Damos las gracias al generoso apoyo de la Fundación Nacional de Ciencia (CARRERA CBET-1055379) y los Institutos Nacionales de Salud (1RC4EY021357, 1R01EY019951). También reconocemos el apoyo del Consejo de Becas de China a Song Wei.

1. Configuración del sistema <ol><li> PAOM Subsistema <ol><li> Fuente de iluminación: un láser Nd: YAG (SPOT-10-100, Elforlight Ltd, UK: 20 μJ / impulso; 2 nseg duración del pulso; 30 kHz máxima tasa de repetición de pulso). </li><li> El láser de salida a 1064 nm de doble frecuencia a 532 nm con un beta-bario-borato (BBO) crystal (Castech, San Jose, CA). Después de escisión adicional por un espejo línea de láser, 532 nm de luz se entrega a través de una fibra óptica de modo único (P1-460A-FC-5, Thorlabs), y 1064 nm láser se registra mediante un fotodiodo (DET10A, Thorlabs), que desencadena PA adquisición de la señal. </li><li> La luz de láser que sale de la fibra óptica de modo único se entrega en la retina por un galvanómetro (GM, QS-7, Nutfield Tecnología) y una configuración de telescopio (f1 = 75 mm y f2 = 14 mm, Edmund Optics) 6. </li><li> Un transductor aguja fuera de foco (40-MHz frecuencia central, ancho de banda de 16 MHz, 0,4 x 0,4 mm 2 tamaño del elemento activo, NIH Recursos Center para las tecnologías de transductor ultrasónico, la University of Southern California) se coloca en contacto con el párpado para detectar las señales PA generadas a partir de la retina. Gel de ultrasonidos (Sonotech) se aplica entre la punta del transductor y el párpado animal para acoplamiento acústico. </li><li> La señal PA es amplificada por dos amplificadores (ZFL-500LN +, mini-circuitos, y 5073PR, Olympus), y se digitaliza mediante una tarjeta de adquisición de datos (CS14200, Gage Aplicada). </li></ol></li><li> SD-OCT Subsistema <ol><li> Fuente de luz baja coherencia: una banda ancha super-luminiscente diodo (IPSDD0804, InPhenix, centro de longitud de onda: 840 nm; 6 dB Ancho de banda: 50 nm), que determina la resolución axial de 6 micras. </li><li> La luz infrarroja cercana se divide para hacer referencia brazo y el brazo de la muestra por un 50 × 50 personalizado de modo único acoplador de fibra (Óptica OZ). </li><li> Después de combinar con PAOM luz que ilumina por un espejo caliente (FM02, Thorlabs), el brazo de octubre muestra comparte el mismo análisis y entrega wi ópticaª PAOM 6. </li><li> Un espectrómetro de fabricación casera se utiliza para registrar las señales de interferencia SD-octubre, cuando una línea de exploración CCD cámara (Aviiva SM2, e2v) permite una tasa de una línea de 24 kHz. Diseño de espectrómetros típicos se pueden encontrar de varias literaturas previamente publicados 11 y fibra acoplados a espectrómetros SD-OCT se encuentra ahora disponible en el mercado. La sensibilidad SD-OCT se mide a ser mejor que 90 dB. </li></ol></li><li> Esquema de exploración <ol><li> Fast 2-D exploración de trama del galvanómetro está controlado por una tarjeta de salida analógica (PCI-6731, National Instruments), que también activa tanto el PAOM disparos láser y la adquisición de la señal de octubre espectrómetro. Como resultado de ello, las adquisiciones de datos en PAOM y subsistemas de octubre están sincronizados. </li><li> La adquisición PAOM se dispara mediante una grabación fotodiodo secuencia PAOM láser (véase 1.1.2). </li><li> 3-D imágenes volumétricas o 2-D imágenes del fondo de ojo se construyen a partir de 256 B-scan imágenes (256 líneas A por BA escanear la imagen). </li></ol></li></ol> 2. Sistema de alineación <ol><li> Maximizar la eficiencia de frecuencia de duplicación del cristal BBO y la eficiencia de acoplamiento de la fibra óptica de modo único. Use gafas de LG3 (Thorlabs) para la protección personal cuando se optimiza la luz PAOM esclarecedor. </li><li> Colimar la salida del láser de fibra de PAOM a 2,0 mm de diámetro. </li><li> Alinear las luces de iluminación combinados de PAOM y SD OCT-ser coaxial. </li><li> Ajuste la luz de excitación PAOM a ~ 40 nJ / pulso y SD OCT-sondeo luz a ~ 0,8 mW, ambos de los cuales son reportados a ser seguro para los ojos 6,12. </li></ol> 3. In vivo de imágenes de retina Multimodal <ol><li> Transferir la rata a una caja de polipropileno transparente, y anestesiar al animal por una mezcla de aire isoflurano (Phoenix Pharmaceutical, Inc.) y normal a una concentración de 1,5% y una velocidad de flujo de 2,0 litros / min durante 10 min. </li><li> Reprime la rata anestesiada en una homemade soporte con cinco ejes libertad ajustable (Figura 1), y mantener su temperatura corporal a ~ 37 ° C mediante una almohadilla de calefacción (Repti Therm, laboratorios Zoomed, Inc.). Mantener la anestesia por inhalación de gas de aire de mezcla de isoflurano y normal con 1,0% de concentración y 1,5 litros / min de flujo de todo el experimento. </li><li> Cortar la pestaña usando unas tijeras quirúrgicas, dilatar las pupilas con tropicamida 1% solución oftálmica, y paralizar el músculo del esfínter del iris usando 0,5% solución oftálmica de clorhidrato de tetracaína. Aplicar gotas de lágrima artificial (Systane, Alcon Laboratories, Inc.) para el ojo de la rata cada dos minutos para evitar la deshidratación córnea y la formación de cataratas. Controlar la frecuencia cardíaca animal, la respiración y la oxigenación de la sangre por un oxímetro de pulso (8.600 V, Nonin Medical, MN) durante la exploración. </li><li> Encienda el SD-OCT luz que ilumina y compruebe la luz de sondeo para ser ~ 0,8 mW. </li><li> Activar el escaneo galvanómetro. Alinee la entrega irradiación SD-OCT luzen la retina de rata y de identificar la región de la retina de interés (ROI) mediante el ajuste del soporte de los animales de cinco ejes. Aquí, el disco óptico es colocado intencionadamente en el centro del campo de visión, mientras que el retorno de la inversión debe ser seleccionado basándose en los requisitos de investigación diferentes. </li><li> Ajustar aún más el soporte de animal para optimizar las cualidades SD-octubre de formación de imágenes de sección transversal de la retina en ambas direcciones de exploración (por conmutación de la dirección de exploración de trama) hasta que el mejor enfoque óptico se alcanza. </li><li> Preparar el transductor aguja en una plataforma ajustable de cinco ejes, se aplica una gota de gel de ultrasonido para la punta del transductor, y suavemente en contacto con la punta del transductor en el párpado animal. </li><li> Establecer láser PAOM al modo de disparo externa, inicie el escaneo galvanómetro, y activar visualización en tiempo real de PAOM imagen transversal de la retina animal. Ajustar cuidadosamente la orientación del transductor hasta que la imagen PAOM tiene la mejor relación señal-ruido (SNR) y, mientras tanto, muestra un uniformemente distributed patrones PA amplitud en los dos sentidos de exploración. </li><li> Establezca los parámetros de escaneado, y llevar a cabo la retina de imágenes simultáneas SD-OCT y PAOM. Reconstruir las imágenes tridimensionales de SD-OCT PAOM y off-line. Nuestros códigos de reconstrucción fueron escritos en Matlab y visualización tridimensional se logró uing un freeware (Volview, Kitware). El algoritmo para SD-octubre reconstrucción se puede encontrar en la referencia. 11 y el algoritmo de reconstrucción para PAOM se puede encontrar en la referencia. 6 y ref. 13. Si es necesario, repita los procedimientos de 3,7) -3,9). </li><li> Después de experimentar, apagar la luz SD-OCT sondaje, retirar al animal de soporte inmediato, y mantenerlo caliente hasta que se despierta de forma natural. Mantenga al animal en un ambiente oscuro durante una hora adicional para los ojos para recuperarse. La duración completa experimental, incluyendo la anestesia animal y la obtención de imágenes, es menos de 30 min para un operador experimentado. </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. 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Todos los procedimientos experimentales con animales fueron aprobados por el Cuidado de Animales institucional y el empleo Comisión de la Universidad Northwestern.

A continuación, presentamos una instrucción detallada sobre simultáneo de imágenes in vivo la retina de los ojos de rata utilizando PAOM combinado con SD-PTU. Óptica de dispersión basada en SD-OCT es, quizás, la clínica &quot;patrón oro&quot; para imágenes de la retina 3, sin embargo, no es sensible para detectar la absorción óptica en la retina. El PAOM nuevo desarrollo es la única óptica de absorción a base técnica de imagen oftálmica que proporciona las propiedades óptic...

integrated photoacoustic ophthalmoscopy and spectral-domain optical coherence tomography

Tanto el diagnóstico clínico y la investigación fundamental de las enfermedades oculares más importantes se benefician enormemente de diversas tecnologías no invasivas de imagen oftálmicos. Existentes modalidades de imágenes retinianas, como la fotografía del fondo 1, oftalmoscopio láser confocal de barrido (CSLO) 2, y la tomografía de coherencia óptica (OCT) 3, tienen contribuciones importantes en el seguimiento de la enfermedad y las progresiones inicios y el desarrollo de nuevas estrategias terapéuticas. Sin embargo, se basan predominantemente en el back-reflejadas fotones de la retina. Como consecuencia, las propiedades de absorción óptica de la retina, que normalmente se asocian fuertemente con el estado de la fisiopatología de retina, son inaccesibles por las tecnologías de imágenes tradicionales. Oftalmoscopia fotoacústica (PAOM) es una modalidad emergente imagen de la retina que permite la detección de los contrastes de absorción óptica en el ojo con una alta sensibilidad 4-7. En PAOM nanpulsos osecond láser se entrega a través de la pupila y explorado a través de la parte posterior del ojo para inducir fotoacústicos (PA), señales que son detectadas por un transductor ultrasónico desenfocada unido al párpado. Debido a la fuerte absorción óptica de la hemoglobina y la melanina, PAOM es capaz de forma no invasiva de imágenes de las vasculaturas la retina y la coroides y el epitelio pigmentario de la retina (RPE) de melanina en contrastes altos 6,7. Más importante, basado en el bien desarrollado espectroscópico fotoacústica imágenes 5,8, PAOM tiene el potencial para mapear la saturación de oxígeno de la hemoglobina en los vasos retinianos, que puede ser crítica en el estudio de la fisiología y patología de varias enfermedades que causan ceguera 9 como la retinopatía diabética y neovascular relacionada con la edad degeneración macular. Además, al ser la única existente óptico de absorción basada en modalidad de imagen oftálmica, PAOM se puede integrar con bien establecida clínica oftálmica imágenes TEchniques posible obtener evaluaciones anatómicas y funcionales más amplias del ojo sobre la base de múltiples contrastes ópticos 6,10. En este trabajo, integramos PAOM y espectral dominio-OCT (SD-OCT) para imágenes in vivo de manera simultánea en la retina de rata, donde se revelan tanto la absorción óptica y propiedades de dispersión de la retina. La configuración del sistema, la alineación del sistema y adquisición de imágenes se presentan.

La Figura 2 muestra las imágenes 2-D del fondo de ojo de SD-OCT y PAOM adquiridas simultáneamente en una rata albina (A y B) y una rata pigmentada (C y D), respectivamente. En las imágenes SD-octubre del fondo de ojo (Figuras 2A y 2C), vasos de la retina tienen una apariencia oscura debido a la absorción de la hemoglobina de sondear la luz. Además de vasos de la retina (RV en la Figura 2B), PAOM visualiza las vasculaturas coroideas...

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

Fotoacústica oftalmología (PAOM), una modalidad de formación de imágenes ópticas de absorción basado, proporciona la evaluación complementaria de la retina a las tecnologías de formación de imágenes disponibles en la actualidad oftálmicas. Se describe el uso de PAOM integrado con dominio espectral tomografía de coherencia óptica (SD-OCT) para el uso simultáneo de imágenes multimodal de la retina en ratas.

La oftalmoscopia integrado fotoacústica y Spectral-tomografía de coherencia óptica de dominio

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 vistas.  Northwestern University. Fotoacústica oftalmología (PAOM), una modalidad de formación de imágenes ópticas de absorción basado, proporciona la evaluación complementaria de la retina a las tecnologías de formación de imágenes disponibles en la actualidad oftálmicas. Se describe el uso de PAOM integrado con dominio espectral tomografía de coherencia óptica (SD-OCT) para el uso simultáneo de imágenes multimodal de la retina en ratas.

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.

Tridimensional óptico resolución microscopía fotoacústica

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

Investigación

Bioingeniería

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.

La detección y aislamiento de las células de melanoma circulantes mediante Flujometría 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.

La síntesis in vitro de Espaciado nativo, y colágeno fibroso Long Long segmentaria Espaciado

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.

Cistografía 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.

Análisis preparación y fotoacústica de Vehículos celulares que contienen 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.

Tomografía Computarizada e Imagen Óptico de Acoplamiento-Osteogénesis angiogénesis para evaluar la integración de craneales hueso autoinjertos y aloinjertos

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.

Microscopía fotoacústica de resolución óptica y acústica conmutable para<em&gt; In Vivo</em&gt; Imágenes de vasos sanguíneos de pequeños animales

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 de tomografía fotoacústica compacta de alto rendimiento para<em&gt; In Vivo</em&gt; Imágenes de cerebro para animales pequeños

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 volumétrica retina imágenes por exploración oblicua oftalmoscopia de láser (oSLO) y tomografía de coherencia 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.

Monitoreo no destructivos del desarrollo Degradable andamio base ingeniería de tejido vascular mediante tomografía de coherencia óptica

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