Este trabajo fue apoyado en parte por los Institutos Nacionales de Salud Subvenciones HL-085 887, HL-092992, S10RR027535 y MOP-93676.

1. Disección de Buques e Imagen <ol><li> Tejido de cosecha de cerdos juveniles nacionales como se describe en Martens et al 27. Coloque ventrículos derecho porcina cosechadas en un polidimetilsiloxano (PDMS) plato de disección de fondo que contiene tamponada con HEPES solución salina fisiológica (PSS). <ol><li> Con la ayuda de un estereomicroscopio, diseccionar y retirar un segmento de la arteria descendente anterior izquierda coronaria (~ 8 mm de longitud, 0,5 mm de diámetro) del tejido circundante utilizando fórceps y tijeras de primavera mediante la eliminación de la segmento de vaso de las capas de tejido cardiaco de los alrededores. NOTA: Tenga cuidado de no perforar la pared del vaso. </li><li> Coloque un bloque de PDMS (1 x 0,5 x 0,5 cm) en el plato de disección, y el uso de una aguja para fijarlo a la parte inferior. Cortar un alambre de tungsteno de aproximadamente 40 m de diámetro en doce ~ segmentos de longitud 0,3 cm, formando microalfileres, y colocarlos en el plato. Con unas pinzas, sujete un extremo del segmento del vaso al bloque con una MICROPIn. </li><li> Inserte con cuidado las pequeñas tijeras de primavera en la luz del vaso, y cortar longitudinalmente en un lado de la embarcación para abrirlo por completo. Oriente el segmento de vaso abierto con el endotelio hacia arriba. </li><li> Utilice los microalfileres restantes para asegurar las fronteras del segmento de vaso abierto para bloquear de manera que el recipiente forma un rectángulo plano. NOTA: Micropin tapas deben estar dobladas a 90 ° y se insertan hasta que quede nivelado con la superficie del bloque. Se debe tener cuidado para estirar la preparación buque sin estirar demasiado; anchura final debe ser ~ 1,5 x el ancho de partida sin estirar. </li><li> Preparar un volumen pequeño (~ 1 ml) de Fluo-4 AM solución de carga mediante la mezcla de Fluo-4 a.m. (10 mM) disuelto en DMSO con plurónico (0,03%) en un tamponada con HEPES PSS (que contiene en mM: 134 NaCl, KCl 6 , 1 de MgCl, 10 HEPES, 10 glucosa), e insertar todo el bloque en la solución de carga durante aproximadamente 40 min a temperatura ambiente en la oscuridad. </li><li> Después de cargar, lavar el bloque en tampón HEPESPSS durante 5-10 min. </li><li> Monte el bloque a 50-100 micras espaciadores de espesor en una cámara inferior cubre objetos que contiene tamponada con HEPES PSS. NOTA: Los clavos metálicos se pueden utilizar como separadores y asegurar el segmento de vaso esté hacia abajo y no tocar los espaciadores. </li><li> Coloque la cámara en el escenario de un microscopio invertido equipado para imagen confocal, y se centran en la capa de células endoteliales. </li><li> Secuencias de imágenes de lapso de tiempo de captura de fluorescencia relativa basal durante ~ 3 min a 20 aumentos y una velocidad de ~ 8 fotogramas por segundo utilizando la imagen confocal software de secuencia de adquisición. </li><li> Después de ~ 3 min de grabación de fluorescencia basal, reemplazar tamponada con HEPES solución de PSS con el mismo volumen (~ 1 ml) de la sustancia P (100 pM) disuelto en tampón HEPES PSS, y récord para un addtional 3 min. </li></ol></li></ol> 2. Análisis Automatizado <ol><li> Render secuencia (s) imagen de la actividad de calcio fluorescente del software de adquisición confocal como 8 bits, grayscale &#39;. tif&#39; archivos de formato sin información de escala. </li><li> Abrir ImageJ, haga clic en &quot;abierto&quot; en el menú archivo y seleccionar la secuencia de imágenes apropiado en la ventana del explorador para ver la secuencia (s) imagen en ImageJ. </li><li> Determinar un diámetro ROI apropiada mediante el uso de la herramienta de ROI rectangular para estimar los límites superior e inferior de la propagación espacial de la actividad dentro de la secuencia (s) de imagen. NOTA: El diámetro rectangular media es una buena opción para diámetro ROI. </li><li> Cree una nueva carpeta en el disco duro del ordenador, y agregar la secuencia (s) imagen en el directorio de la carpeta. </li><li> En ImageJ, haga clic en la ventana de &#39;plugins&#39;, a continuación, haga clic en &#39;LC_Pro&#39; para iniciar el análisis. </li><li> Introduzca el valor de retorno de la inversión de diámetro y seleccione el valor umbral del filtro (ya sea 0.05 o 0.01). </li><li> Haga clic en la casilla de verificación &#39;tratamiento contra las drogas&#39; y escriba los valores de los puntos de tiempo se añadió el fármaco inmediatamente antes y después (en segundos). </li><li> Haga clic en &#39;ok&#39;, yentrar en el directorio de secuencias de imágenes en la ventana del explorador de archivos. </li></ol> 3. Salida gráfica <ol><li> Descargar R versión 3.0.2 de <a href="http://www.r-project.org/">http://www.r-project.org/</a> . </li><li> Abra la versión de 32 bits de R. </li><li> Haga clic en &quot;Archivo&quot;, luego &quot;guión abierto &#39;y seleccione el script de R&#39; traceplot.R &#39;para la generación de informes de experimentación gráfica. </li><li> Instale la calibración, y los paquetes gplots seleccionando &quot;paquetes&quot;, &quot;instalar paquetes&quot;, y la selección de los paquetes appropirate. </li><li> Haga clic en &#39;Archivo&#39; y luego &#39;cambio de directorio &quot;de comandos, y el uso de la ventana del explorador para seleccionar el directorio que corresponde a la salida análisis LC_Pro. </li><li> Haga clic en la ventana de guión y luego el &#39;run all&#39; comando para ejecutar el script. </li></ol>

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Los autores no tienen nada que revelar.

Descifrando complejos Ca 2 + señales a nivel celular y pluricelular requerirá enfoques experimentales y analíticos rigurosos. Aquí, un enfoque se describe en el cual las secuencias de imagen confocal resueltos de Ca 2 + dependientes de fluorescencia se someten a un análisis automatizado que identifica y cuantifica Ca 2 + señales estadísticamente relevantes dentro de campos celulares intactas en el caso concreto que se presentan, un segmento de la arteria se aisló de corazón de c...

Ca 2 + es un segundo mensajero ubicua molécula de señalización y citosólica de Ca 2 + niveles están muy regulados. Intracelular de Ca 2 + señales son complejas e incluyen transitorios aislados, oscilaciones y ondas que se propagan 1-4. Control espacial y temporal de Ca 2 + se cree que subyacen especificidad señal fisiológica, y por lo tanto el análisis de Ca 2 + patrones de señales es de gran interés para los investigadores en múltiples campos 5. Colorantes Ca 2 + indicador como Fluo-4 y Fura-2 se emplean comúnmente para medir Ca 2 + señales intracelulares con microscopía de fluorescencia 5-12. Por lo general, temporales Ca 2 + señales se evalúan los cambios dependientes del tiempo en medio de fluorescencia dentro de un área definida por el usuario, o región de interés (ROI) 5,6,13 - 16. Actualmente, el análisis ROI manual es a la vez tiempo y mano de obra enintensivo, ya que requiere que los usuarios identifiquen muchas regiones de interés y realizar cálculos repetitivos 17-19. Estas técnicas también pueden estar sujetos a considerables errores de los usuarios, incluida la introducción de modos de señal artificiales y la exclusión de las señales difusas 18,20 baja amplitud o. Algoritmos automatizados de detección de ROI se han aplicado anteriormente usando una variedad de métodos estadísticos para determinar la colocación óptima retorno de la inversión, pero por lo general se han limitado al análisis de exploración de línea o pseudo-line imágenes de exploración, lo que restringe el análisis a una sola dimensión espacial en el tiempo 17, 19-22. Además, muchos algoritmos existentes no son suficientes para abarcar la diversidad de Ca 2 + de liberación eventos que van desde, transientes periódicos localizados a las ondas que se propagan a 23,24. Evaluación integral de Ca 2 + señales fisiológicas a menudo se complica aún más por la presencia de Artif imagen significativaacto que confunde señal a ruido de la discriminación en muchos sistemas experimentales. Anteriormente, una solución algoritmo de detección de ROI automatizado para Ca 2 + señal de detección de transitorios, implementado como un plug-in para el software ImageJ NIH (Institutos Nacionales de Salud, Bethesda, MD), fue desarrollado y validado 25,26. Este algoritmo, llamado LC_Pro, fue diseñado para identificar y analizar los ROIs que abarca Ca 2 + transitorios de señal en secuencias de imágenes de lapso de tiempo de dos dimensiones. Aquí se proporciona un protocolo experimental práctico y demostración representativa de una aplicación del algoritmo en el endotelio de la arteria coronaria porcina, con post-procesamiento adicional utilizando el código abierto estadística software de procesamiento de R para generar una salida gráfica utilizable.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissection dish</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#08-772-70</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polydimethylsiloxane (PDMS)</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#NC9644388</td>
			<td>elastomer kit, must be molded into dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES-buffered PSS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">#H3375-250G</td>
			<td>HEPES acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">stereomicroscope</td>
			<td style="width:71px;">Nikon Inst.</td>
			<td style="width:119px;">#MNA42000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#11223-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">spring scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tungsten wire</td>
			<td style="width:71px;">Scientific Inst Svcs</td>
			<td style="width:119px;">#406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 AM</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#F-14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pluronic F-127</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#P3000MP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">metal pins</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cover-glass bottom chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Custom designed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spinning disc confocal microscope</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td style="width:119px;">RS-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imageJ software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/download.html">download at: http://rsbweb.nih.gov/ij/download.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC_Pro plugin for imageJ</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html">download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://www.r-project.org/">download at: http://www.r-project.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R traceplot script</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing">download at: https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing</a></td>
		</tr>
	</tbody>
</table>

automated analysis of dynamic ca2+ signals in image sequences

Intracelular de Ca 2 + señales se estudian comúnmente con colorantes fluorescentes Ca 2 + indicadores y técnicas de microscopía. Sin embargo, el análisis cuantitativo de Ca 2 + de datos de formación de imágenes es mucho tiempo y sujetos a sesgo. Algoritmos de análisis de señales automatizadas basadas en región de interés (ROI) de detección han sido implementados para las mediciones de barrido línea unidimensional, pero no hay ningún algoritmo actual que integra la identificación optimizado y el análisis de rendimiento de la inversión en secuencias de imágenes bidimensionales. Aquí se describe un algoritmo para la rápida adquisición y el análisis de rendimiento de la inversión en secuencias de imágenes. Utiliza elipses ajustan a las señales filtradas de ruido con el fin de determinar la colocación óptima retorno de la inversión, y calcula Ca 2 + parámetros de la señal de amplitud, duración y amplitud espacial. Este algoritmo fue implementado como un plugin disponible gratuitamente para ImageJ (NIH) de software. Junto con guiones escritos para el análisis de código abierto de software de procesamiento estadístico R,este enfoque proporciona una tubería de alta capacidad para llevar a cabo el análisis estadístico rápida de la producción experimental. Los autores sugieren que el uso de este protocolo de análisis dará lugar a una caracterización más completa y no sesgada de fisiológica Ca 2 + señalización.

Un algoritmo personalizado, LC_Pro, fue desarrollado e implementado con el fin de realizar el análisis automatizado de Ca 2 + en la dinámica de las secuencias de imagen confocal. Como se muestra en la Figura 1, el algoritmo utiliza los módulos de procesamiento secuencial que A) detectar y yacimientos de huellas de dinámica Ca 2 + cambiar encima estadística (p &lt;0,01) el ruido, B) definir regiones de interés (ROI) de forma automática en los centros del sitio activo, y C) el...

Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Aquí una novela región de interés protocolo de análisis basado en la clasificación de las elipses de mejor ajuste asignados a las regiones de la señal positiva dentro de secuencias de imágenes lapso de tiempo de dos dimensiones se demuestra. Este algoritmo puede permitir a los investigadores a analizar exhaustivamente las señales + Ca 2 fisiológicas con intervención mínima del usuario y el sesgo.

Análisis automatizado de Dinámica Ca<sup&gt; 2 +</sup&gt; Las señales en secuencias de imágenes

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

automated-analysis-of-dynamic-ca2-signals-in-image-sequences

Research

JoVE Journal

Biology

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

17.0K vistas.  University of South Alabama.  Aquí una novela región de interés protocolo de análisis basado en la clasificación de las elipses de mejor ajuste asignados a las regiones de la señal positiva dentro de secuencias de imágenes lapso de tiempo de dos dimensiones se demuestra. Este algoritmo puede permitir a los investigadores a analizar exhaustivamente las señales + Ca 2 fisiológicas con intervención mínima del usuario y el sesgo. 

Video: Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Aplicación de curado con luz Resina Adhesivo Dental para los electrodos de montaje o sondas de microdiálisis en experimentos crónica

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Investigación

Biología

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semi-automatizado de análisis óptico latido de los corazones pequeños

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robótica y Análisis de Imagen Dinámica de Estudios de la expresión génica en los tejidos vegetales

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Desarrollo de la imagen y el análisis automatizado de pantallas de química de pez cebra.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de Imagen Ca 2 + Señalización en el esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash etiquetado y análisis de imágenes de las proteínas en las células del sensor Tetracysteine

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medición Espacial y Temporal Ca<sup&gt; 2 +</sup&gt; Las señales en plantas de Arabidopsis

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Análisis automatizado de Dinámica Ca

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