Este proyecto fue financiado por el NIH / NINDS (# 1R01-NS54252), NSF (# 0,905,041, # 0,079,874), el Programa de Tecnología Avanzada de Texas (# ATP-000,512-0.146-2001, # ATP-000512-0261-2001), Texas A &amp; M Research Foundation, el Departamento de Ciencias de la Computación e Ingeniería de la Universidad Texas A &amp; M, y 3Scan. Nos gustaría agradecer a Bernard Mesa (Tecnologías de Micro Star) para la consulta y apoyo técnico para la instrumentación KESM. Las partes principales del diseño e implementación de la KESM fue realizado por Bruce H. McCormick, quien murió en 2007.

1. Preparación de la muestra: Golgi-Cox <ol><li> Este protocolo sigue de cerca el protocolo de Mayerich et al. 7. </li><li> El ratón C57BL/6J es profundamente anestesiados con anestesia inhalatoria con isoflurano y luego decapitado. </li><li> El cerebro es removido y colocado en una solución de fijación de Golgi-Cox (1% de cromato de potasio, dicromato de potasio al 1% y 1% de cloruro de mercurio en agua desionizada). </li><li> El cerebro es la izquierda en la solución de Golgi-Cox en la oscuridad, a temperatura ambiente, durante 10 a 16 semanas. </li><li> Los cerebros se enjuagan en agua desionizada durante la noche en la oscuridad. </li><li> El cerebro enjuagado se sumerge en una solución de amonio al 5% de hidróxido en agua desionizada durante 7 a 10 días en la oscuridad a temperatura ambiente. Esta vez fue una larga preparación para asegurar la infiltración de todo el cerebro, de manera que el precipitado negro tuvieron la oportunidad de forma completamente en el tejido. </li><li> El cerebro se aclara de nuevo con agua desionizada a temperatura ambiente durante 4 horas y tgallina deshidratados en una serie gradual de alcoholes etílico: 50% y 70% en el refrigerador (4 ° C), y el 85%, 95% (3 cambios), y el 100% (4 a 5) cambios en la temperatura ambiente. El cerebro se deja en cada solución durante 24 horas. </li><li> El cerebro deshidratado se pone entonces en acetona (3 a 4 cambios, cada uno por un día), seguido por una mezcla de acetona con Araldite Araldite a acetona proporción de 1:2, 1:1, 2:1, y finalmente en 100% araldite (3 cambios, cada vez durante la noche), en el refrigerador (4 ° C). </li><li> Finalmente, el cerebro trata está integrado en el 100% Araldite y se calienta a 60 ° C durante 3 días (cf. Protocolo de incrustación de Abbott y Sotelo 2). Si las muestras están incrustadas en LR White continuación las muestras se transfieren de la solución de etanol últimos 100% a través de tres cambios de LR solución Blanca que cada uno mantenerse en el refrigerador durante la noche, que es un procedimiento estándar antes de la polimerización. Tres cambios se realizan para garantizar la infiltración total. La muestra se tgallina transferido a un nuevo blanco LR que se polimeriza en un recipiente cerrado a 60 ° C durante 24 horas, que es la cantidad necesaria de tiempo y temperatura para la polimerización adecuada. </li><li> Fig. 1 muestra un cerebro de Golgi-Cox manchada incrustado en Araldite. </li><li> Primero la muestra curada se monta en el anillo de muestra de metal con epoxi, y los lados de la manzana recortada como sea necesario (ver fig. 2). </li></ol> 2. Preparación de la muestra: Nissl <ol><li> El ratón está profundamente anestesiados con ketamina y xilacina (1,7 mg de ketamina / xilazina 0,26 mg por cada 20 gramos de peso corporal) inyecta por vía intraperitoneal y perfundidos transcardially con 50 ml de la temperatura ambiente de buffer fosfato salino (pH 7,4), seguido por 250 ml de habitación temperatura de 10% neutral formalina tamponada (pH 7,4). y, finalmente, con 3,0 cc de pura tinta china. </li><li> Perfusión de todo el cuerpo con solución salina y fijador es necesario para limpiar la sangre del sistema cardiovascular y para fijar el ti<html> TEMAS. Perfusión con tinta china es necesaria para llenar completamente la vasculatura del sistema cardiovascular. </li><li> El cerebro resultante se deshidrata a través de una serie de alcoholes clasificado de etilo (25% -100%) y luego incorporados en plásticos araldite siguiendo el protocolo de 1,8-1,9 por encima. Si LR blanco es el medio de inclusión que el protocolo de 1,10 mencionado anteriormente. </li></ol> 4. Preparación de la muestra: las especies genéricas, los órganos de genéricos <ol><li> Para el caso genérico, la fijación se puede hacer con cualquiera de 10% neutral paraformaldehido tamponado% de formalina o 4. </li><li> Para el volumen de tejido pequeños, la difusión en lugar de la perfusión se recomienda para la fijación y la tinción. En el caso de los pequeños organismos u órganos, coloración también se puede hacer durante el proceso de deshidratación. </li><li> El protocolo de incorporación es el mismo que el anterior, aunque los tiempos para la infiltración de una solución puede ser reducido por los órganos u organismos que son mucho más pequeños que el cerebro del ratón entero. </li></ol>jove_title &quot;KESM&gt; 5. configuración y la imagen <ol><li> Apague la bomba, a su vez en el escenario, a su vez en la cámara, a su vez en la iluminación. </li><li> Levante un cuchillo y objetiva. </li><li> Instale el anillo de la muestra. </li><li> Medir la dimensión de muestras y crear un archivo de configuración de la aplicación Etapa KESM Controller2. </li><li> Inicio KESM Controller2 etapa e iniciar la etapa. </li><li> La cuchilla inferior / objetivo, a su vez en la bomba. </li><li> Enfoque objetivo y la cámara, girando la perilla de enfoque en el tren óptico y el ajuste de campo de la cámara de vista en relación con el filo de la navaja. </li><li> Pulse en [Ir] para iniciar la formación de imágenes. </li><li> Vea las figuras. 8.3. Véase el punto 9, 7 para los detalles técnicos. </li></ol> 6. Procesamiento de imágenes y preparación de datos <ol><li> Copia de la pila de KESM ejecutable del procesador en la carpeta de datos en la carpeta de archivo de configuración (00000, 00001, etc.) </li><li> Ejecutar el procesador de la pila KESM para eliminar los artefactos de iluminación y normalizar la intensidad de fondo en todos loslas imágenes. Repita la operación para todas las subcarpetas de datos. </li><li> Prepare las baldosas de escala múltiple del conjunto de datos y cargar y configurar los archivos en el servidor web del cerebro KESM Atlas. </li><li> Ver fig. 9. </li></ol> 7. Visualización de datos y análisis <ol><li> Visualización utilizando el atlas del cerebro KESM. Ir al <a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a> y seleccione el atlas correspondiente. <ol><li> Navegar como en los mapas de Google. </li><li> Acercar y alejar al igual que en los mapas de Google. </li><li> Para ir a una profundidad diferente, seleccione la profundidad de ir a cada paso en el menú desplegable, a continuación, haga clic en cualquiera [+] o [-] para aumentar o disminuir la profundidad z. </li><li> Ajustar el número de superposición utilizando el menú desplegable. </li><li> Ajustar el intervalo de superposición utilizando el menú desplegable. </li><li> Ver fig. 11. </li></ol></li><li> Visualización mediante MeVisLab. <ol><li> Lanzamiento MeVisLab. </li><li> Crear un nuevo proyecto. </li><li> En el siguiente, los nuevos módulos se pueden crear por tymesa de ping en el nombre del módulo en el cuadro de comandos. </li><li> Crear un módulo [Compose3Dfrom2DFiles], e ir a la carpeta deseada. Introduzca la cadena de archivo y haga clic en [GetFileList]. Asegúrese de que las imágenes seleccionadas pueden caber en la memoria. Haga clic en [Create3D] para crear volumen. </li><li> Crear un módulo [SetWorldMatrix], y establecer la &quot;escala&quot; en &quot;Transforma Primaria&quot; x, y, z con el tamaño adecuado voxel (0,6, 0,7, 1,0 para un objetivo 0.45NA 10X). Enlace [Compose3Dfrom2DFiles] a [SetWorldMatrix]. </li><li> Establecer la matriz y se transforma en &quot;composición de la matriz&quot; a &quot;Ignore&quot;, &quot;Ninguna&quot;, &quot;Adelante&quot;. </li><li> Crear un módulo [Arithmetic1] y establecer la &quot;función&quot; a &quot;Invertir (Max-IMG)&quot;. Enlace [SetWorldMatrix] a [Arithmetic1]. </li><li> Crear un módulo [View3D] y conectar [Arithmetic1]. </li><li> En View3D, mientras que el botón derecho del ratón se presiona, mueve el ratón para ajustar el umbral. Puede recortar las regiones, la orientación del cambio (movimiento de mouse botón izquierdo del ratón mientras se pulsa), iluminación de cambio (renderin volumeng o MIP), activar / desactivar el fondo, etc </li><li> Ver fig. 10. </li></ol></li></ol> 8. Los resultados representativos: A continuación, presentamos los datos de todo el cerebro y los detalles. Higos. 12-15 muestran a todo el cerebro tinta china, Golgi, Nissl y conjuntos de datos de 1, 4, 3. <img alt="Figura 1" src="/files/ftp_upload/3248/3248fig1.jpg" /> Figura 1. Cerebro de ratón fija, se tiñe y embebidos <img alt="Figura 2" src="/files/ftp_upload/3248/3248fig2.jpg" /> Figura 2. Muestras de cerebro de ratón integrado montado en el anillo de la muestra. <img alt="Figura 3" src="/files/ftp_upload/3248/3248fig3.jpg" /> Figura 3. El filo del cuchillo Microscopio de Barrido (KESM). Una foto de la KESM se muestra con sus principales componentes marcados: (1) de montaje de alta velocidad de la línea de barrido de la cámara, (2) objetivo del microscopio, (3) cuchilla de diamante y el colimador de luz, (4) de la SPECimen tanque (para obtener imágenes de la inmersión en agua), (5) de tres ejes de aire de precisión teniendo etapa, (6) blanco-luz del iluminador microscopio, (7) la bomba de agua (en la parte posterior) para la extracción de tejido seccionado, (8) PC servidor para el control de la etapa y la adquisición de imágenes (9), base de granito, y (10) puente de granito. <img alt="Figura 4" src="/files/ftp_upload/3248/3248fig4.jpg" /> Figura 4. Principios de imágenes de la KESM. El director de operaciones de KESM se ilustra. El objetivo y el cuchillo se mantiene en su lugar, mientras que la muestra colocada en la etapa de movimientos de posicionamiento (flecha con línea continua) en la resolución de 20 nm y la velocidad de desplazamiento de 1-5, y se raspó contra la cuchilla de diamante (5 mm de ancho para el objetivo de 10X), generando una lámina delgada que fluye sobre el cuchillo (flecha con línea continua). Línea de exploración de imágenes se realiza cerca de la punta del cuchillo. <img alt="Figura 5" src="/files/ftp_upload/3248/3248fig5.jpg" /> Figura 5. KESM cámara y objetivo de los controles de enfoque. <img alt="Figura 6" src="/files/ftp_upload/3248/3248fig6.jpg" /> Figura 6. Asamblea KESM cuchillo y controles. <img alt="Figura 7" src="/files/ftp_upload/3248/3248fig7.jpg" /> Figura 7. Inicial centrado a través del puerto de observación. <img alt="Figura 8" src="/files/ftp_upload/3248/3248fig8.jpg" /> Figura 8. Controlador KESM Etapa 2 de la aplicación (de pantalla). <img alt="Figura 9" src="/files/ftp_upload/3248/3248fig9.jpg" /> Figura 9. Pila KESM procesador de aplicaciones (screenshot). <img alt="Figura 10" src="/files/ftp_upload/3248/3248fig10.jpg" /> Figura 10. MeVisLab aplicación (captura de pantalla). <img alt="Figura 11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; Figura 11 KESM Atlas del Cerebro:. Web-interfaz (pantalla). <img alt="Figura 12" src="/files/ftp_upload/3248/3248fig12.jpg" /> Figura 12. KESM todo el cerebro de datos con tinta china. Visualizaciones volumen de datos KESM pilas se muestran para el conjunto de datos vascular. (A) Close-up de los datos vascular. Ancho ~ 100 um. (Bd) de tres vistas estándar de toda la vasculatura del cerebro del ratón (submuestras de datos de alta resolución). Ancho ~ 10mm. <img alt="Figura 13" src="/files/ftp_upload/3248/3248fig13.jpg" /> Figura 13. KESM todo el cerebro del ratón de datos de Golgi. <img alt="Figura 14" src="/files/ftp_upload/3248/3248fig14.jpg" /> Figura 14. KESM Golgi detalles de los datos. <img alt="Figura 15" src="/files/ftp_upload/3248/3248fig15.jpg" /> Figura 15. KESM todo el cerebro de datos Nissl. (A) Primer plano de la Ndatos ISSL. ~ 300 um 3. (Bd) de tres puntos de vista estándar de la totalidad del cerebro del ratón de datos Nissl (submuestras de datos de alta resolución). Sección transversal se muestra una visión clara de las estructuras internas.

<ol>
	<li>Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+whole+small+animal+brains+with+the+knife-edge+scanning+microscope.">High-throughput imaging of whole small animal brains with the knife-edge scanning microscope.</a> Neuroscience Meeting Planner. Program No. 504.2, (2008).</li><li>Abbott, L. C., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+catecholaminergic+innervation+in+weaver+and+normal+mouse+cerebellar+cortices.">Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices.</a> Journal of Comparative Neurology. 426, 316-329 (2000).</li><li>Choe, Y., Abbott, L. C., Miller, D. E., Han, D., Yang, H. -. F., Chung, J. R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging,+analysis,+and+integration+of+mouse+brain+networks.">Multiscale imaging, analysis, and integration of mouse brain networks.</a> Neuroscience Meeting Planner. Program No. 516.3, (2010).</li><li>Choe, Y., Han, D., Huang, P. -. S., Keyser, J., Kwon, J., Mayerich, D., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+submicrometer+scans+of+mouse+brain+microstructure:+Neurons+and+vasculatures.">Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures.</a> Neuroscience Meeting Planner. Program No. 389.10, (2009).</li><li>Choe, Y., Abbott, L. C., Han, D., Huang, P. S., Keyser, J., Kwon, J., Mayerich, D., Melek, Z., McCormick, B. H., Ravi Rao, A., Cecchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy:+High-throughput+imaging+and+analysis+of+massive+volumes+of+biological+microstructures.">Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures.</a> High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications. , 11-37 (2008).</li><li>Choe, Y., Abbott, L. C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A. M., Fiorito, F., Edelman, D. B., McKinstry, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+out+the+octopus+connectome+at+submicron+resolution+using+the+knife-edge+scanning+microscope.">Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope.</a> BMC Neuroscience. 11, 136-136 (2010).</li><li>Mayerich, D., Abbott, L. C., McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy+for+imaging+and+reconstruction+of+three-dimensional+anatomical+structures+of+the+mouse+brain.">Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain.</a> Journal of Microscopy. 231, 134-143 (2008).</li><li>Mayerich, D., Kwon, J., Sung, C., Abbott, L. C., Keyser, J., Choe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+macro-scale+Transmission+Imaging+of+Microvascular+Networks+Using+KESM.">Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM.</a> Biomedical Optics Express. 2, 2888-2896 (2008).</li><li>McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Brain+Tissue+Scanner.">Development of the Brain Tissue Scanner.</a> Technical Report. , (2002).</li><li>Sporns, O., Tononi, G., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+connectome:+A+structural+description+of+the+human+brain.">The human connectome: A structural description of the human brain.</a> PLoS Computational Biology. 1, 42-42 (2005).</li></ol>

Todd Huffman es con 3Scan, una empresa comercial que fabrica y comercializa la tecnología KESM (patente de EE.UU. 6.744.572 #).

El KESM permite un estudio submicrométricas a nivel de grandes volúmenes de muestras biológicas (~ 1 cm 3). Este tipo de volumen es suficiente para almacenar toda órganos de animales pequeños, como el cerebro, pulmones, corazón, riñones, etc escaneadas las imágenes de los órganos pueden proporcionar información sin precedentes cuantitativa sobre la organización estructural de estos órganos, y permitir el modelado computacional de diversos funcionales aspectos de estos órganos, incluyendo la dinámica del circuito y de la red, propiedades eléctricas, de fluidos y la dinámica del flujo de aire, y la dinámica muscular. El protocolo de preparación de muestras y el protocolo de post-análisis detallados en este artículo se espera para ayudar a grupos al margen de la investigación para tener un acceso más fácil a la KESM y los datos resultantes. El protocolo de operación KESM ayudará a los investigadores externos a apreciar los beneficios y limitaciones de esta técnica de imagen única.

A correction was made to <a href="http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

A correction was made to <a href="http://http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

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

specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy

Los principales avances en alto rendimiento, de alta resolución, técnicas de microscopía 3D han permitido la adquisición de grandes volúmenes de datos neuroanatómicos a una resolución de submicrométricas. Uno de los primeros instrumentos como la producción de todo el cerebro escala de datos es el microscopio de filo de la navaja (KESM) 7, 5, 9, desarrollada y alojada en el laboratorio de los autores. KESM se ha utilizado para la sección y el cerebro de toda la imagen del ratón con una resolución de submicrométricas, revelando los detalles intrincados de las redes neuronales (Golgi) 1, 4, 8, redes vasculares (tinta china) 1, 4, y la distribución de las células del cuerpo (Nissl) 3 . El uso de KESM no se limita al ratón ni el cerebro. Hemos logrado imágenes del cerebro del pulpo 6, pulmón de ratón, y el cerebro de la rata. En estos momentos estamos trabajando en conjunto embriones de peces cebra. Datos como estos pueden contribuir enormemente a la investigación conectómica 10, de la micro y la investigación hemodinámica, y la investigación estereología por disposicionesDing an. exacta verdad-terreno En este artículo vamos a describir la tubería, incluyendo la preparación de muestras (fijación, tinción, y la inclusión), configuración y puesta en marcha KESM, la sección y la imagen con la KESM, procesamiento de imágenes, preparación de datos, y visualización de datos y análisis. Se hará hincapié en la preparación de muestras y la visualización / análisis de los datos obtenidos KESM. Esperamos que el protocolo detallado que se presenta en este artículo para ayudar a ampliar el acceso a KESM y aumentar su utilización.

Watch this Scientific Journal Video about Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy at JoVE.com

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

El proceso completo, desde la preparación del cerebro muestra de imágenes en serie de seccionamiento con el microscopio de filo de la navaja, a la visualización y análisis de datos se describe. Esta técnica se utiliza actualmente para obtener datos del cerebro del ratón, pero es aplicable a otros órganos, otras especies.

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 ...

specimen-preparation-imaging-analysis-protocols-for-knife-edge

Research

JoVE Journal

Bioengineering

17.5K vistas.  Texas A&M University. El proceso completo, desde la preparación del cerebro muestra de imágenes en serie de seccionamiento con el microscopio de filo de la navaja, a la visualización y análisis de datos se describe. Esta técnica se utiliza actualmente para obtener datos del cerebro del ratón, pero es aplicable a otros órganos, otras especies.

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

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

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Inmovilización de bacterias para tratamiento de imágenes de microscopía de fuerza atómica

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

Video-velocidad de barrido y microscopía confocal Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Creación de degradados adhesivas y soluble para la migración celular con imágenes de microscopía de fluorescencia

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

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., ...

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 ...

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

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

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 ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Fuerza Atómica Microscopía Imaging y Fuerza Espectroscopia de admitidos Bilayers lípidos

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Correlativos luz microscopia electrónica (CLEM) para el seguimiento y proyección de imagen proteína Viral asociada a estructuras en las células inmovilizadas de Cryo

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizar la formación de adherencias en las células por medio de avanzados Spinning microscopía de fluorescencia de reflexión interna Total de disco

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...