Los autores agradecen la lectura de cerca de Dr. Lynnea Brumbaugh del manuscrito. Este trabajo fue patrocinado por los Institutos Nacionales de Salud subvenciones R01 EB000712, R01 EB008085, R01 CA134539, CA136398 U54, y DK02057933 5P60. Prof. Lihong V. Wang tiene un interés financiero en Microphotoacoustics, Inc. y Endra, Inc., que, sin embargo, no apoyó este trabajo.

1. La configuración del sistema <ol><li> Radiación óptica <ol><li> Fuente de radiación óptica: un bombeado por diodos de estado sólido, láser pulsado (INNOSLAB, Edgewave) y un láser de colorante (CBR-D, Sirah). </li><li> El rayo láser de salida (ancho de pulso: 7 ns) se centra por una lente condensadora (LA1131, Thorlabs) para pasar a través de un orificio de 50 micras (P50C, Thorlabs). </li><li> El orificio se coloca un poco lejos del foco de la lente condensadora para que coincida con el diámetro del agujero con el diámetro del haz de modo fundamental para la eficacia de filtrado espacial. </li><li> El haz de filtrado se ve atenuado por un filtro de densidad neutra (NDC-50C-2M, Thorlabs) y luego, junto a una fibra óptica monomodo (P1-460A-FC-2, Thorlabs). </li><li> La salida de la fibra llena la abertura posterior de un objetivo de microscopio (RMS4X, Thorlabs) para lograr un enfoque óptico de difracción limitada de ~ 2.6 micras, con la longitud de onda de 570 nm. </li></ol></li><li> La detección ultrasónica <ol><li> Transductor de ultrasonidos: 50-MHz frecuencia central (V214-BB-RM, de Olympus NDT). </li><li> El transductor ultrasónico está conectado a un combinador de fabricación casera acústico-ópticos del haz de ultrasonidos para la detección de 11, que está alineado coaxialmente con la difracción limitada irradiación óptica. </li><li> Una cavidad esférica es tierra de la parte inferior del combinador para producir una lente acústica. Esta lente acústica tiene una apertura numérica de 0,5 en agua y da un diámetro acústica focal de 43 micras, con la frecuencia de 50 MHz central. </li><li> Los focos ópticos y acústicos están alineados confocally para maximizar la sensibilidad de detección. </li></ol></li><li> Acoplamiento acústico <ol><li> Acoplamiento ultrasónico seco se utiliza para evitar sumergirse en el agua los animales de experimentación, que fue utilizado a principios de los sistemas de imagen fotoacústica 12. </li><li> Una ventana de imagen se abre en la parte inferior de una placa de Petri (9 cm de diámetro) y está sellado con una membrana de polietileno de ultrasonidos y ópticamente transparente. </li><li> Gel de ultrasonidos (imagen clara y SonoTech) entre la membrana de polietileno y el objeto a ser fotografiado parejas de la onda generada fotoacústica del objeto a la placa de Petri, y el agua desionizada en la placa de Petri parejas más la onda a la sumergida O-PAM cabezal de exposición . </li></ol></li><li> Electrónica <ol><li> La señal fotoacústica detectado por el transductor ultrasónico es amplificada por dos amplificadores en cascada (ZFL 500LN, Mini-Circuits) </li><li> La señal amplificada es digitalizada por una adquisición de 14-bit de datos (DAQ) (CompuScope 14200, Gage Ciencias Aplicadas) a una velocidad de muestreo de 200 MS / s. </li></ol></li><li> Escaneo esquema <ol><li> De dos dimensiones (2-D) de trama de exploración de la cabeza o-PAM imágenes a lo largo de la horizontal (xy) avión es controlado por un ordenador personal, lo que provoca tanto la tarjeta DAQ y el láser de bombeo. La señal de disparo está sincronizado con la señal de reloj de salida de la tarjeta DAQ. </li><li> El eje rápido del escáner 2-D se define como la dirección de la exploración de la sección transversal (B-scan). </li><li> Una secuencia de imágenes B-scan puede ser adquirido mediante la traducción de la cabeza de imágenes a lo largo del eje lento para formar una imagen volumétrica, que puede ser visto ya sea en forma directa en 3-D de representación o en una proyección máxima de 2-D amplitud (PAM) de la imagen . </li></ol></li></ol> 2. Sistema de alineación <ol><li> El uso de pulso-eco de ultrasonido y un reflector de ultrasonidos para determinar la posición del plano focal acústico (es decir, el tiempo de retardo de la señal de disparo al máximo pulso-eco de la señal ultrasónica). Este paso es necesario para llevar a cabo una sola vez cuando se crea el sistema de O-PAM. </li><li> Maximizar la eficiencia de acoplamiento de la fibra de modo único. </li><li> Aplique el gel de ultrasonido en la parte superior de un objeto ópticamente absorbente (por ejemplo, un trozo de cinta negro) y con cuidado colocar debajo de la ventana de imagen en la placa de Petri con agua destilada. </li><li> Baje la cabeza de imágenes en el agua, y eliminar las burbujas de aire atrapadas bajo la lente acústica. </li><li> Ajuste el cabezal de exposición hasta que la señal fotoacústica del objeto absorbente es desde el plano focal acústico, que puede ser juzgado por la demora acústica. </li><li> Ajustar la posición vertical (es decir, la posición z) del objetivo de microscopio para maximizar la amplitud de la señal fotoacústica generada a partir del objeto plano. La amplitud de la señal al máximo sugiere que el enfoque óptico está alineado con el enfoque acústico en la dirección vertical. </li><li> Ajustar la posición horizontal (es decir, posiciones, X e Y) del objetivo del microscopio hasta que la señal fotoacústica generada a partir de la meta muestra un patrón simétrico. La simetría sugiere que el enfoque óptico está alineado con el enfoque acústico en la dirección horizontal. </li><li> Repita los pasos 2.6 y 2.7 hasta que la señal fotoacústica se ha optimizado tanto en la simetría y la amplitud. </li></ol> 3. Un SA<html> procedimiento en MPLE experimental in vivo O-PAM de oreja de ratón vasculatura <ol><li> Este paso no es necesario para ratones desnudos. Anestesiar al animal con una inyección intraperitoneal de un cóctel [receta: 1 ml de ketamina (100 mg / ml), 0,1 ml de xilazina (100 mg / ml), y 8,9 ml de solución salina, la dosis: 0,1 ml/10 g]. Afeitarse el cabello en el oído y, además, depilarse el vello residual con crema Surgi (Categoría #: 82565, American International Industries) antes de la limpieza con agua destilada. Tenga en cuenta que la depilación tan poco podría irritar la piel y el sistema vascular por lo tanto es mejor realizar 24 horas antes del experimento planeado. </li><li> Encienda el sistema láser fotoacústica, y compruebe la alineación del sistema. </li><li> Anestesiar el ratón con el 3% isoflurano vaporizado por la inhalación de gas (la tasa de flujo típica es 1,0 a 1,5 l / min, dependiendo del peso corporal del animal), y mantener la anestesia con isoflurano al 1% durante todo el experimento. Grado médico de aire se recomienda como la inhalación de gas para mantener el ratón en el estado fisiológico normal. </li><li> Transferir el ratón a una etapa estereotáctica, y controlar su temperatura corporal a 37 º C con una almohadilla térmica. </li><li> Aplanar la oreja de ratón en un plato de plástico y aplicar una capa de gel de ultrasonido en la parte superior de la oreja. Evitar que queden atrapadas burbujas de aire en el interior del gel. Luego, coloque el oído en la ventana de imagen y levante lentamente la etapa de los animales hasta que los contactos gel para ultrasonidos de la parte inferior de la membrana de polietileno. De contacto blandas es necesario porque presionar la oreja contra la membrana puede afectar el flujo de sangre en el oído. </li><li> Abrazadera de un oxímetro de pulso en la pierna o la cola del ratón para controlar su estado fisiológico, y aplique una pomada para los ojos para evitar la sequedad y los daños accidentales de láser en los ojos del ratón. </li><li> Baje el cabezal de exposición hasta que la lente acústica se sumerge en agua desionizada, y eliminar las burbujas de aire atrapadas bajo la lente acústica. </li><li> Compruebe la fluencia del láser para asegurarse de que está dentro de los estándares de seguridad del láser de la American National Standards Institute 13. La fluencia del láser no debe superar los 20 mJ / cm 2, que se traduce en 80 nJ de la energía láser de pulso cuando el rayo láser se enfoca a 150 m por debajo de la superficie de la piel. </li><li> Ajuste el láser para el modo de disparo externo y comenzar a escanear el juicio. Ajustar la posición z de la cabeza la imagen hasta la señal más fuerte fotoacústica es desde el plano focal acústico. </li><li> Establecer los parámetros correctos de escaneo e iniciar la adquisición de la imagen formal. </li><li> Después del experimento, apague el láser, levantar la cabeza imágenes de la agua desionizada, baje la etapa animal a soltar el ratón, limpiar la oreja de ratón con agua destilada, a su vez del sistema de anestesia y el control de temperatura, y descargar el ratón desde la etapa de estereotáctica. </li><li> Si las imágenes repetitivas es necesario, poner el ratón en una incubadora con la temperatura ambiente a 37 ° C. Devolver el ratón para la instalación de animales después de que se despierta de forma natural. De lo contrario, siga los protocolos para la eutanasia de los animales y disponer de ella. </li></ol> 4. Funcionales o-PAM de total concentración y saturación de oxígeno de la hemoglobina <ol><li> Oxihemoglobina (HbO 2) y desoxihemoglobina (HBR) son las dos formas principales de la hemoglobina, la fuente endógena fotoacústica predominante en el rango espectral visible. HbO 2 y HBr tienen un espectro de absorción óptica distinta y por lo tanto puede ser espectralmente diferenciados para cuantificar tanto la concentración total (HBT) y la saturación de oxígeno (SO 2) de la hemoglobina 5. Aquí hay dos guías para ayudar a seleccionar longitudes de onda ópticas adecuadas para el SO 2 medidas: </li><li> Longitudes de onda se debe seleccionar en el Q-banda del espectro de absorción de la hemoglobina (es decir, 550-600 nm) para garantizar una suficiente relación señal-ruido y una penetración adecuada. </li><li> Longitudes de onda donde los coeficientes de absorción de HbO 2 y HBr tiene una marcada diferencia (por ejemplo, HBR-561 nm dominante y HBO 2-dominante, 578 nm) se recomiendan. </li></ol> Además de SO 2, HBT se puede calcular mediante la adición de [HBR] y [HbO 2] juntos, o que se puede medir directamente en longitudes de onda isosbestic de los espectros del coeficiente de extinción molar de la hemoglobina (por ejemplo, 530 nm, 545 nm, 570 nm, y 584 nm) 14, en Harvard Business Review y HBO 2 tienen los mismos coeficientes de extinción molar. 5. Los resultados representativos: Muestra en la Figura 1 es la anatomía vascular y SO 2 en una oreja de ratón vivo desnudo fotografiado por doble longitud de onda (561 y 570 nm) o PAM. La imagen típica de tiempo de adquisición de doble longitud de onda por lo que 2 mediciones de una región de interés (tamaño de la imagen: 10 mm x 10 mm, tamaño de paso: 2,5 m x 5 m) es de ~ 80 min. p_upload/2729/2729fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. En vivo óptico resolución de la microscopía fotoacústica. Imágenes de mapas de (A) la concentración de hemoglobina total que muestra la anatomía vascular (adquirida a 570 nm) y (B) la saturación de oxígeno de la hemoglobina (adquirida en 561 nm y 570 nm) en una oreja de ratón desnudo. (C) Detalle de la región en caja en el panel (A). La barra de escala en el panel (A) se aplica a ambos (A) y (B).

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Todos los procedimientos experimentales con animales se llevaron a cabo de conformidad con el protocolo de animales de laboratorio aprobado por la Escuela de Medicina de la Comisión de Estudios de Animales de la Universidad de Washington en St. Louis.

En este vídeo, que proporciona una instrucción detallada sobre los protocolos experimentales de O-PAM, incluyendo la configuración del sistema, la alineación del sistema y los procedimientos típicos experimental. Sin etiquetas, no invasivo O-PAM ha permitido a los estudios de funcionamiento microvascular y el metabolismo de forma capilar única y por lo tanto tiene el potencial para ampliar nuestra comprensión de la microcirculación relacionados con la fisiología y la patología. Microphotoacoustics es actualmen...

Hecho en casa acústico-ópticos combinador de haz: <ul><li> en ángulo recto del prisma (NT32-545, Edmund Optics) </li><li> romboidal prisma (NT49-419, Edmund Optics) </li><li> aceite de silicona (1000cSt, Productos Clearco) </li><li> O-PAM del sistema (Microphotoacoustics) </li></ul>

three-dimensional optical-resolution photoacoustic microscopy

Microscopía óptica, proporcionando valiosa información a nivel celular y orgánulos, ha sido ampliamente reconocida como una tecnología biomédica. Como los pilares de la in vivo de tres dimensiones (3-D) microscopía óptica, microscopía de fluorescencia single-/multi-photon y tomografía de coherencia óptica (OCT) han demostrado su sensibilidad a los contrastes extraordinaria dispersión de la fluorescencia y la óptica, respectivamente. Sin embargo, el contraste de absorción óptica de tejidos biológicos, que codifica la información esencial fisiológico / patológico, aún no ha sido evaluables. La aparición de photoacoustics biomédica ha conducido a una nueva rama de la microscopía óptica óptico resolución microscopía fotoacústica (O-PAM) 1, donde se concentra la radiación óptica hasta el límite de difracción para lograr un celular o subcelular resolución de 2 nivel lateral. Como un valioso complemento de las actuales tecnologías de microscopía óptica, O-PAM lleva en al menos dos novedades. Lo primero y más importante aún, O-PAM detecta contrastes ópticos de absorción con una extraordinaria sensibilidad (es decir, 100%). La combinación de O-PAM con microscopía de fluorescencia 3 o con óptica de dispersión basada en 04 de octubre (o ambos), provee información integral propiedades ópticas de los tejidos biológicos. En segundo lugar, O-PAM codifica la absorción óptica en ondas acústicas, a diferencia de los procesos de pura óptica en microscopía de fluorescencia y octubre, y justifica sin detección. La detección acústica de O-PAM mitiga los efectos de la dispersión óptica en la degradación de la señal y elimina de manera natural las posibles interferencias (por ejemplo, crosstalks) entre la excitación y la detección, que es un problema común en la microscopía de fluorescencia debido a la coincidencia entre la excitación y el espectro de fluorescencia. Única para obtener imágenes de absorción óptica, O-PAM ha demostrado amplia gama de aplicaciones biomédicas, desde su invención, incluyendo, pero no limitado a, neurología 5, 6, oftalmología 7, 8, 9 biología vascular, dermatología y 10. En este video, les enseñamos a la configuración del sistema y la alineación de la O-PAM, así como los procedimientos experimentales para imágenes in vivo microvascular funcional.

Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

Three-dimensional Optical-resolution Photoacoustic Microscopy

Resolución óptica de microscopía fotoacústica (O-PAM) es una tecnología emergente capaz de absorción de imágenes ópticas contrastes<em&gt; In vivo</em&gt; Con resolución celular y la sensibilidad. Aquí, le ofrecemos una instrucción visualiza en los protocolos experimentales de O-PAM, incluyendo la alineación del sistema de configuración del sistema, típico<em&gt; In vivo</em&gt; Procedimientos experimentales y los sistemas de neuroimagen funcional.

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 ...

three-dimensional-optical-resolution-photoacoustic-microscopy

Research

JoVE Journal

Bioengineering

17.7K vistas.  Washington University in St. Louis. Resolución óptica de microscopía fotoacústica (O-PAM) es una tecnología emergente capaz de absorción de imágenes ópticas contrastes In vivo Con resolución celular y la sensibilidad. Aquí, le ofrecemos una instrucción visualiza en los protocolos experimentales de O-PAM, incluyendo la alineación del sistema de configuración del sistema, típico In vivo Procedimientos experimentales y los sistemas de neuroimagen funcional.

Video: Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Los hidrogeles de alginato para la cultura del órgano en tres dimensiones de los ovarios y oviductos

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Investigación

Bioingeniería

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impresión plana y tridimensional de tintas conductoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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 ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigación De La Separación De Flujo Tridimensional Inducida Por Un Pólipo Modelo De Pliegue Vocal

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

El cartílago humano Tissue Fabrication Usando tridimensional Tecnología de impresión inyección de tinta

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análisis de la migración de células dentro de una matriz de colágeno tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingeniería tridimensionales epiteliales tejidos embebidos dentro de la matriz extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

La fabricación de dispositivos de microfluidos basados ​​en papel tridimensional para inmunoensayos

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.