I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

1. Preparación de plántulas <ol><li> Esterilizar las semillas. <ol><li> Preparar la solución de esterilización mediante la adición de 500 l NaClO (cloro disponible: 5,0%) y 1 l 10% de Triton X-100 a 500 l de agua estéril. </li><li> Lugar ca. 10 transgénico A. thaliana semillas que llevan GFP-PATROL1 8 en un tubo de 1,5 ml. </li><li> Añadir 1 ml solución de etanol al 70%, y mezclar bien invirtiendo cinco veces. Dejar durante 1 min. </li><li> Tenga en cuenta las semillas se hunden hasta el fondo del tubo. En una cabina de flujo laminar limpio, retire suavemente el etanol al 70% utilizando una micropipeta, y añadir 1 ml de solución de esterilización. Mezclar bien por inversión cinco veces y se deja durante 5 min. </li><li> Lavar las semillas. Sigue trabajando en condiciones asépticas en un banco limpio, retire con cuidado la solución con una micropipeta, y añadir 1 ml de agua estéril. Repita esto cinco veces. Las semillas esterilizadas se pueden almacenar en agua estéril a 4 ° C durante 2 días. </li></ol></li><li> Asi quew las semillas esterilizadas en un gellan 0,5% de goma solidificado media Murashige y Skoog placa de medio (pH 5,8) 9. Cinta de la tapa sobre la placa usando dos capas de cinta quirúrgica. </li><li> Incubar la placa en la oscuridad a 4 ° C con una cámara de almacenamiento en frío, O / N. </li><li> La transferencia de la placa en una cámara de crecimiento fijado en 23,5 ° C, con un ciclo de luz-oscuridad / 12-hr 12 hr utilizando 100 mol m -2 s -1 luces blancas, y se incuba durante 7 días. Las plántulas con cotiledones de alrededor de 1 mm de largo y luego se pueden cosechar. </li></ol> 2. Sky gota Montaje de Cotiledón Especímenes NOTA: Un factor importante en la preparación de muestras para la observación VAEM es evitar la inclusión de burbujas de aire entre la muestra y la cubierta de vidrio. Burbujas reducen en gran medida la calidad de imagen de VAEM al causar diferencias en el índice de refracción. Un método simple, lo que hemos llamado &quot;caída del cielo &#39;de montaje, se puede utilizar para evitar burbujasentre la A. cotiledones thaliana y la cubierta de vidrio. Esto se debe hacer inmediatamente antes de la observación. <ol><li> Coloque 30 l de tampón basal [5 mM 2- (N morfolino) etanosulfónico ácido-Tris (Tris-MES) a pH 6,5, KCl 50 mM, 100 mM CaCl 2] en el centro de un portaobjetos de vidrio (tamaño: 76 × 26 mm, espesor: 1,0 a 1,2 mm). </li><li> Retirar un cotiledón de una plántula de 7 días de edad, con unas tijeras de disección. Flotador el cotiledón con la cara de observación arriba en la caída de tampón basal (Figura 1, paso 1). </li><li> Ponga 30 l de tampón basal en el centro de una cubierta de vidrio (tamaño: 18 × 18 mm, espesor: 0,12-0,17 mm) (Figura 1, paso 2). Gire la cubierta de vidrio boca abajo con suavidad. La tensión superficial evitará la caída de tampón de caerse. Sostenga la cubierta de vidrio con pinzas en un borde. Coloque el borde opuesto sobre el portaobjetos de vidrio de modo que la caída de tampón es de aproximadamente por encima del cotiledón. </li><li>Con el borde de la cubierta de vidrio aún en la diapositiva, y todavía con el borde opuesto con pinzas, ajustar la posición de la caída de amortiguación bajo la cubierta de vidrio para que sea directamente sobre la muestra cotiledones (Figura 1, paso 3). Dejar de lado la cubierta de vidrio. Montar una gota en la muestra resultante en una preparación sin burbujas de aire. </li><li> Limpie el exceso de buffer usando tejidos sin pelusa. Observar la preparación inmediatamente (ver paso 3). NOTA: No hay necesidad de sellar las muestras. </li></ol> 3. VAEM Observación y Movie Adquisición NOTA: El sistema de microscopio TIRF 9 utilizado en el presente estudio se describe como sigue: un microscopio invertido está equipado con una unidad de TIRF y una lente objetivo TIRF con una apertura numérica de 1,49. Para el control informatizado del ángulo de entrada del láser, se utiliza una caja de control. Proteína verde fluorescente (GFP) se excita con un 488 nm láser semiconductor de bombeo óptico, y tque la fluorescencia se detecta a través de un filtro de paso de banda 510 a 550 nm para evitar que la autofluorescencia de los cloroplastos. El valor máximo medido de la potencia de salida de la fibra es 13,0 a 13,5 mW. Para la detección, un electrón multiplicando dispositivo de carga acoplada (EM-CCD) sistema de cabezal de la cámara y una unidad de cambio de magnificación de la cámara de montaje C se utilizan. <ol><li> Calibrar centrar el láser y el enfoque de acuerdo con las instrucciones del fabricante. NOTA: Este paso es crucial para las observaciones VAEM precisas. Se recomienda encarecidamente que los controles periódicos de los procedimientos de ajuste de trayectoria láser, apropiado a su microscopio, se llevan a cabo. <ol><li> Determinar la posición central iluminada con una trayectoria de la luz sin lente objetivo en el techo de la sala de microscopía. Para marcar la posición central, poner un sello de color en el techo (Figura 2, paso 1, 2). </li><li> Ilumine el techo con una lente objetivo (Figura 2, paso 3, 4). Mueva el illuminated región a la posición central (Figura 2, paso 5). Enfoque el láser (figura 2, paso 6). Ajustar la posición del láser enfocado hacia el centro (figura 2, paso 7). </li></ol></li><li> Establecer un ejemplar en la platina del microscopio y seleccionar celdas de observación utilizando iluminación de campo brillante. </li><li> Compruebe que la proteína fluorescente se puede observar en las células, y fijó la posición de la z eje x en la superficie celular mediante la iluminación de epifluorescencia (Figura 3A). </li><li> Realizar observaciones VAEM. <ol><li> Inclinar el ángulo de entrada del haz de láser gradualmente con la caja del controlador. Al mismo tiempo, controlar la imagen en vivo con cuidado. Inicialmente, la imagen será borrosa (Figura 3B). </li><li> A medida que el ángulo aumenta láser, la imagen VAEM será menos borrosa, finalmente producir un imagen clara (Figura 3C y D). En este punto, dejar de Increacantar el ángulo láser. Si se pierde la señal de fluorescencia, disminuya a un ángulo menos profundo. </li><li> Ajustar el ángulo de entrada de láser para obtener una mejor imagen. Puesta a punto de la posición del eje x z también puede mejorar la imagen. Si es necesario, ajuste los parámetros ópticos (energía láser, longitud de onda y de conjunto de filtros) y parámetros del sensor de imagen [tamaño de imagen, tiempo de exposición, ganancia, digitalizador y (EM) de ganancia de electrones multiplicando]. NOTA: En el caso de los equipos microscopio TIRF ha descrito anteriormente, los parámetros representativos son los siguientes. Ampliación de la lente justo en frente de la cámara: 2X; Salida de láser: 1,0 mW; Tamaño de imagen: 512 × 512 píxeles; Tiempo de exposición: 100 ms; Ganancia: 5 ×; Digitalizador: 11 MHz; y EM ganancia: 100. </li></ol></li><li> Adquirir una película como una de varias páginas TIFF archivo de imagen utilizando el software microscopio comercial. En este caso, la película es de puntos GFP-etiquetados parpadeantes en la superficie de las células de los estomas. NOTA: las condiciones de adquisición de imágenes representativas son como folmínimos. Modo de adquisición: Corriente de RAM; Número de imágenes: 600; y el tamaño de varias páginas TIFF: ~ 302 MB. </li></ol> 4. Análisis quimógrafo para la cuantificación de GFP-etiquetados Dot Residencia Tiempo Usando Fiji Software <ol><li> Instale Fiji (&#39;Fiji es Justo ImageJ&#39;) de software 10 usando las instrucciones de los autores (http://fiji.sc/Fiji). </li><li> Ejecute Fiji y abra el archivo TIFF de varias páginas adquirida mediante el menú Fiji &quot;Archivo-Abrir&quot;. </li><li> Coloque una línea en el sitio de interés, utilizando la herramienta de menú de la barra de Fiji &quot;Herramienta de selección Línea Recta&quot;. Opcionalmente, utilice la &quot;Herramienta de selección de línea segmentada&quot; o &quot;Herramienta de selección Línea a mano alzada&quot;. En los resultados presentados aquí, una línea recta de 100 pixeles (correspondientes a 8,0 M) se colocó en una célula subsidiaria (Figura 4A). </li><li> Hacer una imagen quimógrafo usando el menú de Fiji &quot;Imagen-Pilas-Dynamic Reslice </em&gt; &quot;(http://fiji.sc/Dynamic_Reslice) (Figura 4B). Opcionalmente, &quot;Flip vertical&quot; y &quot;Girar 90 grados&quot; en una ventana de la casilla de verificación también están disponibles (no se utiliza en los resultados presentados aquí). La x y eje y en la imagen quimógrafo indican la posición de la línea (100 píxeles correspondiente a 8,0 micras) y el tiempo de observación (600 píxeles correspondientes a 60 seg), respectivamente. Si la imagen quimógrafo no es satisfactoria, la posición y el tipo (recta, segmentados y mano alzada) de la línea en la imagen de varias páginas se pueden cambiar. Como los cambios en la formación, la imagen quimógrafo cambia dinámicamente. Guarda una imagen de quimógrafo como un archivo TIFF usando el menú de Fiji &quot;Archivo-Guardar como-Tiff&quot;. </li><li> Mida la longitud de la burbuja en la orientación del eje de tiempo. <ol><li> Para llevar a cabo la reducción de ruido, aplicar un filtro gaussiano a las imágenes quimógrafo utilizando el menú de Fiji &quot;Process-Filtros-Gaussian Blur&quot;. El &quot;Sigma (Radius) &quot;parámetro es sintonizable (1 radio de píxeles se utiliza para los resultados presentados). </li><li> Segmento de las regiones de señal de umbral, utilizando el menú de Fiji &quot;Imagen-Adjust-Umbral&quot;. NOTA: Hay varios algoritmos de umbral para elegir. En los resultados presentados, el algoritmo &quot;Yen&quot; fue elegido (Figura 4C). </li><li> Prepárese para medir el tiempo (y) eje x longitud de la burbuja con el menú &quot;Analizar-Set Mediciones&quot;. Compruebe el &quot;rectángulo delimitador&quot; en la ventana &quot;Medidas Set&quot;. Lo ideal sería que el área de juego debe ser lo más pequeño posible, para excluir el ruido. En este caso, el área mínima se fijó en 50 píxeles. Medir el tiempo (y) eje x longitud de la burbuja con el menú &quot;Analizar-Analizar Partículas&quot;. Para obtener los valores de los resultados y demostrar la credibilidad de las regiones de medición, compruebe los &quot;Mostrar resultados&quot; y &quot;Añadir a Gerente &lt;/ em&gt; &quot;cajas. </li><li> Los valores en la columna &quot;Altura&quot; son el tiempo (y) eje x longitudes para la mancha de medición (Figura 4D). Guarde los valores utilizando el menú de la tabla Resultados &quot;Archivo-Guardar como&quot; y calcular y procesar el conjunto de datos de las duraciones imagen blob utilizando un paquete estadístico y / o software de hoja de cálculo (Figura 4E). </li></ol></li></ol>

<ol>
	<li>Konopka, C. A., Bednarek, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+dynamics+and+functional+redundancy+of+the+Arabidopsis+dynamin-related+isoforms+DRP1A+and+DRP1C+during+plant+development.">Comparison of the dynamics and functional redundancy of the Arabidopsis dynamin-related isoforms DRP1A and DRP1C during plant development.</a> Plant Physiol. 147 (4), 1590-1602 (2008).</li><li>Fujimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+dynamin-related+proteins+DRP2B+and+DRP1A+participate+together+in+clathrin-coated+vesicle+formation+during+endocytosis.">Arabidopsis dynamin-related proteins DRP2B and DRP1A participate together in clathrin-coated vesicle formation during endocytosis.</a> Proc. Natl. Acad. Sci. USA. 107 (13), 6094-6099 (2010).</li><li>Higaki, T., Sano, T., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+microfilament+dynamics+and+actin+side-binding+proteins+in+plants.">Actin microfilament dynamics and actin side-binding proteins in plants.</a> Curr. Opin. Plant Biol. 10 (6), 549-556 (2007).</li><li>Rosero, A., &#381;&#225;rsky, V., Cvr&#269;kov&#225;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtFH1+formin+mutation+affects+actin+filament+and+microtubule+dynamics+in+Arabidopsis+thaliana.">AtFH1 formin mutation affects actin filament and microtubule dynamics in Arabidopsis thaliana.</a> J. Exp. Bot. 64 (2), 585-597 (2013).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+internal+reflection+fluorescence+microscopy+in+cell+biology.">Total internal reflection fluorescence microscopy in cell biology.</a> Traffic. 2 (11), 764-774 (2001).</li><li>Konopka, C. A., Bednarek, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable-angle+epifluorescence+microscopy:+a+new+way+to+look+at+protein+dynamics+in+the+plant+cell+cortex.">Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex.</a> Plant J. 53 (1), 186-196 (2008).</li><li>Wan, Y., Ash, W. M., Fan, L., Hao, H., Kim, M. K., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable-angle+total+internal+reflection+fluorescence+microscopy+of+intact+cells+of+Arabidopsis+thaliana.">Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana.</a> Plant Methods. 7 (27), (2011).</li><li>Hashimoto-Sugimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Munc13-like+protein+in+Arabidopsis+mediates+H+-ATPase+translocation+that+is+essential+for+stomatal+responses.">A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses.</a> Nat. Commun. 4, 2215 (2013).</li><li>Higaki, T., Hashimoto-Sugimoto, M., Akita, K., Iba, K., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+environmental+responses+of+PATROL1+in+Arabidopsis+subsidiary+cells.">Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells.</a> Plant Cell Physiol. 55 (4), (2014).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li><li>Chebli, Y., Kaneda, M., Zerzour, R., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+wall+of+the+Arabidopsis+pollen+tube-spatial+distribution,+recycling,+and+network+formation+of+polysaccharides.">The cell wall of the Arabidopsis pollen tube-spatial distribution, recycling, and network formation of polysaccharides.</a> Plant Physiol. 160 (4), 1940-1955 (2012).</li><li>Fujimoto, M., Suda, Y., Vernhettes, S., Nakano, A., Ueda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+3-kinase+and+4-kinase+have+distinct+roles+in+intracellular+trafficking+of+cellulose+synthase+complexes+in+Arabidopsis+thaliana.">Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.</a> Plant Cell Physiol. 56 (2), 287-298 (2015).</li><li>Li, X., Wang, X., Yang, Y., Li, R., He, Q., Fang, X., Luu, D. T., Maurel, C., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+PIP2;+1+dynamics+and+partitioning+reveals+multiple+modes+of+Arabidopsis+plasma+membrane+aquaporin+regulation.">Single-molecule analysis of PIP2; 1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation.</a> Plant Cell. 23 (10), 3780-3797 (2011).</li><li>Li, R., Liu, P., Wan, Y., Chen, T., Wang, Q., Mettbach, U., Baluska, F., Samaj, J., Fang, X., Lucas, W. J., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+membrane+microdomain-associated+protein,+Arabidopsis+Flot1,+is+involved+in+a+clathrin-independent+endocytic+pathway+and+is+required+for+seedling+development.">A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.</a> Plant Cell. 24 (5), 2105-2122 (2012).</li><li>Staiger, C. J., Sheahan, M. B., Khurana, P., Wang, X., McCurdy, D. W., Blanchoin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+filament+dynamics+are+dominated+by+rapid+growth+and+severing+activity+in+the+Arabidopsis+cortical+array.">Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array.</a> J. Cell Biol. 184 (2), 269-280 (2009).</li><li>Ueda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-dependent+endoplasmic+reticulum+motility+and+F-actin+organization+in+plant+cells.">Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.</a> Proc. Natl. Acad. Sci. USA. 107 (15), 6894-6899 (2010).</li></ol>

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En este artículo de vídeo, los protocolos se dan para el seguimiento y la medición del comportamiento dinámico de puntos GFP-PATROL1 en el complejo de los estomas de Arabidopsis thaliana. Como se muestra aquí, la observación VAEM es una poderosa herramienta para imágenes en vivo de las superficies celulares de las plantas. Bajo las condiciones experimentales utilizadas aquí para la supervisión GFP-PATROL1, había muy poco photobleaching fluorescencia en la muestra utilizada para 1 min de captura de ví...

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-73</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TIRF unit</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX3-RFAEVAW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TIRF objective lens&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">UAPON 100 &#215; OTIRF&#160;</td>
			<td>NA = 1.49</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser angle control box</td>
			<td style="width:71px;">Chuo Seiki</td>
			<td style="width:119px;">QT-AK</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optically pumped semiconductor laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">SapphireTM LP USB 488-20 CDRH Laser</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">510&#8211;550 nm band-pass filter</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-FBNA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EM CCD camera</td>
			<td style="width:71px;">Hamamatsu Photonics</td>
			<td style="width:119px;">ImagEM C9100-13</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C-mount camera magnification change unit&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-TVCAC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaMorph software</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;">MetaMorph version 7.7.11.0</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">TIRF microscopy manual</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">AX7385</td>
			<td style="width:167px;">Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immersion oil</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">Immersion Oil Typr-F</td>
			<td>ne = 1.518 (23 degrees)</td>
		</tr>
	</tbody>
</table>

real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

En este artículo de vídeo, los protocolos para VAEM observación de GFP-PATROL1 en A. Se proporcionan células complejas thaliana cotiledones estomas. Montaje caída del cielo es un método de preparación simple que puede ayudar a reducir la aparición de burbujas de aire en los preparativos VAEM de A. cotiledones thaliana (Figura 1). Inclinación excesiva del láser de entrada y / o z-posicionamiento de muestras para VAEM proporcionará una imagen clara. Si eso su...

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

El objetivo de este protocolo es demostrar cómo monitorear la dinámica de proteínas con fluorescencia de etiquetado en las superficies celulares de las plantas con la microscopía de epifluorescencia de ángulo variable, mostrando parpadear puntos de PATROL1 GFP-etiquetados, una proteína tráfico de membrana, en la corteza celular del complejo estomático en Arabidopsis thaliana.

Imágenes en tiempo real de la Planta Dinámica superficie celular con Variable de ángulo epifluorescencia Microscopía

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Research

JoVE Journal

Biology

8.9K vistas.  The University of Tokyo.  El objetivo de este protocolo es demostrar cómo monitorear la dinámica de proteínas con fluorescencia de etiquetado en las superficies celulares de las plantas con la microscopía de epifluorescencia de ángulo variable, mostrando parpadear puntos de PATROL1 GFP-etiquetados, una proteína tráfico de membrana, en la corteza celular del complejo estomático en Arabidopsis thaliana. 

Video: Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imágenes de células vivas de la F-actina Dynamics a través de Microscopía Fluorescente Speckle (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Investigación

Biología

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Concentración, la carga, y la alineación de una célula de ultracentrifugación analítica de muestras

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidez detección basada en la interacción extracelular (AVEXIS) para la detección escalable de extracelular de baja afinidad interacciones ligando-receptor

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Tandem congelación de alta presión y la rápida congelación de sustitución de tejidos vegetales para Microscopía Electrónica de Transmisión

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Mediciones en tiempo real de proteínas de membrana: Receptor interacciones utilizando resonancia de plasmones superficiales (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Bienes Análisis Tiempo de perfil metabólico en<em&gt; Ex Vivo</em&gt; Culturas ratón intestinal Cripta organoides

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

La visualización de frecuencia Stromule con microscopía de fluorescencia

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Microscopía de luz Hoja de fluorescencia de las raíces de plantas que crecen en la superficie de un gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Flash-y-Freeze: una técnica novedosa para capturar la dinámica de la membrana con Microscopía Electrónica

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Agregación del Receptor de superficie celular microscopia confocal revela a través espectroscopia de la correlación de imagen

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...