DR reconoce el apoyo de la Fundación Alemana de Investigación (DFG) Beca de Investigación (RA 1848/1) y el Consejo Europeo de Investigación Starting Grant. VN reconoce el apoyo financiero del Premio al Investigador Avanzada Consejo Europeo de Investigación e Innovación de la BMBF en el Premio de Medicina.

1. Optoacústica Caracterización del detector de π-FBG <ol><li> Preparación de una esfera microscópica en suspensión en agar: <ol><li> Mezclar polvo de agar (1,3% en peso) con agua destilada en un vaso de precipitados de vidrio. Utilice un dispositivo magnético-agitador de placa caliente para calentar la solución cercana a la temperatura de ebullición y se disuelve el agar en polvo hasta que la solución se vuelva clara y libre de burbujas de aire. Alternativamente, la solución de agar puede ser calentado usando un horno de microondas convencional con agitación a cabo manualmente usando un agitador de vidrio. Vierta la solución caliente en un molde de plástico, por ejemplo, la jeringa con la punta cortada. </li><li> Rocíe una pequeña cantidad de esferas microscópicas en la solución de agar y esperar hasta que la solución se solidifica completamente. Tome el fantasma de agar sólido del molde empujando el émbolo. </li><li> Ver el fantasma bajo un microscopio estereoscópico cortar un pequeño trozo de agar que contiene una sola esfera microscópica. </li><li> Repita el paso 1.1.1 y añade to la solución de agar agar la pieza sólida que contiene la esfera microscópica sola. </li><li> Después de la solidificación, corte el fantasma de agar con el microscopio de tal manera que la esfera microscópica se encuentra cerca de la superficie del fantasma. </li></ol></li><li> Medición optoacústica <ol><li> Utilice dos soportes de fibra de ranura en V para mantener la fibra firmemente en ambos lados de la π-FBG, y conectado a su titular a una etapa de ordenador que funciona en tres dimensiones (XYZ), de traducción. Asegúrese de que la fibra se sumerge para permitir la propagación de los ultrasonidos. </li><li> Encontrar la localización aproximada del elemento de π-FBG de detección mediante la iluminación de diferentes partes de la fibra con la alta potencia del haz láser de nanosegundos-pulso. La absorción óptica del recubrimiento, sin embargo débil, creará una señal cuando la iluminación se realiza en el π-FBG. </li><li> Coloque la esfera microscópica agar embebido directamente debajo del π-FBG. La esfera microscópica debe ser visible a simple vista. </li><li> Uso de la etapa de traducción, realice una exploración 2D del π-FBG en el plano paralelo al suelo para encontrar la ubicación en la que la señal de la esfera microscópica es más fuerte y su tiempo de retardo correspondiente es más corto. </li><li> Realice últimos ajustes a la iluminación para proporcionar la máxima potencia a la esfera microscópica. </li><li> Uso de la etapa de traducción realizar una exploración 3D del π-FBG y registrar la señal para cada posición. </li><li> Para obtener la respuesta de frecuencia dependiente espacialmente del detector de ultrasonidos, realizar la transformada de Fourier de la señal de ultrasonido de dominio de tiempo registrada. </li></ol></li></ol> 2. Estimación de la robustez y la sensibilidad del rendimiento del detector de π-FBG <ol><li> Utilice dos soportes de fibra de ranura en V para mantener la fibra firmemente en ambos lados de la π-FBG y sumergir el π-FBG. </li><li> Coloque un plato oscuro o una varilla de grafito robusta para enfrentar el π-FBG y iluminarlo ingenioh la alta potencia nanosegundo pulsos de rayo láser para crear un campo acústico de fuerte. </li><li> Coloca una bomba de agua en el interior del depósito de agua y vuelva a encenderlo para crear variaciones rápidas en las condiciones ambientales. </li><li> Para estimar la robustez del sistema, medir la salida con el circuito de bloqueo girado tanto dentro como fuera. Cuando no se realiza ninguna de bloqueo, no es posible detectar con precisión la señal de ultrasonidos. </li><li> Encienda la bomba de suministro de agua. </li><li> Para estimar el beneficio de la sensibilidad debido a la alta coherencia de la fuente, sustituir el láser de impulsos de banda ancha con una fuente de baja coherencia y repetir la medición acústica. Se espera que una disminución de más de un orden de magnitud en la sensibilidad cuando se utiliza la fuente de baja coherencia. </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+transducers+for+pulse-echo+medical+imaging.">Ultrasound transducers for pulse-echo medical imaging.</a> IEEE Transactions on Biomedical Engineering. 30, 453-481 (1983).</li><li>Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+opto-acoustic+tomography+of+deep-seated+fluorescent+proteins+in+vivo.">Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo.</a> Nature Photon. 3, 412-417 (2009).</li><li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+deeper+than+microscopy:+the+optical+imaging+frontier+in+biology.">Going deeper than microscopy: the optical imaging frontier in biology.</a> Nature Methods. 7, 603-614 (2010).</li><li>Wang, L. 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Los autores declaran que no tienen intereses financieros en competencia.

En conclusión, se introduce un nuevo método óptico para la detección de ultrasonidos, que se basa en una combinación de una interferometría π-FBG y el pulso. La técnica es especialmente adecuado para aplicaciones de imagen optoacústica debido a la transparencia del elemento de detección, que permite patrones de iluminación objeto casi arbitrarios. En contraste, los detectores convencionales de ultrasonido piezoeléctricos basados ​​son opacos y por lo tanto bloquean algunos de los caminos ópticos al obje...

Detectores de ultrasonido juegan un papel clave en muchas aplicaciones de formación de imágenes. Convencionalmente, el ultrasonido se detecta por transductores piezoeléctricos, que transforman las ondas de presión en señales de tensión 1. En las imágenes optoacústica, el ultrasonido se genera a través de un proceso de expansión térmica al iluminar el objeto con alta potencia de luz modulada 2-6. Aunque transductores piezoeléctricos son el método de elección en aplicaciones optoacústica, su uso a menudo impide la miniaturización principalmente porque los transductores piezoeléctricos miniaturizados a menudo se caracterizan por una baja sensibilidad. Además, dado que los transductores piezoeléctricos son ópticamente opaca, pueden interferir gravemente con suministro de luz al objeto fotografiado, lo que limita las posibilidades de configuraciones de formación de imágenes utilizables. Luz que se retrodispersada desde el objeto hasta el transductor también puede limitar la detección apropiada de ultrasonido y complicar el diseño del sistema de formación de imágenes debido a parasit inducida ópticamenteIC señales en el transductor 7. Detectores ópticos de ultrasonido han sido reconocidos como una posible alternativa a los transductores piezoeléctricos que ofrece muchos beneficios en los escenarios de proyección de imagen optoacústica 8-12: A menudo son transparentes y pueden ser generalmente miniaturizados sin pérdida de sensibilidad. El principio de funcionamiento de los detectores ópticos es la detección interferométrica de la deformación minutos creado en el medio óptico debido a la presencia de ultrasonidos. A menudo, los resonadores ópticos se utilizan para mejorar la sensibilidad de detección por captura de luz en el medio perturbado para duraciones prolongadas, lo que aumenta el efecto de la deformación en la fase de la señal óptica. En esos casos, los sistemas de detección óptica se basan en variaciones de monitoreo en la longitud de onda de resonancia, que se refieren directamente a la estructura deformaciones en el resonador. Por lo general, se utilizan estrecho ancho de línea de onda continua (CW) técnicas en las que un láser CW está sintonizado a thlongitud de onda de resonancia de correo. Pequeños cambios en la resonancia de longitud de onda cambian la posición relativa de la longitud de onda del láser dentro de la resonancia, causando por lo tanto variaciones en la intensidad de la transmisión / luz láser reflejada, que puede ser controlado fácilmente. Sin embargo, si los cambios de resonancia son demasiado fuertes, por ejemplo, debido a las grandes variaciones en la presión, la temperatura o vibraciones, la resonancia puede cambiar completamente lejos de la longitud de onda del láser, saturando eficazmente el detector 13. Interferometría de impulsos 14 ofrece una solución para la limitación de la saturación de la señal y permite la detección de ultrasonidos bajo condiciones ambientales volátiles. En contraste a la estrechez linewidth esquemas de CW, la interferometría de impulsos utiliza una fuente de impulsos de banda ancha para iluminar el resonador. En este caso, el resonador actúa como un filtro de paso de banda, la transmisión de sólo aquellas longitudes de onda que corresponden a su frecuencia de resonancia, mientras que la resonancia se desplaza unre detectado mediante la medición de las variaciones de longitud de onda en la señal óptica en la salida del resonador, por ejemplo, utilizando un interferómetro de Mach-Zehnder bloqueado para cuadratura 14,15. Un circuito de rearme automático se utiliza para restaurar de inmediato el punto de trabajo del interferómetro en el caso de que se pierda debido a la variación extrema en las condiciones ambientales. Debido a la relativamente amplio ancho de banda de la fuente, la longitud de onda de resonancia se mantiene dentro de la banda de sistema de iluminación, incluso bajo fuertes perturbaciones, lo que permite el funcionamiento del detector estable incluso bajo condiciones ambientales duras. El uso de una fuente coherente para ser interrogado, pulsos ópticos es decir, facilita la detección de bajo ruido. El sistema de interferometría impulso correspondiente usado en nuestros experimentos se muestra en la Figura 1. El láser de pulso utilizado para la interrogación produjo 90 pulsos FSEC a una tasa de repetición de 100 MHz con una potencia de salida de 60 mW y la anchura espectral de más de 100nm. El filtro óptico tenía una anchura espectral FWHM de aproximadamente 0,4 nm y se sintoniza a la frecuencia de la resonancia. Tras el filtro, se utilizó un amplificador óptico para compensar la pérdida significativa en el filtrado. Filtrado adicional se aplicó después de la etapa de amplificación para reducir la emisión espontánea amplificada desde el amplificador. El resonador usado en nuestros experimentos es una fibra pi-cambiado de fase rejilla de Bragg (FBG-π) 8, fabricado por Teraxion Inc. En particular, para la aplicación médica de detección de ultrasonidos, π-FBG tienen la ventaja de ser todos los componentes de fibra, y por lo tanto robusto y pequeño. Figura 2 muestra una comparación entre las dimensiones de la fibra óptica utilizada en este trabajo y un 15 MHz de ultrasonido intravascular miniaturizado (IVUS) transductor piezoeléctrico. Algunos enfoques alternativos basados ​​en resonancia de detección, tales como resonadores de micro anillo fabricadas en guías de ondas planares, requieren fibras de acoplamiento en el componente dede entrada y de salida, ya sea que conduce a dispositivos más frágiles o dificultando la miniaturización. Por el contrario, π-FBGs son en fibra de componentes, y no requieren de acoplamiento de fibra adicional. La resonancia en π-FBG es creado por el desplazamiento de fase PI en su centro; la luz está atrapado alrededor del desplazamiento de fase PI sobre la porción de la fibra que es considerablemente más corta que la longitud de la propia rejilla. En nuestros experimentos, el π-FBG tenía una longitud de 4 mm y coeficiente de acoplamiento de κ = 2 mm -1 y su sensibilidad fue distribuida de manera no uniforme a lo largo de su longitud, con la sensibilidad exponencialmente decreciente desde el centro de la rejilla con una tasa de κ . El ancho completo-media-máxima (FWHM) de la distribución de sensibilidad (SD) fue de aproximadamente 350 micras. La anchura de resonancia de la rejilla se determina tanto por su longitud y su coeficiente de acoplamiento de acuerdo con la siguiente ecuación: <img alt="Ecuación 1" fo:content-width="1.5in" src= &quot;/ Files/ftp_upload/50847/50847eq1.jpg&quot; width = &quot;150&quot; /&gt; (1) donde λ es la longitud de onda de resonancia y n eff es el índice de refracción efectivo de la modo guiado en la fibra 8. Para evaluar si el detector de π-FBG es apropiada para aplicaciones de imagen, su respuesta espacialmente dependiente necesita ser medido sobre una banda de frecuencia amplia. Sin embargo, esta tarea es extremadamente difícil cuando se utilizan técnicas acústicas convencionales. Por lo tanto, empleamos un método para la caracterización optoacústica detector de ultrasonidos 16 en la que una esfera microscópica oscuro embebido en agar transparente sirve como una fuente de punto optoacústica. En nuestro experimento, la esfera microscópica tiene un diámetro de aproximadamente 100 micras y se ilumina con pulsos ópticos de nanosegundos alta potencia con una tasa de repetición de 10 Hz, duración de pulso de aproximadamente 8 nseg, y potencia media de 200 mW. La energía óptica depositado en el SPH microscópicaEres genera señales de ultrasonidos de banda ancha debido al efecto optoacústica. El detector de π-FBG se traduce relativamente a la esfera microscópica para obtener su respuesta acústica espacialmente dependiente. Figura 3 muestra una ilustración del experimento optoacústica. Generalmente, esta técnica se puede emplear para caracterizar diferentes tipos de detectores de ultrasonido.

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			<td height="43" style="height:43px;width:216px;">&#960;-FBG</td>
			<td style="width:71px;">Teraxion Inc.</td>
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			<td style="width:167px;">&#160;Custom made device</td>
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			<td height="43" style="height:43px;width:216px;">microscopic sphere</td>
			<td style="width:71px;">Cospheric LLC</td>
			<td style="width:119px;">BKPMS 90-106um- 10g</td>
			<td style="width:167px;">100 &#181;m polyethylene microsphere</td>
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			<td height="64" style="height:64px;width:216px;">Femto-second pulse laser used for interrogation&#160;</td>
			<td style="width:71px;">Menlo Systems GmbH</td>
			<td style="width:119px;">T-Light Femtosecond Laser</td>
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			<td height="64" style="height:64px;width:216px;">Optical filter</td>
			<td style="width:71px;">Optoplex Corporation</td>
			<td style="width:119px;">2-Port Optical Tunable Filter (50 GHz)</td>
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			<td height="64" style="height:64px;width:216px;">Optical amplifier</td>
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			<td style="width:119px;">AEDFA-PM-PA-35-B-FC</td>
			<td style="width:167px;">Benchtop 35dB Gain Pre Amp Polarization Maintaining EDFA&#160;</td>
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			<td height="232" style="height:232px;width:216px;">50/50 coupler</td>
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			<td style="width:119px;">FUSED-22-1550-8/125-50/50-3S3S3S3S-3-0.5-PM</td>
			<td style="width:167px;">Fused 2x2 fiber splitter with 0.5 meter long, 3mm OD PVC jacketed 1550nm 8/125&#956; PM fiber 
			pigtails, 50/50 split ratio in the slow axes of PM fibers and with super FC/PC connectors on all 
			ports.</td>
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			<td height="43" style="height:43px;width:216px;">Fiber holder</td>
			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">T711/M-250</td>
			<td style="width:167px;">Metric, Post-Mountable Fiber Clamp, 250 &#181;m&#160;</td>
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			<td height="43" style="height:43px;width:216px;">Agar for microbiology</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">05039-500G</td>
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			<td height="64" style="height:64px;width:216px;">Nano-second pulse laser used for generating the optoacoustic signals</td>
			<td style="width:71px;">Opotek</td>
			<td style="width:119px;">VIBRANT Arrow 532 type I</td>
			<td style="width:167px;"></td>
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			<td height="43" style="height:43px;width:216px;">Graphite rod</td>
			<td style="width:71px;">&#160;Faber-Castell</td>
			<td style="width:119px;">120700</td>
			<td style="width:167px;">Faber-Castell Pencil Leads - 0.7 mm</td>
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wideband optical detector of ultrasound for medical imaging applications

Los sensores ópticos de ultrasonido son una alternativa prometedora a las técnicas piezoeléctricas, como se ha demostrado recientemente en el campo de la imagen optoacústica. En aplicaciones médicas, una de las principales limitaciones de la tecnología de detección óptica es su susceptibilidad a las condiciones ambientales, por ejemplo, cambios en la presión y la temperatura, lo que puede saturar la detección. Además, el entorno clínico a menudo impone límites estrictos sobre el tamaño y la robustez del sensor. En este trabajo, la combinación de la interferometría de pulso y sensores ópticos basados ​​en fibra se ha demostrado para la detección de los ultrasonidos. Interferometría de pulsos permite un rendimiento robusto del sistema de lectura en la presencia de variaciones rápidas en las condiciones ambientales, mientras que el uso de la tecnología totalmente de fibra óptica conduce a un elemento de detección mecánicamente flexible, compatible con muy exigentes aplicaciones médicas tales como la imagen intravascular. Con el fin de lograr una longitud del sensor corto, unase utiliza la fibra desplazado-pi-fase de rejilla de Bragg, que actúa como un resonador de retención de la luz a través de una longitud efectiva de 350 micras. Para habilitar gran ancho de banda, el sensor se utiliza para la detección sideway de ultrasonido, que es altamente beneficioso en geometrías circulares de imagen como la imagen intravascular. Una configuración de imagen optoacústica se utiliza para determinar la respuesta del sensor para fuentes puntuales acústicas en diferentes posiciones.

Las figuras 4a y 4b muestran, respectivamente, las señales y su correspondiente espectros de la esfera microscópica a una distancia de 1 mm de la fibra para tres desplazamientos del centro de la π-FBG. Los desplazamientos se dan en la dirección z, como se muestra en la Figura 3. Claramente, la sensibilidad del detector óptico a los ultrasonidos de alta frecuencia (f&gt; 6 MHz) es anisotrópico y es mayor cuando el centro de la π-FBG es directamente encima de la es...

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Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Detección óptica de la ecografía no es práctico en muchos escenarios de proyección de imagen, ya que a menudo requiere de condiciones de estabilidad ambiental. Se demuestra una técnica óptica para la detección de los ultrasonidos en ambientes volátiles con niveles de miniaturización y sensibilidad apropiadas para la imagen optoacústica en escenarios restrictivos, por ejemplo, aplicaciones intravasculares.

Banda ancha Detector óptico de ultrasonido para aplicaciones de imágenes médicas

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

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JoVE Journal

Bioengineering

10.7K vistas.  Technical University of Munich and Helmholtz Center Munich.  Detección óptica de la ecografía no es práctico en muchos escenarios de proyección de imagen, ya que a menudo requiere de condiciones de estabilidad ambiental. Se demuestra una técnica óptica para la detección de los ultrasonidos en ambientes volátiles con niveles de miniaturización y sensibilidad apropiadas para la imagen optoacústica en escenarios restrictivos, por ejemplo, aplicaciones intravasculares. 

Video: Wideband Optical Detector of Ultrasound for Medical Imaging Applications

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

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Frecuencia de imagen óptica de dominio de<em&gt; Ex vivo</em&gt; Piezas de resección pulmonar: Obtención de One to One de imagen para estudio histopatológico de correlación

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Preparación de Mica Soportado bicapa lipídica de Alta Resolución Microscopía Óptica de Imagen

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofuncionalizadas prusiana nanopartículas azul para Multimodal Molecular Aplicaciones de imágenes

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoreo en tiempo real de Ultrasonido de alta intensidad (HIFU) Ablación de<em&gt; In Vitro</em&gt; Canine hígados Uso Movimiento armónico Imaging para Ultrasonido Focalizado (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

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 ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

De alto rendimiento de identificación de bacterias polímeros repelentes para dispositivos médicos

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal Therapy and Surgery) project. One of the aims of this project is to engineer novel, real-time image processing methods for intra-operative use in the treatment of congenital birth defects, such as spina bifida and the twin-to-twin transfusion syndrome. The developed target allows for the sterility-preserving optical distortion calibration of endoscopes within a few minutes. Good optical distortion calibration and compensation are important for mitigating undesirable effects like radial distortions, which not only hamper accurate imaging using existing endoscopic technology during fetal surgery, but also make acquired images less suitable for potentially very useful image computing applications, like real-time mosaicing. In this paper proposes a novel fabrication method to create an affordable, sterilizable calibration target suitable for use in a clinical setup. This method involves etching a calibration pattern by laser cutting a sandblasted stainless steel sheet. This target was validated using the camera calibration module provided by OpenCV, a state-of-the-art software library popular in the computer vision community.

This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

Objetivo esterilizable de calidad médica para Fluid-sumergido Fetoscopio óptico Distorsión de calibración

We have developed a calibration target for use with fluid-immersed endoscopes within the context of the GIFT-Surg (Guided Instrumentation for Fetal ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

Dispositivo de proyección de imagen de vivo a largo plazo para mejor manipulación Experimental de larvas de pez cebra

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Imágenes ópticas de miocitos ventriculares murinos aislados

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...