (2011).</li><li>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=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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+distribution+and+consumption+within+the+retina+in+vascularised+and+avascular+retinas+and+in+animal+models+of+retinal+disease.">Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease.</a> Prog. Retin. Eye Res. 20 (2), 175-208 (2001).</li><li>Song, W., Wei, Q., Liu, T., Kuai, D., Burke, J. M., 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=Integrating+photoacoustic+ophthalmoscopy+with+scanning+laser+ophthalmoscopy,+optical+coherence+tomography,+and+fluorescein+angiography+for+a+multimodal+retinal+imaging+platform.">Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform.</a> J. Biomed. Opt. 17 (6), 061206 (2012).</li><li>Mark, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brezinski Optical Coherence Tomography: Principles and Applications. , (2006).</li><li>Hu, S., Rao, B., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+photoacoustic+ophthalmic+angiography.">Label-free photoacoustic ophthalmic angiography.</a> Opt. Lett. 35 (1), 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>

Tutte le procedure sperimentali sono stati approvati dalla cura degli animali e del Comitato Istituzionale uso della Northwestern University.

Qui, vi presentiamo un istruzioni dettagliate su simultanea in vivo imaging della retina degli occhi di ratto utilizzando PAOM combinato con SD-OCT. Ottico-scattering basata SD-OCT è forse la clinica &quot;gold standard&quot; per imaging della retina 3, tuttavia, non è sensibile per rilevare l&#39;assorbimento ottico nella retina. Il recente sviluppo PAOM è l&#39;unica ottica di assorbimento a base di modalità di imaging oftalmico che fornisce le proprietà di assorbimento ottico della retina <su...

integrated photoacoustic ophthalmoscopy and spectral-domain optical coherence tomography

Sia la diagnosi clinica e di indagine fondamentale delle malattie oculari gravi trarre grandi vantaggi dalle tecnologie oftalmiche varie non invasive di imaging. Gli attuali modalità di imaging della retina, come la fotografia del fondo oculare 1, oftalmoscopia scansione laser confocale (cSLO) 2, e la tomografia a coerenza ottica (OCT) 3, hanno contribuito in modo significativo nel monitoraggio insorgenze della malattia e progressioni, e lo sviluppo di nuove strategie terapeutiche. Tuttavia, essi prevalentemente affidamento sulle back-fotoni riflessi dalla retina. Di conseguenza, le proprietà di assorbimento ottico della retina, che sono di solito fortemente associati con lo stato fisiopatologia retinica, sono accessibili dalle tecnologie di imaging tradizionali. Oftalmoscopia fotoacustica (PAOM) è una modalità emergente imaging della retina che permette di rilevare i contrasti di assorbimento ottico nell&#39;occhio con una sensibilità elevata 4-7. In PAOM nanimpulsi laser osecond vengono forniti attraverso la pupilla e la scansione attraverso l&#39;occhio posteriore per indurre fotoacustica (PA), i segnali che vengono rilevati da un trasduttore ad ultrasuoni non a fuoco attaccato alla palpebra. A causa del forte assorbimento ottico di emoglobina e melanina, PAOM è in grado di non-invasivo imaging delle vasculatures retina e della coroide, e dell&#39;epitelio pigmentato retinico (RPE) melanina ad alto contrasto 6,7. Ancora più importante, basato sul ben sviluppato spettroscopica di imaging fotoacustica 5,8, PAOM ha il potenziale per mappare la saturazione di ossigeno dell&#39;emoglobina in vasi retinici, che può essere critica nello studio della fisiologia e patologia delle diverse malattie cecità 9 come la retinopatia diabetica e neovascolare legata all&#39;età degenerazione maculare. Inoltre, essendo l&#39;unico esistente assorbimento ottico-oftalmico a base di modalità di imaging, PAOM può essere integrato con consolidata clinica oftalmica Imaging techniques per ottenere valutazioni anatomiche e funzionali più complete degli occhi in base a molteplici contrasti optical 6,10. In questo lavoro, integriamo PAOM e spettrali nel dominio OCT (SD-OCT) per la realizzazione contemporanea in vivo imaging della retina di ratto, in cui si rivelano sia assorbimento ottico e proprietà di diffusione della retina. La configurazione del sistema, l&#39;allineamento del sistema di acquisizione e di imaging sono presentati.

La figura 2 mostra le immagini 2-D fundus di SD-OCT e PAOM acquisite simultaneamente in un ratto albino (A e B) e un ratto pigmentato (C e D), rispettivamente. Nelle SD-ottobre le immagini del fondo oculare (figure 2A e 2C), vasi retinici hanno aspetto scuro a causa dell&#39;assorbimento di emoglobina di sondare luce. Oltre a vasi retinici (RV in Figura 2B), PAOM visualizza le vasculatures coroide (CV in Figura 2B) in a...

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

Fotoacustica oftalmologia (PAOM), un assorbimento ottico basato modalità di imaging, fornisce la valutazione complementare della retina alle tecnologie attualmente disponibili oftalmici imaging. Riportiamo l&#39;utilizzo di PAOM integrato con-dominio spettrale tomografia a coerenza ottica (SD-OCT) per il controllo simultaneo di imaging multimodale retinica nel ratto.

Integrato oftalmoscopia fotoacustico e Spectral-dominio 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 Views.  Northwestern University. Fotoacustica oftalmologia (PAOM), un assorbimento ottico basato modalità di imaging, fornisce la valutazione complementare della retina alle tecnologie attualmente disponibili oftalmici imaging. Riportiamo l'utilizzo di PAOM integrato con-dominio spettrale tomografia a coerenza ottica (SD-OCT) per il controllo simultaneo di imaging multimodale retinica nel ratto.

Video: 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.

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

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.

Rilevazione e isolamento delle cellule del melanoma circolanti con flussimetria fotoacustico

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 sintesi in vitro di Native, Spaziatura fibrosa lungo e segmentale collagene spaziatura lungo

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.

Cistografia fotoacustico

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

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.

Preparazione e fotoacustico Analisi dei veicoli cellulari contenenti oro Nanorods

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.

Tomografia Computerizzata e Optical Imaging di osteogenesi-angiogenesi Accoppiamento per valutare l&#39;integrazione di craniche Bone Autoinnesti e Alloinnesti

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.

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

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

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.

Un sistema compatto di Tomografia Photoacoustico ad alte prestazioni per<em&gt; In Vivo</em&gt; Imaging cerebrale di piccole dimensioni

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 Imaging retinico volumetrica da Oblique scansione Laser oftalmoscopia (oSLO) e tomografia a coerenza ottica (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.

Non distruttive monitoraggio dello sviluppo del vaso sanguigno tessutale basati su Scaffold degradabile usando la tomografia a coerenza ottica

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