Damos las gracias a Marek Weiler para la realización de los experimentos fantasmas. Este trabajo fue apoyado por el Consejo Europeo de Investigación (ERC Starting Grant 309495: NeoNaNo), el Estado federal alemán de Renania del Norte Westfalia (NRW; High-Tech.NRW/EU-Ziel 2-Programm (EFRE); ForSaTum), el alemán Ministerio de Educación e Investigación (BMBF) (programas de financiación de hígado virtual (0.315.743), LungSys (0315415C), LungSys2 (0316042F), Photonik Forschung Deutschland (13N13355)), la Universidad RWTH Aachen (I 3 Fondo Semilla TM), e Investigación de Philips (Aquisgrán, Alemania).

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Felix Gremse es fundador y propietario de Gremse-IT, una compañía de lanzamiento que ofrece software y servicios para el análisis de imágenes médicas en colaboración con Philips y el Departamento de Imagen Molecular Experimental de la Universidad RWTH Aachen.

Describimos y aplicamos un protocolo para multimodal de imágenes μCT-FMT. Utilizamos FMT disponible en el mercado y ampliamente utilizado y dispositivos μCT 3,11,15 - 17,21. Mientras que el protocolo requiere una FMT específica, el μCT puede ser sustituido por otro μCT con una funcionalidad similar y parámetros de análisis comparables, por ejemplo, el campo de visión debe ser lo suficientemente grande como para cubrir la cama ratón incluyendo los marcadores. La FMT se ha utilizado para el análisis de biodistribución sin combinarla con μCT o MRI 21, sin embargo, los datos anatómica es beneficioso para aumentar la reproducibilidad porque la segmentación se puede basar en los límites de órganos que son visibles en los datos μCT 10. Mientras que los dispositivos μCT-FMT integrados se han desarrollado 2,7, éstos aún no están disponibles comercialmente. Además, el uso de dos dispositivos separados permite la tubería, es decir., El siguiente ca ratónn ser fotografiado en el μCT mientras que el primer ratón se encuentra todavía en la FMT, para aumentar el rendimiento. Para reducir la carga de trabajo manual, llevamos a cabo la detección de marcadores automatizado y la fusión. Además, la forma de ratón se segmenta automáticamente esta información y mejora significativamente la 11,13,22 reconstrucción de fluorescencia. Para la reconstrucción de fluorescencia cuantitativa, se necesitan mapas de absorción y dispersión 13,23. Obtenemos el mapa de dispersión por la segmentación automatizado de los datos μCT y asignar coeficientes de dispersión conocidos de varios tipos de tejidos (pulmón, hueso, piel, grasa y tejido blando restante) 24. Posteriormente, se reconstruye un mapa de absorción de los datos en bruto óptico que es particularmente importante para los órganos bien perfundidos, tales como el corazón y el hígado 13,20. Escaneado de varios ratones en múltiples puntos de tiempo se traduce rápidamente en un gran número de conjuntos de datos para ser analizados. Para Biodisestudios tribución, varios órganos deben ser segmentados para cada exploración μCT-FMT. Por desgracia, las segmentaciones no pueden ser reutilizados, ya que el ratón está recién colocado en el lecho del ratón repetidamente. Utilizamos una herramienta para la segmentación interactiva, desarrollada en nuestro instituto, sin embargo, otras herramientas pueden también ser apropiados 25. Generamos segmentaciones-vóxel sabio, ya que estos se ajustan mejor a los órganos complejos de formas simples tales como elipses y cubos 26. Segmentación todo animal automatizada sería útil para reducir aún más la carga de trabajo manual de 27, sino una herramienta de segmentación interactiva seguiría siendo necesario para corregir errores de segmentación. Además, las herramientas de segmentación automatizada difícilmente pueden anticipar casos especiales como patologías correctamente. Desde que usamos exploraciones μCT nativos, algunos órganos como el bazo son muy difíciles de segmento incluso manualmente. Los medios de contraste se ayudan, pero hay problemas con la tolerabilidad y es difícil de mantenina distribución de agente de contraste constante en toda la imagen longitudinal. Nuestro estudio fantasma muestra que la localización de la señal se mejora cuando se utiliza la información de la forma para la reconstrucción de fluorescencia. In vivo, una mejora similar es evidente para el punto de tiempo temprano (15 min después de la inyección), cuando una gran cantidad de la sonda ya está en el vejiga urinaria. La sonda de unión a hidroxiapatita se acumula en los huesos y las articulaciones. Es notable lo rápido que se produce este, es decir, la señal ya es claramente visible en la columna 15 min después de la inyección. Esto probablemente es causada por el bajo peso molecular de la sonda, lo que permite la extravasación rápido y difusión a las regiones de destino. La sonda se une covalentemente a su hidroxiapatita diana y la sonda no unido se excreta. Para los puntos de tiempo posteriores, entre 6 h y 24 h después de la inyección, la intensidad de la señal en la columna vertebral se mantiene relativamente estable, probablemente, porque casi no re luzdolores profundamente en el ratón para blanquear la fluorescencia. Para nuestro estudio, hemos utilizado el canal de 750 nm, lo que resulta en una baja fluorescencia de fondo como es evidente para los escáneres adquiridos antes de la inyección. En longitudes de onda más bajas, más la señal de fondo se puede esperar 28. En resumen, se describe un protocolo de imagen multimodal para dispositivos FMT y μCT disponibles en el mercado. Se demuestra que la combinación proporciona beneficios para la reconstrucción de fluorescencia. Se ilustra cómo las curvas de biodistribución se extraen de la gran cantidad de datos de imagen por medio de la segmentación del órgano interactivo y procesamiento por lotes. Creemos que este flujo de trabajo estandarizado puede ser útil para el desarrollo de medicamentos y otros estudios de imagen utilizando sondas marcadas con fluorescencia.

Tomografía de fluorescencia mediada, también llamada tomografía de fluorescencia molecular (FMT), es una técnica prometedora para evaluar cuantitativamente la distribución de fluorescencia en los tejidos difusos, tales como ratones anestesiados o incluso tejidos del cuerpo humano, por ejemplo, senos o articulaciones de los dedos. En contraste con las técnicas de microscopía no invasivos, que permiten imágenes de objetivos superficiales en resolución subcelular 1, FMT permite la reconstrucción tridimensional de fuentes fluorescentes en profundidades de varios centímetros, aunque a menor resolución 2. Muchas sondas fluorescentes específicas están disponibles para la angiogénesis imagen, apoptosis, inflamación, y otros 2 - 5. Algunas sondas son activable, por ejemplo., Mediante escisión específica de la enzima que conduce a unquenching de fluorocromos. Por otra parte, los genes indicadores que expresan proteínas fluorescentes se pueden obtener imágenes, por ejemplo, para rastrear la migración de células del tumor 6. FMT beneficia considerablemente de la combinación con una modalidad anatómica, por ejemplo, μCT 2,7 o RM 8. Mientras que los dispositivos FMT independientes están disponibles comercialmente 9, las imágenes de fluorescencia son difíciles de interpretar, sin información de referencia anatómica. Recientemente hemos sido capaces de demostrar, que los datos de imagen anatómica fundido permite un análisis más robusto 10. Los datos anatómicos también se pueden utilizar para proporcionar conocimiento previo, tal como la forma exterior del ratón, lo cual es importante para el modelado óptico de fluorescencia reconstrucción precisa y 11. Además, los mapas de dispersión y absorción ópticos pueden estimarse utilizando la segmentación de tipos de tejido y mediante la asignación de coeficientes específicos de clase 12,13. Para la luz del infrarrojo cercano, la hemoglobina es el absorbente principal en ratones, además de la melanina y la piel 14. Dado que el volumen de sangre relativa varía regionalmente por órdenes de magnitud, un mapa de absorción es particularmente importante para Quanfluorescencia reconstrucción cuanti- 13. Una ventaja de utilizar dispositivos de imagen no invasivas es que los ratones se pueden obtener imágenes longitudinalmente, es decir, en múltiples puntos de tiempo. Esto es importante para evaluar el comportamiento dinámico de sondas, es decir, su acumulación de destino, biodistribución y la excreción 10,15, o para evaluar la progresión de la enfermedad 16. Cuando la formación de imágenes de varios ratones en múltiples puntos de tiempo, una gran cantidad de conjuntos de datos de imagen surge. Para permitir la comparabilidad, estos deben ser adquiridos de manera sistemática, es decir, con un protocolo bien definido y documentado. El gran número de exploraciones plantea un desafío para el análisis de imágenes, que se requiere para extraer mediciones cuantitativas de los datos de imagen. El objetivo de nuestro estudio es proporcionar una descripción detallada de un protocolo de imágenes μCT-FMT que utilizamos y optimizado a lo largo de varios estudios 10,13,15,17,18. Describimoscómo se generan los conjuntos de datos, procesado, visualizado, y se analizaron. Esto se demuestra utilizando una sonda molecular establecido, OsteoSense, que se une a la hidroxiapatita 19, y se puede utilizar para enfermedades de los huesos y remodelación de imagen 2. Todos los procedimientos con animales fueron aprobados por el comité de evaluación del gobierno en el cuidado de animales.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>FMT (Fluorescence molecular tomography) FMT2500 LX</td>
			<td>PerkinElmer</td>
			<td>FMT2000</td>
			<td>Device for fluorescence molecular tomography</td>
		</tr>
		<tr>
			<td>&#181;CT (micro computed tomography) Tomoscope Duo</td>
			<td>CT Imaging GmbH</td>
			<td>Tomoscope Duo</td>
			<td>Device for micro computed tomography</td>
		</tr>
		<tr>
			<td>Multimodal Mouse Bed</td>
			<td>CT Imaging GmbH</td>
			<td>Experimental builder</td>
			<td>Partially transparent animal holder</td>
		</tr>
		<tr>
			<td>IsoFlo (isoflurane, USP)</td>
			<td>Abbott</td>
			<td>05260-05</td>
			<td>Isoflurane Inhalation anesthesia</td>
		</tr>
		<tr>
			<td>Small animal anesthesia system</td>
			<td>Harvard apparatus</td>
			<td>726419</td>
			<td>Complete Isoflurane Table-Top System</td>
		</tr>
		<tr>
			<td>Chlorophyll-free mouse food</td>
			<td>Ssniff</td>
			<td>E15051</td>
			<td>low chlorophyll / low fluorescence food</td>
		</tr>
		<tr>
			<td>OsteoSense 750EX</td>
			<td>PerkinElmer</td>
			<td>NEV10053EX</td>
			<td>Animal FMT contrast agent</td>
		</tr>
		<tr>
			<td>Portex Fine Bore Polythene Tubing</td>
			<td>Smith medical</td>
			<td>800/100/120</td>
			<td>Tube for injection catheter</td>
		</tr>
		<tr>
			<td>Sterican 30g</td>
			<td>BBraun</td>
			<td>4656300</td>
			<td>Hypodermic needle for catheter</td>
		</tr>
		<tr>
			<td>Imeron</td>
			<td>Altana pharma</td>
			<td>INLA F.1/0203/3.5337.69</td>
			<td>CT contrast agent for the phantom inclusions</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>90-12-36-6</td>
			<td>Agarose for phantom production</td>
		</tr>
		<tr>
			<td>TiO2</td>
			<td>Applichem</td>
			<td>A1900,1000</td>
			<td>Titanium oxyde as phantom scattering agent</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Fluka</td>
			<td>93595</td>
			<td>Trypan blue to adjust phantom light propagation</td>
		</tr>
		<tr>
			<td>Cy7</td>
			<td>Lumiprobe</td>
			<td>15020</td>
			<td>Fluorochrome for the phantom inclusions</td>
		</tr>
		<tr>
			<td>Lipovenoes 20%</td>
			<td>Fresenius Kabi</td>
			<td>3094740</td>
			<td>Lipid emulsion, scattering agent for FMT contrast agents</td>
		</tr>
		<tr>
			<td>Definiens Developer XD Server</td>
			<td>Definiens AG</td>
			<td>Server XD</td>
			<td>Software platform for automated segmentation</td>
		</tr>
		<tr>
			<td>Imalytics Preclinical</td>
			<td>ExMI/Gremse-IT</td>
			<td>Version 2.0.1</td>
			<td>Software for image fusion, reconstruction and analysis</td>
		</tr>
		<tr>
			<td>NVIDIA Geforce Titan</td>
			<td>Asus</td>
			<td>GTXTITAN6GD5</td>
			<td>High end computer graphics card, 6GB Memory</td>
		</tr>
	</tbody>
</table>

hybrid &#181;ct-fmt imaging and image analysis

La fluorescencia mediada por tomografía (FMT) permite la determinación longitudinal y cuantitativa de la distribución de fluorescencia in vivo y se puede utilizar para evaluar la biodistribución de nuevas sondas y para evaluar la progresión de la enfermedad utilizando sondas moleculares establecidas o genes informadores. La combinación con una modalidad anatómica, por ejemplo, micro tomografía computarizada (μCT), es beneficioso para el análisis de imagen y para la reconstrucción de fluorescencia. Se describe un protocolo para multimodal de imágenes μCT-FMT incluyendo los pasos de procesamiento de imágenes necesarias para extraer mediciones cuantitativas. Después de la preparación de los ratones y la realización de la formación de imágenes, los conjuntos de datos multimodales están registrados. Posteriormente, se realiza una mejor reconstrucción de fluorescencia, que tiene en cuenta la forma del ratón. Para el análisis cuantitativo, segmentaciones de órganos se genera en base a los datos anatómicos utilizando nuestra herramienta de segmentación interactiva. Finalmente, el cu biodistribuciónrves se generan utilizando una función de procesamiento por lotes. Mostramos la aplicabilidad del método mediante la evaluación de la biodistribución de una sonda bien conocido que se une a los huesos y las articulaciones.

Se aplicó el protocolo descrito para evaluar la biodistribución de una sonda específica, OsteoSense, que se une a la hidroxiapatita. 3 ratones (C57BL / 6 ApoE - / - AHSG - / - ratones doble knockout, 10 semanas de edad) se obtuvieron imágenes de antes y 15 min, 2 h, 4 h, 6 h, y 24 h después de la inyección iv de 2 nmol OsteoSense. Nuestro software detecta automáticamente los marcadores incorporados en la cama ratón multimodal (Figura 1, Figura 2A, B), lo que permitió la fusión de los datos μCT anatómicas con la reconstrucción de fluorescencia realizado por el FMT (Figura 2C, D). Desde OsteoSense es una sonda con un bajo peso molecular, un excreción renal rápido y por lo tanto, la señal de alto en la vejiga urinaria que se espera. La fusión de la reconstrucción de fluorescencia de la FMT reveló problemas como señal fuera de lugar fuera de la vejiga (Figura 2C, D). Estos problemas se producen porque el FMT no sabe la verdadera forma del ratón y asume una forma de bloque. Oureconstrucción r determina la forma precisa de los datos μCT y genera dispersión y los mapas de absorción 13 con el fin de permitir una reconstrucción más exacta de fluorescencia con una mejor localización de la señal, que es particularmente evidente en la vejiga (Figura 2E, F). Para asignar la fluorescencia reconstruida a regiones apropiadas, que de forma interactiva segmentado varios órganos que utilizan nuestro software (Figura 3). Para cada una de las 18 exploraciones, 7 regiones fueron segmentados basan en los datos, es decir μCT., Corazón, pulmón, hígado, riñones, la columna vertebral, el intestino y la vejiga. Posteriormente, el software se utilizó para calcular la concentración media de fluorescencia para cada una de las regiones 126. Afortunadamente, el software ofrece un modo discontinuo, que calcula todos los valores y las guarda en una sola hoja de cálculo. Para visualizar la distribución de fluorescencia, representaciones en 3D se generaron para cada punto de tiempo,usando entorno de ventanas comparables (Figura 4A-F). Utilizando los valores cuantificados de órganos, la biodistribución se calculó promediando los valores de órganos durante los tres ratones (Figura 4G). Las exploraciones previas, adquiridas antes de la inyección, mostraron señal de fondo insignificante. 15 min después de la inyección, la señal más fuerte apareció en la vejiga urinaria, debido a la excreción renal rápido. En los puntos de tiempo posteriores, la sonda restante había acumulado en los huesos y las articulaciones. <img alt="Figura 1" src="/files/ftp_upload/52770/52770fig1.jpg" /> Figura 1. Multimodal ratón cama. (A) La cama del ratón multimodal contiene dos placas de vidrio acrílico que sujetan firmemente el ratón. El endurecimiento se ajusta con dos tornillos. La cama ratón contiene marcadores (agujeros vacíos) para la fusión de imágenes. Gas anestésico se suministra mediante un tubo flexible que está obsesionado con tsimio. (B) La cama del ratón está unido a un soporte de metal y se mantiene en el centro del pórtico μCT rotativo. (C) Evitar una brecha entre la cama del ratón y el soporte de metal, porque de lo contrario, los marcadores pueden ser asignados de forma incorrecta que lleva a la fusión incorrecta. El tubo de gas anestésico debe ser conectado al conector de tubo. (D) La cama ratón debe ser insertado en el FMT con el frente primero para permitir una fusión correcta automatizado. (E) Los marcadores son visibles para la cámara FMT, que se utiliza para la detección de marcadores automatizado y la fusión. <a href="https://www.jove.com/files/ftp_upload/52770/52770fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52770/52770fig2.jpg" /> Figura 2. Imagen de Fusión y Reconstrucción. (A, B) Marcadores y la forma exterior del mouSE se determina por el algoritmo de segmentación automatizado. (C, D) 15 min después de la inyección de OsteoSense, una cantidad considerable de la sonda ya ha sido excretado en la vejiga urinaria. Después de la fusión de la reconstrucción proporcionada por el proveedor con los datos μCT, los problemas se hacen visibles. La mayor parte de la señal aparece alrededor de la vejiga, pero no dentro de la vejiga y algunos incluso señal aparece en el aire. Esto sucede porque el FMT asume un ratón en forma de bloque. (E, F) Nuestra mejorado la reconstrucción de fluorescencia, usando la forma del ratón derivado de los datos μCT, se traduce en una mejor localización de la fluorescencia dentro de la vejiga. <a href="https://www.jove.com/files/ftp_upload/52770/52770fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52770/52770fig3.jpg" /> Figura 3. interactivo Organ Segmentat. iónico (A) Para cuantificar la distribución de fluorescencia, varios órganos están segmentados: Corazón (rojo), pulmón (rosa), hígado (marrón), estómago (beige), la columna vertebral (morado), los riñones (amarillo), intestino (verde) y la vejiga urinaria (oro). (B) El pulmón, que se contrastó fuertemente en comparación con el tejido circundante, se segmenta usando umbralización y llenado región. (C) de la Ronda órganos, como la vejiga, los riñones y el corazón están segmentados utilizando &quot;garabatos&quot;. (D) Los órganos con una forma más compleja, por ejemplo, el hígado y el estómago están segmentados de forma incremental utilizando garabatos. Para el segmento de la columna vertebral, un umbral alto se aplica a todos los segmentos de huesos. Entonces algunos huesos, por ejemplo., Las costillas, se cortan, hasta que la columna vertebral se mantiene. <a href="https://www.jove.com/files/ftp_upload/52770/52770fig3large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> Figura 4. La biodistribución. Para evaluar la biodistribución, los ratones se escanean en varios puntos temporales (AF). (A) La detección previa, antes de la inyección, muestra poca señal de fondo en el canal 750 nm. (B) 15 min después de la inyección, una cantidad considerable de la sonda ya está en la vejiga urinaria. (C) En el punto de tiempo de 2 h, el ratón había orinado, lo que resulta en algunos fluorescencia fuera del ratón. En los puntos de tiempo posteriores (DF), la señal aparece predominantemente en los huesos y articulaciones, es decir., En la columna vertebral y las rodillas. (G) La concentración de fluorescencia cuantificada se muestra para los órganos seleccionados.

Watch this Scientific Journal Video about Hybrid ÂµCT-FMT imaging and image analysis at JoVE.com

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Híbrido de imágenes μCT-FMT y análisis de imágenes

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Research

JoVE Journal

Bioengineering

Hybrid &#181;CT-FMT imaging and image analysis

13.0K vistas.  RWTH Aachen University. We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (µCT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Video: Hybrid  &#181;CT-FMT imaging and image analysis

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Preparación de las muestras, imágenes y análisis de protocolos para la microscopía de barrido filo de la navaja

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

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 ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolución espacio-temporal Análisis de la Dinámica del receptor por Casa molécula de microscopía de fluorescencia

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Levitación magnética Junto con Imaging portátil y Análisis para el diagnóstico de enfermedades

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

Cuantificar distancias intermembrana con dilataciones de imagen Serial

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

Analizador de Subestructuras: Un flujo de trabajo amigable para el usuario para la exploración rápida y el análisis preciso de los cuerpos celulares en imágenes de microscopía de fluorescencia

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

Imágenes y cuantificación del área de microburbujas de movimiento rápido mediante una cámara de alta velocidad y análisis de imágenes

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

Citometría de flujo de alta dimensionalidad para el análisis de la función inmunitaria de tejidos de implantes disecados

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Area-based Image Analysis Algorithm for Quantification of Macrophage-fibroblast Cocultures

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either fluorescence detection approaches, such as immunofluorescent staining or transfection with fluorescent proteins or edge detection techniques, which are often error-prone due to noise and other non-idealities in the image background. 
We designed a new algorithm that could accurately count and distinguish macrophages and fibroblasts, cells of different phenotypes that often colocalize during tissue regeneration. MATLAB was used to implement the algorithm, which differentiated distinct cell types based on differences in height from the background. A primary algorithm was developed using an area-based method to account for variations in cell size/structure and high-density seeding conditions. 
Non-idealities in cell structures were accounted for with a secondary, iterative algorithm utilizing internal parameters such as cell coverage computed using experimental data for a given cell type. Finally, an analysis of coculture environments was carried out using an isolation algorithm in which various cell types were selectively excluded based on the evaluation of relative height differences within the image. This approach was found to accurately count cells within a 5% error margin for monocultured cells and within a 10% error margin for cocultured cells.

We present a method, which utilizes a generalizable area-based image analysis approach to identify cell counts. Analysis of different cell populations exploited the significant cell height and structure differences between distinct cell types within an adaptive algorithm.

Algoritmo de análisis de imágenes basado en áreas para la cuantificación de cocultivos de macrófagos y fibroblastos

Quantification of cells is necessary for a wide range of biological and biochemical studies. Conventional image analysis of cells typically employs either ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Sistema de análisis de células híbridas para evaluar cambios estructurales y contráctiles de cardiomiocitos humanos derivados de iPSC para la evaluación preclínica del riesgo cardíaco

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...