Name: Video: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00
Duration: 14 min 10 s

Los autores desean agradecer a Thorlabs por su apoyo a este proyecto. AJN desea agradecer el apoyo de una beca de la NSF Graduate. Este trabajo fue parcialmente financiado por los Institutos Nacionales de Salud a través del Nuevo Director de los NIH Programa Premio a la Innovación, el número de concesión una DP2 OD007096-01. Información sobre el Programa Innovator Award Nuevo en <a href="http://nihroadmap.nih.gov/newinnovator/">http://nihroadmap.nih.gov/newinnovator/</a> . Los autores desean agradecer a Tom Hayes para el uso del laboratorio de Harvard Electronics.

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	<li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , 985-985 (2006).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nature Reviews Molecular Cell Biology. 2, 444-456 (2001).</li><li>Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+photobleaching+analysis+for+the+study+of+cellular+dynamics.">Fluorescence photobleaching analysis for the study of cellular dynamics.</a> European Biophysics Journal. 31, 36-51 (2002).</li><li>Stephens, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Microscopy+Techniques+for+Live+Cell+Imaging.">Light Microscopy Techniques for Live Cell Imaging.</a> Science. 300, 82-86 (2003).</li><li>McMahon, A., Supatto, W., Fraser, S. E., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Analyses+of+Drosophila+Gastrulation+Provide+Insights+into+Collective+Cell+Migration.">Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration.</a> Science. 322, 1546-1550 (2008).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled+controls+cell+polarity+during+Xenopus+gastrulation.">Dishevelled controls cell polarity during Xenopus gastrulation.</a> Nature. 405, 81-85 (2000).</li><li>Laemmel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+Confocal+Fluorescence+Microscopy+(Cell-viZio)+Facilitates+Extended+Imaging+in+the+Field+of+Microcirculation.">Fibered Confocal Fluorescence Microscopy (Cell-viZio) Facilitates Extended Imaging in the Field of Microcirculation.</a> Journal of Vascular Research. 41, 400-411 (2004).</li><li>Moussata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+confocal+laser+endomicroscopy.">The confocal laser endomicroscopy.</a> Acta Endoscopica. 39, 448-451 (2010).</li><li>Dunbar, K., Canto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+endomicroscopy.">Confocal endomicroscopy.</a> Current Opinion in Gastroenterology. 24, 631-637 (2008).</li><li>Udovich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+background+and+transmission+characteristics+of+fiber+optic+imaging+bundles.">Spectral background and transmission characteristics of fiber optic imaging bundles.</a> Applied optics. 47, 4560-4568 (2008).</li></ol>

La producción de este video este video fue patrocinado por Thorlabs, Inc.

Este sistema de imágenes de vídeo de tipo hace uso de un espejo de funcionamiento galvanómetros de resonancia alrededor de 8 kHz. Espejos de resonancia puede ser muy fuerte cuando se ejecuta a plena potencia, y su tono alto puede ser molesto o incluso peligroso en los tiempos de exposición suficiente. Aunque no se han presentado aquí, se recomienda proteger el espejo resonante galvanométrico dentro de una caja transparente para reducir significativamente el volumen del sistema y / o usar equipo de protección adecuada para los oídos, como tapones para los oídos. El espejo galvanómetros de resonancia escanea en un patrón sinusoidal. Sin embargo, las tarjetas de framegrabber leer en señal de asumir una frecuencia de barrido completamente lineal en las direcciones horizontal y vertical. Desde un barrido sinusoidal se ralentiza en los bordes de la exploración, los artefactos de compresión de imágenes se puede observar a lo largo del eje rápido de la imagen (horizontal). Una forma de minimizar este problema es conducir a propósito del espejo resonante galvo rango de lectura significativamente mayor que eldiámetro de la lente de relé. Al hacer esto, sólo la barra central de casi lineal de la trama de lectura sinusoidal recorrerá la muestra, reduciendo al mínimo las distorsiones de la imagen. Otro enfoque sería para procesar imágenes post-recogida para alinear el eje rápido. Esto se puede lograr mediante imágenes de un patrón conocido fluorescentes (como una rejilla) y el uso de las dimensiones de la forma conocida para crear una secuencia de comandos de procesamiento que unwarps las imágenes recogidas. Este sistema de escaneo en particular fue diseñada con el propósito de imágenes in vivo, que requiere a menudo una posición vertical orientada al video-tasa de microscopio. Para los experimentos de imagen celular, microscopios invertidos son más habitual. El diseño que aquí se presenta se pueden cambiar fácilmente para construir tal un microscopio invertido, todo lo que se requiere es una rotación de los últimos 2 espejo &quot;de diámetro. En lugar de orientar el espejo para dirigir el haz de exploración hacia abajo, el espejo puede dirigir el haz hacia arriba. La colocación de la lente del objetivo unot la misma distancia del espejo, junto con una etapa de la muestra que permite obtener imágenes en una geometría invertida. Si el sistema de imagen se construye exclusivamente para obtener imágenes de microendoscópica, no hay ninguna razón para &quot;doblar&quot; el diseño del microscopio vertical en absoluto. En cambio, el sistema de análisis completo se puede construir sobre una placa horizontal con un solo lente objetivo del paralelo orientado a la mesa óptica. Tenga en cuenta que el microscopio en esta versión utiliza una configuración de orificio fijo, mientras que esta prevé la construcción de mayor simplicidad y facilidad de adaptación, los usuarios que desean un sistema más versátil podría considerar la incorporación de un orificio variable, como se puede encontrar en la mayoría de los microscopios confocal comercial. Al permitir al usuario ajustar el tamaño del agujero de alfiler para compensar las muestras de mayor o menor intensidad de las emisiones, lo que permite al usuario optimizar mejor el equilibrio entre la fuerza de la señal y la resolución para una muestra dada. La chOZ de la fibra de la imagen seleccionada para el microscopio es importante. Recomendamos el uso de fibras de Sumitomo coherente de la imagen debido a su espaciamiento núcleo de la fibra y bajos en relación con autofluorescencia. Fibras de la imagen fabricado por Fujikura se ha encontrado que tienen altas cantidades de autofluorescencia 10, que puede abrumar a las débiles señales de fluorescencia de una muestra de la sensibilidad y el límite máximo de la microendoscope. Fibras Sumitomo manufacturados, tales como la 8-30N utiliza en esta configuración en particular, tienen niveles mucho más bajos que sus equivalentes de autofluorescencia Fujikura. Mientras haces de fibra de sanguijuelas puede ser considerado atractivo para microendoscopy, su diseño general, los lugares los núcleos individuales de fibra demasiado lejos, lo que significa que los núcleos de fibra de baja densidad de los objetos de la muestra, dejando fuera a importantes regiones de interés potencial. Por último, cabe señalar que mientras que el microscopio se describe aquí será útil en una variedad de ensayos in vitro e in vivo de solicomplementos y se pueden crear para una fracción del costo de un sistema comercial completa, que no tiene características como la detección de la luz transmitida, un ocular para ver, o una trayectoria del haz de no confocal de campo amplio de epifluorescencia. Si bien es posible construir un sistema con estas características desde el principio, los lectores que deseen un sistema que desee modificar un sistema comercial existente para satisfacer sus necesidades en lugar de iniciar una completamente nueva construcción.

<table border="1"><tbody><tr><td> Nombre de la pieza </td><td> Fabricante </td><td> Número del artículo </td><td> Especificaciones </td><td> Cantidad </td></tr><tr><td> 515 nm banda del filtro de paso </td><td> Chroma </td><td> HQ515/50M </td><td> 46 FWHM </td><td> 1 </td></tr><tr><td> Lente acromática Doublet 25,4 mm Dia. x 50mm FL, revestimiento MgF2 </td><td> Edmund Óptica </td><td> NT49-766 </td><td></td><td> 1 </td></tr><tr><td> Lente acromática Doublet 25,4 mm Dia. x 76,2 mm FL, revestimiento MgF2 </td><td> Edmund Óptica </td><td> NT49-768 </td><td></td><td> 1 </td></tr><tr><td> Lente acromática Doublet 25,4 mm Dia. x 88.9mm FL, revestimiento MgF2 </td><td> Edmund Óptica </td><td> NT49-769 </td><td></td><td> 2 </td></tr><tr><td> Dia doblete acromático lente de 50 mm. x 300mm FL, revestimiento MgF2 </td><td> Edmund Óptica </td><td> NT45-179 </td&gt; <td></td><td> 1 </td></tr><tr><td> 8 kHz de frecuencia R Scanner óptico de alta </td><td> Electro-Optical Products Corporation (EOPC) </td><td> SC-30 </td><td> 8 kHz </td><td> 1 </td></tr><tr><td> AGC conductor </td><td> Electro-Optical Products Corporation (EOPC) </td><td> ACG: 8K </td><td></td><td></td></tr><tr><td> H7422-PA módulo fotosensor </td><td> Hamamatsu </td><td> H7422-PA </td><td> Limitación de corriente recomendados </td><td> 1 </td></tr><tr><td> M9012 Alimentación </td><td> Hamamatsu </td><td> M9012 </td><td> Para su uso con H7422-PA </td><td> 1 </td></tr><tr><td> HC PL APO CS Objetivo </td><td> Leica </td><td> 11506284 </td><td> 10x/0.40 </td><td> 1 </td></tr><tr><td> Solios eA / XA Framegrabber tarjeta </td><td> Matrox </td><td> Solios eA / XA </td><td> MIL software necesario; OEM interconexiones recomienda </td><td> 1 </td></tr><tr><td> Fuente de alimentación 12V </td><td&gt; Meanwell </td><td> LPV-100-12 </td><td> 12 V, 8.5A </td><td> 1 </td></tr><tr><td> Microscopio 5x lente del objetivo </td><td> Newport </td><td> M-5X </td><td> NA 0.10, longitud 25,4 mm Focal </td><td> 1 </td></tr><tr><td> Fibra imagen coherente </td><td> Sumitomo </td><td> 8-30N </td><td></td><td> 1 </td></tr><tr><td> 1 / 4 &quot;-20 tapón de rosca y Kit de hardware </td><td> Thorlabs </td><td> HW-KIT2 </td><td></td><td> 1 </td></tr><tr><td> 100 micras montado Pinhole </td><td> Thorlabs </td><td> P100S </td><td> Ideal para la construcción de filtros espaciales </td><td> 1 </td></tr><tr><td> Jaula 30 mm Cubo Clamp </td><td> Thorlabs </td><td> B6C </td><td></td><td> 1 </td></tr><tr><td> Jaula 30 mm Sistema de Cubo, 4-Way </td><td> Thorlabs </td><td> C4W </td><td></td><td> 1 </td></tr><tr><td> 406 nm, de 5 MW, B Pin Code, diodo láser de fibra SM Pigtailed, FC / PC </td><td> Thorlabs </td><td> LPS-406-FC </td><td> Obsoletas de productos y fue sustituido por LP405-SF10 </td><td> 1 </td></tr><tr><td> De 5 minutos de epoxy, 1 onza </td><td> Thorlabs </td><td> G14250 </td><td></td><td> 1 </td></tr><tr><td> 6 Eje cinemática óptica Monte </td><td> Thorlabs </td><td> K6X </td><td></td><td> 1 </td></tr><tr><td> 8-32 tapón de rosca y Kit de hardware </td><td> Thorlabs </td><td> HW-KIT1 </td><td></td><td> 1 </td></tr><tr><td> 8-32 tornillo de fijación y kit de hardware </td><td> Thorlabs </td><td> HW-KIT3 </td><td></td><td> 1 </td></tr><tr><td> Adaptador de Exteriores e Interior Temas RMS Temas SM1 </td><td> Thorlabs </td><td> SM1A4 </td><td></td><td> 1 </td></tr><tr><td> Adj. FC / PC y el colimador FC / APC, f = 2,0 mm, ARC: 400-600 nm </td><td> Thorlabs </td><td> CFC-2X-A </td><td> f = 2,0 mm </td><td> 1 </td></tr><tr><td> Fibra ajustable Adaptador colimador, roscadas SM1 </td><td> Thorlabs </td><td> AD9.5F </td><td></td><td> 1 </td></tr><tr><td> Breadboard aluminio, 12 &quot;x 18&quot; x 1 / 2 &quot; </td><td> Thorlabs </td><td> MB1218 </td><td> 1 / 4 &quot;-20 rosca </td><td> 2 </td></tr><tr><td> Diodo láser de sobremesa / controlador TEC </td><td> Thorlabs </td><td> ITC4001 </td><td> 1 A/96 W </td><td> 1 </td></tr><tr><td> DMLP 425 nm de longitud de paso de espejo dicroico </td><td> Thorlabs </td><td> DMLP425 </td><td></td><td> 1 </td></tr><tr><td> Cinemática de montaje para Ø1 &quot;Óptica </td><td> Thorlabs </td><td> KM100 </td><td></td><td> 3 </td></tr><tr><td> LD / TEC Monte de diodos láser de fibra Thorlabs Pigtailed </td><td> Thorlabs </td><td> LM9LP </td><td></td><td> 1 </td></tr><tr><td> Montaje de la lente óptica de Ø18 mm </td><td> Thorlabs </td><td> LMR18 </td><td> Un anillo de retención incluida </td><td> 1 </td></tr><tr><td> Se monta la lente de 2 &quot;Óptica </td><td> Thorlabs </td><td> LMR2S </td><td> Con rosca interna y externa, incluido el anillo de retención </td><td> 2 </td></tr><tr><td> Mini Serie Jaula conjunto de la varilla, 6 &quot;mm de largo, Ø4, Cantidad 1. </td><td> Thorlabs </td><td> SR6 </td><td></td><td> 4 </td></tr><tr><td> Ø1.0 &quot;Post Pillar pedestal, 8-32 Grifos, 1&quot; de largo </td><td> Thorlabs </td><td> RS1P8E </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Post Pilar de extensión, longitud = 0,5 </td><td> Thorlabs </td><td> RS05 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Post Pilar de extensión, longitud = 0,75&quot; </td><td> Thorlabs </td><td> RS075 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Protegido espejo de plata, 3,2 mm de espesor </td><td> Thorlabs </td><td> ME1-P01 </td><td></td><td> 1 </td></tr><tr><td> Ø1 &quot;SM1 rotación ajustable elemento de enfoque, L = 1&quot; </td><td> Thorlabs </td><td> SM1V10 </td&gt; <td></td><td> 1 </td></tr><tr><td> Ø2 &quot;Protegido espejo de plata, 3,2 mm de espesor </td><td> Thorlabs </td><td> ME2-P01 </td><td></td><td> 2 </td></tr><tr><td> P100S - Ø100 m Montado Pinhole </td><td> Thorlabs </td><td> P100S </td><td></td><td> 1 </td></tr><tr><td> Polaris baja deriva Ø1 &quot;cinemática montura del espejo </td><td> Thorlabs </td><td> POLARIS-K1 </td><td> Baja deriva </td><td> 1 </td></tr><tr><td> SM1 lente de tubo, L = 1 &quot; </td><td> Thorlabs </td><td> SM1L-10 </td><td> Un anillo de retención incluida </td><td> 4 </td></tr><tr><td> SM1 roscadas de 30 mm Placa de Cage, 0.35 &quot;de espesor </td><td> Thorlabs </td><td> CP02 </td><td></td><td> 2 </td></tr><tr><td> SM1 a M25 adaptador roscado de componentes ópticos </td><td> Thorlabs </td><td> SM1A24 </td><td> Externos e internos SM1 Temas Temas M25.5x0.5 </td><td> 1 </td></tr><tr><td> Diam pequeño rayoeter Galvo sistema </td><td> Thorlabs </td><td> GVSM001 </td><td></td><td> 1 </td></tr><tr><td> Tenedor pequeño de sujeción </td><td> Thorlabs </td><td> CF125 </td><td> 1 / 25 &quot;contrataladrarse ranura, universal </td><td> 15 </td></tr><tr><td> Sistema de filtro espacial </td><td> Thorlabs </td><td> KT310 </td><td> Pinhole se vende por separado </td><td> 1 </td></tr><tr><td> TE-enfriado de montaje para 5,6 y 9 mm Láser </td><td> Thorlabs </td><td> TCLDM9 </td><td></td><td> 1 </td></tr><tr><td> Soporte vertical para Ayudas para diseño </td><td> Thorlabs </td><td> VB01 </td><td> Cada </td><td> 2 </td></tr><tr><td> Plan-Apochromat </td><td> Zeiss </td><td> 1101-957 </td><td> 20x/0.75 NA </td><td> 1 </td></tr></tbody></table>

video-rate scanning confocal microscopy and microendoscopy

La microscopía confocal se ha convertido en una herramienta indispensable en la biología y las ciencias biomédicas, lo que permite una rápida y de alta sensibilidad y alta resolución de seccionamiento óptico de los sistemas complejos. La microscopía confocal se utiliza habitualmente, por ejemplo, para estudiar los objetivos específicos celulares 1, la dinámica del monitor en las células vivas 2-4, y visualizar la evolución en tres dimensiones de organismos enteros 5,6. Extensiones de los sistemas de visualización de imagen confocal, como microendoscopes confocal, permite imágenes de alta resolución en vivo 7 y actualmente se aplican a las imágenes de enfermedades y diagnóstico en la práctica clínica 8,9. La microscopía confocal ofrece en tres dimensiones resolución mediante la creación de las llamadas &quot;secciones ópticas&quot; utilizando sencilla óptica geométrica. En una norma de amplio campo del microscopio de fluorescencia generada a partir de una muestra es recogida por una lente objetivo y transmitida directamente a un detector. Mientras que aceptarpoder de las muestras de imágenes finas, las muestras de espesor desdibujado por fluorescencia generada por encima y por debajo del plano focal del objetivo. En contraste, la microscopía confocal permite el corte virtual, ópticas de las muestras, el rechazo fuera del foco de luz para construir de alta resolución representaciones tridimensionales de las muestras. Microscopios confocal lograr esta hazaña con una apertura confocal en la trayectoria del haz de detección. La fluorescencia de una muestra recogida por el objetivo se retransmiten a través de los espejos de exploración ya través del espejo primario dicroicas, un espejo cuidadosamente seleccionados para reflejar longitudes de onda cortas, como el haz de excitación láser al pasar el tiempo, Stokes-cambió de emisión de fluorescencia. Esta señal de fluorescencia de onda larga se pasa a un par de lentes a cada lado de un agujero de alfiler que se coloca en un plano exactamente conjugado con el plano focal de la lente del objetivo. Fotones recogidos en el volumen focal del objeto son colimadospor el objetivo y se centran en las lentes confocal a través del agujero. Fluorescencia generada por encima o por debajo del plano focal por lo tanto no se colimado correctamente, y no pasará a través del agujero de alfiler un confocal, la creación de una sección de óptica en la que sólo la luz del foco microscopio es visible. (Fig. 1). Así, el agujero de alfiler actúa efectivamente como una apertura virtual en el plano focal, limitando la emisión detectada a una sola ubicación espacial limitada. Modernos microscopios confocal comerciales ofrecen a los usuarios un funcionamiento totalmente automático, los procedimientos de toma de imágenes antes complejo relativamente sencillo y accesible. A pesar de la flexibilidad y potencia de estos sistemas, comercial microscopios confocal no son adecuados para todas las tareas de visualización de imagen confocal, como muchas aplicaciones de imagen en vivo. Sin la capacidad de crear imágenes de sistemas a medida para satisfacer sus necesidades, experiencias importantes pueden permanecer fuera de las reaccionesh para muchos científicos. En este artículo se proporciona un método paso a paso para la construcción completa de una costumbre, video sistema de tipo de imagen confocal de componentes básicos. El microscopio vertical se construye con un espejo de galvanómetros de resonancia para proporcionar el eje de barrido rápido, mientras que una velocidad estándar de resonancia espejo galvanométrico analizará el eje lento. Para crear un haz de escaneado preciso en el enfoque de la lente objetivo, estos espejos se colocará en los planos telecéntrico llamada con cuatro lentes de relé. Detección confocal se realiza mediante una norma, fuera de la plataforma de tubos fotomultiplicadores (PMT), y las imágenes se capturan y se muestran usando una tarjeta digitalizadora Matrox y el software incluido.

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

La construcción completa de una costumbre, en tiempo real, sistema de microscopía confocal de exploración se describe. Este sistema, que puede ser fácilmente utilizada para microscopía de vídeo de tipo y microendoscopy, permite una gran variedad de geometrías de imágenes y aplicaciones que no son accesibles con los sistemas estándar confocal comercial, a una fracción del costo.

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 ...

video-rate-scanning-confocal-microscopy-and-microendoscopy

Research

JoVE Journal

Bioengineering

27.6K vistas.  Harvard University. La construcción completa de una costumbre, en tiempo real, sistema de microscopía confocal de exploración se describe. Este sistema, que puede ser fácilmente utilizada para microscopía de vídeo de tipo y microendoscopy, permite una gran variedad de geometrías de imágenes y aplicaciones que no son accesibles con los sistemas estándar confocal comercial, a una fracción del costo.

Video: Video-rate Scanning Confocal Microscopy and Microendoscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

De alta resolución de fibra óptica para Microendoscopy In situ Celular imágenes

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Investigación

Bioingeniería

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

Bajo Costo Cryo-microscopio de luz para la luz de la etapa de fabricación correlacionadas / Microscopía Electrónica

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

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 ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Correlativa a la luz y microscopía electrónica de Uso de Quantum Dot nanopartículas

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Multimodal de imágenes y la Plataforma Microendoscopy Espectroscopia de fibra paquete para no invasiva,<em&gt; En Vivo</em&gt; Análisis de Tejidos

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Disección, de exploración y morfométricos microTC Los análisis de la Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

División de célula mamífera en Matrices 3D mediante microscopia de reflexión Confocal cuantitativa

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Multimodal volumétrica retina imágenes por exploración oblicua oftalmoscopia de láser (oSLO) y tomografía de coherencia óptica (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

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 ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Microscopía de Fuerza Atómica y Confocal de Escaneo Láser Casi Simultánea (Conpokal) en Células Vivas

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...