Agradecemos o apoio generoso da National Science Foundation (CARREIRA CBET-1055379) e os Institutos Nacionais de Saúde (1RC4EY021357, 1R01EY019951). Agradecemos também o apoio da China Scholarship Council para Song Wei.

1. Configuração do Sistema <ol><li> PAOM Subsistema <ol><li> Fonte de iluminação: um laser Nd: YAG (SPOT-10-100, Elforlight Ltd, UK: 20 μJ / pulso, 2 ns duração de pulso, 30 kHz máxima taxa de repetição de impulsos). </li><li> O laser de saída em 1064 nm é freqüência dobrada para 532 nm por um beta-borato de bário (BBO) cristal (CasTech, San Jose, CA). Depois da separação ainda mais por um espelho de feixe de laser, 532 nm de luz é entregue através de uma fibra óptica de modo único (P1-460A-FC-5, Thorlabs), e 1064 nm do laser é gravada por um fotodíodo (DET10A, Thorlabs), o que desencadeia PA a aquisição de sinal. </li><li> A luz de laser que sai da fibra óptica de modo único é entregue na retina por um galvanómetro (GM, QS-7, Nutfield Technology) e uma configuração de telescópio (f1 = 75 mm e f2 = 14 mm, Edmund Optics) 6. </li><li> Um transdutor de agulha sem foco (40-MHz frequência central, de 16 MHz de largura de banda, 0,4 × 0,4 mm 2 tamanho elemento ativo, NIH Recursos Center para as Tecnologias de transdutor de ultra-som, a Universidade do Sul da Califórnia) é colocado em contato com a pálpebra para detectar os sinais PA gerados a partir da retina. Gel de ultra-sons (Sonotech) é aplicada entre a ponta do transdutor e da pálpebra animal para acoplamento acústico boa. </li><li> O sinal é amplificado PA por dois amplificadores (ZFL-500LN +, Mini-circuitos, e 5073PR, Olympus), e é digitalizado por uma placa de aquisição de dados (CS14200, Gage Aplicada). </li></ol></li><li> SD-OCT Subsistema <ol><li> Fonte de luz baixa coerência: um diodo luminescente super-banda larga (IPSDD0804, InPhenix; centro de comprimento de onda: 840 nm; 6-dB largura de banda: 50 nm), que determina a resolução axial de 6 m. </li><li> A luz de infravermelho próximo é dividido para referenciar braço e braço de amostra por um 50 × 50 acoplador de fibra de modo único personalizado (OZ Optics). </li><li> Depois de combinar com PAOM luz que ilumina por um espelho quente (FM02, Thorlabs), braço de amostra outubro partes a digitalização mesmo e entrega óptica with PAOM 6. </li><li> Um espectrômetro construídos em casa é usado para gravar os sinais SD-outubro de interferência, onde uma linha de varredura câmera CCD (Aviiva SM2, e2v) permite uma taxa de uma linha de 24 kHz. Projeto de espectrômetros típicos podem ser encontrados a partir de várias literaturas publicadas anteriormente 11 e fibra acoplados SD-outubro espectrômetros estão disponíveis comercialmente. A sensibilidade SD-OCT é medido para ser melhor do que 90 dB. </li></ol></li><li> Esquema de varredura <ol><li> Fast 2-D raster de varrimento do galvanómetro é controlado por uma placa de saída analógica (PCI-6731, National Instruments), que também acciona tanto o PAOM disparo do laser e a aquisição de sinal de outubro espectrómetro. Como resultado, as aquisições de dados em PAOM e subsistemas de outubro estão sincronizados. </li><li> A aquisição de dados PAOM é desencadeada por uma gravação fotodiodo PAOM sequência de laser (ver 1.1.2). </li><li> Imagens em 3-D de volume ou imagens 2-D de fundo de olho são construídos a partir de 256 B-scan imagens (256 linhas por A B-Digitalizar a imagem). </li></ol></li></ol> 2. Alinhamento sistema <ol><li> Maximizar a eficiência de frequência de duplicação do cristal BBO e a eficiência de acoplamento da fibra óptica de modo único. Use óculos de proteção (LG3 Thorlabs) para o pessoal de protecção quando otimizando o PAOM luz iluminadora. </li><li> Colimar o laser de fibra de saída PAOM a 2,0 mm de diâmetro. </li><li> Alinhe as luzes de iluminação combinados de PAOM e SD-OCT para ser coaxial. </li><li> Definir a luz de excitação PAOM em ~ 40 nJ / pulso e SD-OCT sondagem luz em ~ 0,8 mW, ambos os quais são descritos como sendo segura olho 6,12. </li></ol> 3. Imagem in vivo Multimodal Retinal <ol><li> Transferir o rato a uma caixa de polipropileno transparente, e anestesiar os animais com uma mistura de ar com isoflurano (Phoenix farmacêutica, Inc.) e normal a uma concentração de 1,5% e uma taxa de fluxo de 2,0 litros / min, durante 10 min. </li><li> Contenha o rato anestesiado em um homemade suporte, com cinco eixos liberdade ajustável (Figura 1), e manter a sua temperatura corporal em ~ 37 ° C por uma almofada de aquecimento (Repti terma, laboratórios Zoomed, Inc.). Manter a anestesia por inalação de gás de ar isoflurano e normal misturada com a concentração de 1,0% e 1,5 litros / min velocidade de fluxo ao longo da experiência. </li><li> Cortar a pestana usando uma tesoura cirúrgica, dilatar as pupilas com uma solução de 1% Tropicamida oftálmica e paralisam o músculo do esfíncter da íris usando 0,5% solução oftálmica Cloridrato de tetracaína. Aplicar gotas de lágrimas artificiais (Systane, Alcon Laboratories, Inc.) para o olho do rato a cada minuto para evitar a desidratação da córnea e formação de catarata. Monitorar a freqüência cardíaca de animais, respiração e oxigenação do sangue por um oxímetro de pulso (8.600 V, Nonin Medical, MN) durante o exame. </li><li> Ligue SD-OCT luz que ilumina e verifique a luz sondagem para ser ~ mW 0,8. </li><li> Ative a digitalização galvanômetro. Alinhe o SD-OCT entrega luz de irradiaçãosobre a retina de rato e identificar a região da retina de interesse (ROI), ajustando o detentor dos animais de cinco eixos. Aqui, o disco óptico é intencionalmente colocada no centro do campo de visão, enquanto o ROI deve ser seleccionado com base nos requisitos de pesquisa. </li><li> Ajustar ainda mais o suporte de animal para optimizar as qualidades SD-outubro de imagem da secção transversal da retina em ambos os sentidos de digitalização (mudando a direcção de exploração de varredura) até que o melhor foco óptico é alcançado. </li><li> Prepare o transdutor agulha sobre uma plataforma de cinco eixos ajustável, aplicar uma gota de gel de ultra-sons para a ponta do transdutor, e gentilmente contactar a ponta do transdutor para a pálpebra animal. </li><li> Definir PAOM laser para o modo de disparo externo-, iniciar a digitalização galvanômetro, e activar visualização em tempo real de PAOM imagem da secção transversal da retina animal. Cuidadosamente ajustar a orientação do transdutor até que a imagem PAOM tem a melhor relação sinal-ruído (SNR) e, ao mesmo tempo, mostra uma forma uniforme distributed padrões de amplitude PA em ambos os sentidos de digitalização. </li><li> Definir os parâmetros de digitalização, e realizar a gravação simultânea da retina da SD-OCT e PAOM. Reconstruir as imagens tridimensionais de SD-OCT e PAOM off-line. Nossos códigos de reconstrução foram escritos em Matlab e visualização tridimensional foi alcançado uing um freeware (Volview, Kitware). O algoritmo para SD-OCT reconstrução pode ser encontrada na Ref. 11. Eo algoritmo para PAOM reconstrução podem ser encontradas na referência. 6 e Ref. 13. Se necessário, repita os procedimentos de 3,7) -3,9). </li><li> Depois de experimentar, desligar a luz SD-OCT sondagem, remover o animal de titular imediatamente, e mantê-lo aquecido até que acorda naturalmente. Manter o animal em um ambiente escuro por uma hora extra para os olhos para se recuperar. A duração total experimental, incluindo a anestesia dos animais ea aquisição de imagem, é inferior a 30 min, para um operador experimentado. </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. 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. 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Todos os procedimentos com animais experimentais foram aprovados pelo Animal Care Institucional e Comitê de Uso da Northwestern University.

Aqui, apresentamos uma instrução detalhada em simultâneo em imagens da retina in vivo de olhos de ratos usando PAOM combinado com SD-outubro Optical-dispersão baseada SD-OCT é, talvez, o &quot;padrão de ouro&quot; para imagiologia clínica retiniana 3, porém, não é sensível para detectar a absorção óptica na retina. O PAOM recentemente desenvolvido é o único óptico-absorção baseada em modalidade de imagem oftálmico que fornece propriedades de absorção óptica da retina <...

integrated photoacoustic ophthalmoscopy and spectral-domain optical coherence tomography

Tanto o diagnóstico clínico e investigação fundamental de doenças oculares grandes beneficiar muito de diversas tecnologias não invasivas de imagem oftálmicas. Existentes modalidades de imagem da retina, como a retinografia colorida 1, oftalmoscopia confocal a laser (cSLO) 2, e tomografia de coerência óptica (OCT) 3, têm contribuições significativas no acompanhamento inícios de doenças e progressões, e desenvolvimento de novas estratégias terapêuticas. No entanto, eles predominantemente contar com os back-refletidas fótons da retina. Como consequência, as propriedades de absorção ópticas da retina, que são normalmente fortemente associados ao estado fisiopatologia da retina, são inacessíveis pelas tecnologias de imagem tradicionais. Oftalmoscopia fotoacústica (PAOM) é uma modalidade de imagem emergente da retina que permite a detecção dos contrastes de absorção óptica no olho com uma alta sensibilidade 4-7. Em PAOM nanpulsos de laser osecond são entregues através da pupila e digitalizados através do olho posterior para induzir fotoacústica (PA), os sinais que são detectadas por um transdutor de ultra-sons sem foco fixado à pálpebra. Por causa da forte absorção óptica de hemoglobina e a melanina, PAOM é capaz de forma não invasiva de imagem os vasculatures retina e coróide e o epitélio pigmentar da retina (RPE) de melanina em contrastes elevados 6,7. Mais importante ainda, com base no bem desenvolvida espectroscópicos imagiologia fotoacústica 5,8, PAOM tem o potencial para mapear a saturação do oxigénio da hemoglobina em vasos da retina, o que pode ser fundamental para o estudo da fisiologia e da patologia de várias doenças que causam cegueira 9, tais como a retinopatia diabética e degeneração macular neovascular relacionada à idade. Além disso, sendo a única existente modalidade de imagiologia óptica, a absorção baseada em oftálmico, PAOM pode ser integrado com o clínico bem estabelecido oftálmica imaging techniques para conseguir mais abrangentes estudos anatômicos e funcionais do olho baseada em múltiplos contrastes óticos 6,10. Neste trabalho, nós integramos PAOM e espectral de domínio outubro (SD-OCT) para simultaneamente em imagens da retina de ratos in vivo, onde tanto a absorção óptica e propriedades de dispersão da retina são revelados. A configuração do sistema de alinhamento do sistema, e de aquisição de imagem são apresentadas.

A Figura 2 mostra as imagens de 2-D de fundus SD-OCT e PAOM adquirida simultaneamente em um rato albino (A e B) e um rato pigmentado (C e D), respectivamente. Nas imagens SD-OCT fundus (Figuras 2A e 2C), vasos retinianos têm aparência escura devido à absorção de hemoglobina de sondagem luz. Além de vasos da retina (RV na Figura 2B), visualiza-se os PAOM vasculatures coróide (CV na Figura 2B) em olho albino por ca...

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

Oftalmologia fotoacústica (PAOM), uma modalidade de imagem óptico-absorção de base, fornece a avaliação complementar da retina para as tecnologias de imagem disponíveis atualmente oftálmicas. Relata-se o uso de PAOM integrado com espectral-domínio tomografia de coerência óptica (SD-OCT) para imagens multimodal simultânea da retina em ratos.

Oftalmoscopia Fotoacústica integrada e Spectral-domínio Tomografia de Coerência Óptica

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 visualizações.  Northwestern University. Oftalmologia fotoacústica (PAOM), uma modalidade de imagem óptico-absorção de base, fornece a avaliação complementar da retina para as tecnologias de imagem disponíveis atualmente oftálmicas. Relata-se o uso de PAOM integrado com espectral-domínio tomografia de coerência óptica (SD-OCT) para imagens multimodal simultânea da retina em ratos.

Vídeo: 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.

Tridimensional Optical resolução Microscopia Fotoacústica

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

Bioengenharia

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.

Detecção e isolamento de células de melanoma circulantes usando Fluxometria Fotoacústica

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.

Em síntese in vitro de Nativo, Espaçamento fibroso Long e Colágeno Espaçamento segmentar Longo

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 fotoacústica

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.

Preparação e Fotoacústica Análise de Veículos celulares contendo ouro nanobastões

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 Computadorizada e imageamento óptico de acoplamento Osteogenesis-angiogênese para avaliar a integração do osso craniano Autoenxertos e Aloenxertos

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.

Microscopia fotoacústica de resolução acústica e óptica comutável para<em&gt; In Vivo</em&gt; Imagem de Vasculatura de Sangue de Pequeno 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.

Um sistema de tomografia fotoacústica compacta de alto desempenho para<em&gt; In Vivo</em&gt; Imagem de cérebro de animais pequenos

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 volumétrico Retinal Imaging pelo oblíquo digitalização Laser Oftalmoscopia (oSLO) e a tomografia de coerência óptica (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.

Monitoramento não destrutiva de desenvolvimento degradáveis baseados em andaime engenharia de tecidos dos vasos sanguíneos, usando a tomografia de coerência óptica

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