Nous tenons à remercier le généreux soutien de la National Science Foundation (CARRIÈRE CBET-1055379) et le National Institutes of Health (1RC4EY021357, 1R01EY019951). Nous reconnaissons aussi le soutien du Conseil des bourses de Chine à Song Wei.

1. Configuration du système <ol><li> PAOM sous-système <ol><li> Source d&#39;éclairage: un laser Nd: YAG (SPOT-10-100, Elforlight Ltd, UK: 20 pJ / impulsion, 2 ns la durée d&#39;impulsion, 30 kHz maximum le taux de répétition des impulsions). </li><li> La sortie du laser à 1064 nm est doublée en fréquence à 532 nm par un bêta-borate de baryum (BBO) cristal (Castech, San Jose, CA). Après scission en outre par un miroir ligne laser, 532 nm de lumière est fournie par une fibre optique monomode (P1-460A-CF-5, Thorlabs), et 1064 nm laser est enregistrée par une photodiode (DET10A, Thorlabs), qui déclenche l&#39;acquisition du signal PA. </li><li> La lumière laser sortant de la fibre optique monomode est fournie sur la rétine par un galvanomètre (GM, QS-7, Nutfield Technology) et une configuration de télescope (f1 = f2 et 75 mm = 14 mm, Edmund Optics) 6. </li><li> Un transducteur aiguille floue (40-MHz de fréquence centrale, à 16 MHz de bande passante, de 0,4 x 0,4 mm 2 taille de l&#39;élément actif, NIH ressources Center pour les technologies de transducteur à ultrasons, The University of Southern California) est mis en contact avec la paupière pour détecter les signaux générés PA de la rétine. Gel à ultrasons (Sonotech) est appliquée entre la pointe de sonde et de la paupière pour animaux bon couplage acoustique. </li><li> Le signal PA est amplifié par deux amplificateurs (ZFL-500LN +, mini-circuits, et 5073PR, Olympus), et est numérisé par une carte d&#39;acquisition de données (CS14200, Gage Applied). </li></ol></li><li> SD-OCT-système <ol><li> Faible source de lumière cohérence: une large bande super-luminescente diode (IPSDD0804, InPhenix; longueur d&#39;onde centrale: 840 nm; 6-dB Largeur de bande: 50 nm), ce qui détermine la résolution axiale de 6 pm. </li><li> La lumière dans le proche infrarouge est divisé pour référencer le bras et le bras d&#39;un échantillon de 50 x 50 coupleur personnalisée fibre monomode (optique OZ). </li><li> Après avoir combiné avec PAOM lumière d&#39;éclairage par un miroir chaud (FM02, Thorlabs), le bras échantillon octobre partage la même analyse et la livraison wi optiquee PAOM 6. </li><li> Un spectromètre de fabrication artisanale est utilisé pour enregistrer les signaux parasites SD-OCT, où une ligne de balayage CCD (Aviiva SM2, e2v) permet un taux d&#39;A-ligne de 24 kHz. Conception des spectromètres typiques peuvent être trouvées à partir de plusieurs littératures publiées antérieurement 11 et couplé par fibre SD-OCT spectromètres sont maintenant disponibles dans le commerce. La sensibilité SD-OCT est mesuré à plus de 90 dB. </li></ol></li><li> Numérisation Scheme <ol><li> 2-D rapide balayage de trame du galvanomètre est commandé par une carte de sortie analogique (PCI-6731, National Instruments), ce qui déclenche également la fois la mise à feu laser PAOM et l&#39;acquisition du signal de spectromètre octobre. En conséquence, les acquisitions de données dans PAOM et sous-systèmes sont synchronisés octobre. </li><li> L&#39;acquisition PAOM données est déclenchée par un enregistrement photodiode PAOM séquence laser (voir 1.1.2). </li><li> Images 3-D volumétriques ou 2-D des images du fond d&#39;œil sont construits à partir de 256 B-scan images (256 lignes A par B-Analyser l&#39;image). </li></ol></li></ol> 2. Système d&#39;alignement <ol><li> Maximiser l&#39;efficacité de doublage de fréquence du cristal BBO et l&#39;efficacité de couplage de la fibre optique monomode. Porter des lunettes de protection (LG3 Thorlabs) pour protéger les yeux du personnel lors de l&#39;optimisation de la lumière PAOM éclairante. </li><li> Collimater la sortie laser à fibre de PAOM 2,0 mm de diamètre. </li><li> Alignez les feux d&#39;éclairage combinées de PAOM et SD-OCT à être coaxial. </li><li> Réglez la lumière d&#39;excitation PAOM à ~ 40 nJ / impulsion et SD-OCT sonder la lumière à ~ 0,8 mW, qui sont tous deux rapporté danger pour les yeux 6,12. </li></ol> 3. L&#39;imagerie in vivo rétine multimodal <ol><li> Transférer le rat à une boîte de polypropylène transparent, et l&#39;animal anesthésié par un mélange d&#39;air isoflurane (Phoenix pharmaceutique, Inc) et la normale à la concentration de 1,5% et un débit de 2,0 litre / min pendant 10 min. </li><li> Retenez le rat anesthésié dans un homemade support avec cinq axes liberté réglable (figure 1), et maintenir sa température corporelle à ~ 37 ° C par un coussin chauffant (Repti Therm, les laboratoires Zoomed, Inc.) Maintenir l&#39;anesthésie par inhalation de gaz de l&#39;air et de l&#39;isoflurane mixte normal avec une concentration de 1,0% et 1,5 litre / min de débit tout au long de l&#39;expérience. </li><li> Couper le cil en utilisant une paire de ciseaux chirurgicaux, dilater les pupilles avec une solution ophtalmique Tropicamide 1%, et de paralyser le muscle sphincter de l&#39;iris en utilisant 0,5% de solution ophtalmique de chlorhydrate de tétracaïne. Appliquer des larmes artificielles (Systane, Alcon Laboratories, Inc) pour l&#39;oeil de rat à chaque instant pour éviter la déshydratation cornée et la formation de cataracte. Surveiller le rythme cardiaque des animaux, la respiration et l&#39;oxygénation du sang par un oxymètre de pouls (8.600 V, Nonin Medical, MN) lors de l&#39;imagerie. </li><li> Allumez le SD-OCT lumière d&#39;éclairage et de vérifier la lumière de sondage à ~ 0,8 mW. </li><li> Activer le balayage galvanomètre. Aligner la livraison SD-OCT irradiation lumineusesur la rétine de rat et d&#39;identifier la région rétinienne d&#39;intérêt (ROI) en ajustant le détenteur des animaux à cinq axes. Ici, le disque optique est volontairement placé dans le centre du champ de vision, tandis que le retour sur investissement doit être choisi en fonction des besoins de recherche différents. </li><li> En outre d&#39;ajuster le support de l&#39;animal à optimiser les qualités SD-OCT imagerie rétinienne de section dans les deux directions de balayage (par commutation de la direction de balayage de trame) jusqu&#39;à ce que la meilleure mise au point optique est atteint. </li><li> Préparer le transducteur aiguille sur une plate-forme de cinq axes réglable, appliquer une goutte de gel à ultrasons à l&#39;extrémité du capteur et douces au contact de la pointe de transducteur sur la paupière animal. </li><li> Définir laser PAOM au mode de déclenchement externe, démarrer le balayage à galvanomètre, et activer l&#39;affichage en temps réel de PAOM de section transversale sur l&#39;image de la rétine animal. Réglez soigneusement le orientation de la sonde jusqu&#39;à ce que l&#39;image PAOM a le meilleur rapport signal sur bruit (SNR) et, dans l&#39;intervalle, montre un uniforme distributed modèles d&#39;amplitude PA dans les deux directions de balayage. </li><li> Définissez les paramètres de numérisation, et de mener l&#39;imagerie simultanée de la rétine SD-OCT et PAOM. Reconstruire les images tridimensionnelles de SD-OCT et PAOM hors ligne. Nos codes de reconstruction ont été écrit en Matlab et visualisation en trois dimensions a été réalisée nuer un freeware (Volview, Kitware). L&#39;algorithme de reconstruction SD-OCT peut être trouvée dans la référence. 11 et l&#39;algorithme de reconstruction PAOM peuvent être trouvées dans la référence. 6 et Réf. 13. Si nécessaire, répétez la procédure de 3,7) -3,9). </li><li> Après l&#39;expérience, éteindre la lumière SD-OCT sondage, enlever l&#39;animal de support immédiatement, et le garder au chaud jusqu&#39;à ce qu&#39;il se réveille naturellement. Garder l&#39;animal dans un environnement sombre pendant une heure supplémentaire pour les yeux pour récupérer. Toute la durée expérimental, y compris l&#39;anesthésie des animaux et de l&#39;acquisition d&#39;image, est inférieure à 30 min pour un opérateur expérimenté. </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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Toutes les procédures expérimentales chez l&#39;animal ont été approuvés par le soin des animaux et du Comité institutionnel utilisation de la Northwestern University.

Ici, nous présentons une instruction détaillée sur la simultanée en imagerie rétinienne in vivo des yeux de rat en utilisant PAOM combiné avec SD-OCT. Optique de diffusion basée sur SD-OCT est, peut-être, le «gold standard» clinique de l&#39;imagerie rétinienne 3, mais il n&#39;est pas sensible pour détecter l&#39;absorption optique dans la rétine. Le PAOM nouvellement développé est le seul absorption optique à base de modalité d&#39;imagerie ophtalmique qui fournit des prop...

integrated photoacoustic ophthalmoscopy and spectral-domain optical coherence tomography

Tant le diagnostic clinique et fondamentale enquête des maladies oculaires majeurs grandement bénéficier de diverses technologies non invasives d&#39;imagerie ophtalmique. Existants modalités d&#39;imagerie de la rétine, comme la photographie de fundus 1, ophtalmoscopie confocale à balayage laser (ALSC) 2, et la tomographie par cohérence optique (OCT) 3, ont des contributions importantes à la surveillance des maladies et des progressions onsets, et le développement de nouvelles stratégies thérapeutiques. Cependant, elles principalement s&#39;appuyer sur les sauvegardes reflètent photons de la rétine. En conséquence, les propriétés d&#39;absorption optique de la rétine, qui sont généralement fortement liés au statut de la physiopathologie de la rétine, sont inaccessibles par les techniques d&#39;imagerie traditionnelles. Ophtalmoscopie photoacoustique (PAOM) est une modalité d&#39;imagerie rétinienne émergente qui permet la détection des contrastes d&#39;absorption optique de l&#39;œil avec une grande sensibilité 4-7. En PAOM nanosecond impulsions laser sont livrés à travers la pupille et balayé à travers l&#39;oeil postérieur à induire photoacoustique (PA), des signaux qui sont détectés par un capteur ultrasonore non focalisé attaché à la paupière. En raison de la forte absorption optique de l&#39;hémoglobine et la mélanine, PAOM est capable de façon non invasive d&#39;imagerie les vascularisations la rétine et de la choroïde et l&#39;épithélium pigmentaire rétinien (EPR) mélanine à forts contrastes 6,7. Plus important encore, sur la base du bien développé spectroscopique photoacoustique imagerie 5,8, PAOM a le potentiel pour cartographier la saturation en oxygène dans l&#39;hémoglobine des vaisseaux rétiniens, qui peut être critique dans l&#39;étude de la physiologie et la pathologie de plusieurs maladies cécitantes 9 telles que la rétinopathie diabétique et la dégénérescence maculaire néovasculaire liée à l&#39;âge. En outre, étant le seul existant absorption optique à base de modalité d&#39;imagerie ophtalmique, PAOM peut être intégré avec bien établie clinique ophtalmique imagerie techniques pour obtenir plus d&#39;évaluations complètes anatomiques et fonctionnelles de l&#39;oeil basée sur de multiples contrastes optiques 6,10. Dans ce travail, nous intégrons PAOM et domaine spectral OCT (SD-OCT) pour simultanément dans l&#39;imagerie rétinienne in vivo de rat, où à la fois l&#39;absorption optique et des propriétés de diffusion de la rétine sont révélés. La configuration du système du système d&#39;alignement, et l&#39;acquisition d&#39;imagerie sont présentés.

La figure 2 montre les images 2-D du fond d&#39;œil de SD-OCT et PAOM acquises simultanément chez un rat albinos (A et B) et un rat pigmenté (C et D), respectivement. Dans les images rétiniennes SD-OCT (Figures 2A et 2C), vaisseaux rétiniens ont une apparence sombre à cause de l&#39;absorption d&#39;hémoglobine de sondant la lumière. En plus des vaisseaux rétiniens (RV dans la figure 2B), PAOM visualise les vascularisations cho...

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

Photoacoustique ophtalmologie (PAOM), une modalité d&#39;imagerie optique basée sur l&#39;absorption, fournit l&#39;évaluation complémentaire de la rétine aux technologies actuellement disponibles imagerie ophtalmique. Nous rapportons l&#39;aide de PAOM intégré domaine spectral tomographie par cohérence optique (SD-OCT) pour une utilisation simultanée imagerie rétinienne chez le rat multimodal.

Intégré ophtalmoscopie photoacoustique et Spectral-domaine tomographie par cohérence optique

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 vues.  Northwestern University. Photoacoustique ophtalmologie (PAOM), une modalité d'imagerie optique basée sur l'absorption, fournit l'évaluation complémentaire de la rétine aux technologies actuellement disponibles imagerie ophtalmique. Nous rapportons l'aide de PAOM intégré domaine spectral tomographie par cohérence optique (SD-OCT) pour une utilisation simultanée imagerie rétinienne chez le rat multimodal.

Vidéo: 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.

Tridimensionnelle optique résolution microscopie photoacoustique

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

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Bio-ingénierie

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.

Détection et isolement des cellules du mélanome de circulation à l&#39;aide Débitmétrie photoacoustique

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.

En synthèse in vitro de la maternelle, de l&#39;espacement de longue fibreux et segmentaire collagène Espacement long

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.

Cystographie Photoacoustique

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.

Préparation et Photoacoustique Analyse des véhicules cellulaires contenant de l&#39;or 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.

Tomodensitométrie et l&#39;imagerie optique du couplage de l&#39;ostéogenèse-angiogenèse à évaluer l&#39;intégration des crâniens autogreffes et allogreffes

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.

Microscopie photoacoustique à résolution acoustique et optique à commutation commutable pour<em&gt; In vivo</em&gt; Imagerie vasculaire de sang de petit animal

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 système de tomographie photoacoustique compacte haute performance pour<em&gt; In vivo</em&gt; Imagerie du cerveau à petits animaux

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.

Multimodal d’imagerie rétinienne volumétrique Oblique à balayage Laser ophtalmologique (oSLO) et la tomographie en cohérence optique (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.

Contrôles non destructifs suivi du développement de dégradable axée sur l’échafaud tissulaire des vaisseaux sanguins à l’aide de la tomographie à cohérence optique

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