Este trabajo fue apoyado por el Instituto Nacional del Ojo Grant 1 R15 EY13649-01, y gracias a una beca descubrimiento individual de las Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC). Un agradecimiento especial a Deda Gillespie para el mecanizado de metales, y para su consulta en el diseño del dispositivo y la construcción.

<ol>
	<li>Goldreich, D. &. a. m. p. ;. a. m. p., Kanics, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+acuity+is+enhanced+in+blindness.">Tactile acuity is enhanced in blindness.</a> J. Neurosci. 23, 3439-3445 (2003).</li><li>Goldreich, D. &. a. m. p. ;. a. m. p., Kanics, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+blind+and+sighted+humans+on+a+tactile+grating+detection+task.">Performance of blind and sighted humans on a tactile grating detection task.</a> Percept. Psychophys. 68, 1363-1371 (2006).</li><li>Kontsevich, L. L. &. a. m. p. ;. a. m. p., Tyler, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+adaptive+estimation+of+psychometric+slope+and+threshold.">Bayesian adaptive estimation of psychometric slope and threshold.</a> Vision. Res. 39, 2729-2737 (1999).</li><li>Bleyenheuft, Y., Cols, C., Arnould, C. &. a. m. p. ;. a. m. p., Thonnard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+tactile+spatial+resolution+from+6+to+16+years.">Age-related changes in tactile spatial resolution from 6 to 16 years.</a> Somatosens. Mot. Res. 23, 83-87 (2006).</li><li>Johnson, K. O. &. a. m. p. ;. a. m. p., Phillips, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+spatial+resolution.+I.+Two-point+discrimination,+gap+detection,+grating+resolution,+and+letter+recognition.">Tactile spatial resolution. I. Two-point discrimination, gap detection, grating resolution, and letter recognition.</a> J. Neurophysiol. 46, 1177-1192 (1981).</li><li>Gibson, G. O. &. a. m. p. ;. a. m. p., Craig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+force+and+conformance+on+tactile+intensive+and+spatial+sensitivity.">The effect of force and conformance on tactile intensive and spatial sensitivity.</a> Exp. Brain Res. 170, 172-181 (2006).</li><li>Johnson, K. O. &. a. m. p. ;. a. m. p., Phillips, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotating+drum+stimulator+for+scanning+embossed+patterns+and+textures+across+the+skin.">A rotating drum stimulator for scanning embossed patterns and textures across the skin.</a> J. Neurosci. Methods. 22, 221-231 (1988).</li><li>Craig, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grating+orientation+as+a+measure+of+tactile+spatial+acuity.">Grating orientation as a measure of tactile spatial acuity.</a> Somatosens. Mot. Res. 16, 197-206 (1999).</li><li>Killebrew, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dense+array+stimulator+to+generate+arbitrary+spatio-temporal+tactile+stimuli.">A dense array stimulator to generate arbitrary spatio-temporal tactile stimuli.</a> J. Neurosci. Methods. 161, 62-74 (2007).</li></ol>

La entrega controlada de estructura espacial estímulos mecánicos plantea desafíos no se enfrentan en la entrega de estímulos visuales o auditivos, por lo que puede ser el equipo disponible en el mercado (pantallas de ordenador, los altavoces de audio) que se utiliza. Por esta razón, muchos táctil experimentos psicofísicos se siguen haciendo con la entrega de estímulo manual. La entrega de manuales de los estímulos táctiles es mucho tiempo, y requiere mucho cuidado y concentración por parte del investigador. Por ejemplo, Bleyenheuft et al. 4 indica que &quot;la aplicación Manual de aproximadamente 1.2 mm de deformación de piel perpendicular fue utilizado ... el examinador estaba particularmente atento a evitar cualquier esfuerzo de corte entre la piel y la reja que puedan distorsionar la medida. .. &quot; Tal vez debido a la entrega de estímulo manual es relativamente lenta e intensa concentración, muchos estudios con este método han utilizado sí / no (sólo un estímulo por ensayo) en lugar de dos protocolos de intervalo elección forzada. Desafortunadamente, incluso cuando se tiene mucho cuidado, la entrega de estímulo manual de las hojas de muchos parámetros de estímulo no controlado. Estos parámetros incluyen la fuerza de estímulo, la velocidad de aparición, duración y la estabilidad de la superficie de estímulo en la piel. De estos parámetros, quizás el mejor estudiado con respecto a su influencia en el rendimiento es la fuerza del estímulo. Los seres humanos muestran una mejoría progresiva de la discriminación rejilla de orientación en la punta de los dedos como los cambios profundidad de penetración 500-1200 5 micrones, el rendimiento es mejor a la fuerza de 10 gramos que en 50 gramos de fuerza 1, aunque no mejora se observa entre 50 gramos y 200 gramos 6. Para otras aplicaciones, como la detección de rejilla (distinguiendo a suave de una superficie estriada), el aumento de la fuerza aumenta significativamente el rendimiento en todo el rango de 10 a 200 gramos 2,6. Para superar estos retos asociados con las pruebas manuales, se desarrolló el sistema automatizado, TAPS. Nuestro objetivo en los grifos de construcción fue la creación de un dispositivo sencillo, seguro, versátil, eficiente y económico para controlar la aplicación estímulo táctil. El uso controlado de la gravedad es una manera sencilla y segura de aplicar un estímulo táctil. La seguridad está garantizada, porque la fuerza contra la piel no puede exceder de la gravedad. El actuador lineal de gran alcance sólo sirve para la transferencia de la fuerza de gravedad sobre la piel, y luego de retirar el contacto. El dispositivo es muy versátil, ya que se acomoda a cualquier estímulo superficies mecanizadas en (o adjuntas a) los extremos de media pulgada de diámetro, las barras, y puede ser programado para ejecutar una variedad de protocolos psicofísicos. TAPS es eficiente, ya que implementa rápidamente los estímulos, y puede funcionar para ahorrar tiempo los algoritmos de adaptación. Por último, los componentes de los grifos están al alcance de los titulares de pequeñas donaciones. Los motores de un costo aproximado de $ 1,500, los componentes eléctricos (con exclusión de la computadora, pero incluyendo las tarjetas PCI y el controlador de motor paso a paso), aproximadamente 6.000 dólares. Una máquina de mini-molienda y accesorios se pueden comprar por aproximadamente $ 1,000, o un mecánico profesional puede ser contratado a planta de las piezas de estímulo y hacer otras piezas pequeñas. Esperamos que este sistema va a ser replicado por otros, y que sirven para promover el avance hacia pruebas de control táctil, que en los últimos años se han hecho progresos prometedores 5,7,8,9.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Delrin plastic rods 0.5&#x201D; diameter 3&#x201D; long </td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>ZRD-08</td>
<td>Rods from which the stimulus pieces are made</td>
</tr>
<tr>
<td>Chrome-steel bearing, 0.5" bore diameter</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>BR-08-01</td>
<td>Pivot for the rotating rod</td>
</tr>
<tr>
<td>Plastic bearing, 0.5&#x201D; bore, 1.125&#x201D; outer diameter, 0.25&#x201D; thick</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>BRP-08-01</td>
<td>Wheel at end of rotating rod</td>
</tr>
<tr>
<td>Fiberglass curtain, 0.75&#x201D; thick</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>9781T83</td>
<td>Sound absorbing curtain around lower table</td>
</tr>
<tr>
<td>Silicone O-rings 5/16" inside diameter</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>AS568A- 011</td>
<td>To reduce vibration of stimulus pieces</td>
</tr>
<tr>
<td>Anti-Vibration Pads 2"x 2", 93 PSI max </td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>60105K61</td>
<td>Put under the linear actuator</td>
</tr>
<tr>
<td>Rubber sandwich mounts 1/2" H x 3/8" W </td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>9378K11</td>
<td>Put under linear actuator base</td>
</tr>
<tr>
<td>Stepper motor Nema 23, 3-stack </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>S23</td>
<td>For rotating the stimulus disk</td>
</tr>
<tr>
<td>Linear rodless actuator</td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>R2S23N-105A-18-l-M35M</td>
<td>To control rotating bar movement</td>
</tr>
<tr>
<td>1 Hall effect switch &#x201C;normally open&#x201D; config. </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>RP1</td>
<td>Actuator home sensor</td>
</tr>
<tr>
<td>2 Hall effect switches &#x201C;normally closed&#x201D; config. </td>
<td>&nbsp;</td>
<td>Industrial Devices</td>
<td>RP2</td>
<td>Actuator limit sensors</td>
</tr>
<tr>
<td>Micro switch</td>
<td>&nbsp;</td>
<td>Honeywell</td>
<td>FSG15N1A</td>
<td>Finger force sensor</td>
</tr>
<tr>
<td>Retro-reflective sensor</td>
<td>&nbsp;</td>
<td>Honeywell</td>
<td>FE7B-RB6VG-M</td>
<td>Piece position sensor</td>
</tr>
<tr>
<td>Motor controller board, closed-loop control</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-step-4CX</td>
<td>Mediates communication between computer and Nudrive</td>
</tr>
<tr>
<td>Nudrive stepper motor driver</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>Nudrive 4SX-211</td>
<td>Sends command voltages to the linear actuator and disk stepper motors</td>
</tr>
<tr>
<td>Data acquisition board</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>PCI-MIO-16E-1</td>
<td>Reads finger force sensor</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>&nbsp;</td>
<td>Programming language</td>
</tr>
<tr>
<td>Valuemotion library</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>&nbsp;</td>
<td>Motor control routines</td>
</tr>
<tr>
<td>Photoelectric sensor</td>
<td>&nbsp;</td>
<td>Panasonic</td>
<td>PM-K53-C1</td>
<td>Disk home sensor</td>
</tr>
<tr>
<td>Mini milling machine</td>
<td>&nbsp;</td>
<td>Sherline</td>
<td>2010-DRO</td>
<td>For machining the stimulus surfaces</td>
</tr>
<tr>
<td>Ohaus precision mass set</td>
<td>&nbsp;</td>
<td>Edmund Scientific</td>
<td>&nbsp;</td>
<td>Mass on rotating bar</td>
</tr>
<tr>
<td>Parrish Magic Line aluminum cake pan bottom, 9&#x201D; diam.</td>
<td>&nbsp;</td>
<td>Sur La Table</td>
<td>&nbsp;</td>
<td>Rotating disk machined with square cut-outs to hold the stimulus pieces</td>
</tr>		</tbody>
		</table>
		</div>

a tactile automated passive-finger stimulator (taps)

A pesar de las pruebas de agudeza táctil espacial se utilizan en investigación en neurociencias y la evaluación clínica, algunos dispositivos automatizados existentes para la entrega de estímulos controlados estructura espacial de la piel. En consecuencia, los investigadores a menudo se aplican estímulos táctiles de forma manual. La aplicación manual de estímulo es mucho tiempo, requiere mucho cuidado y concentración por parte del investigador, y deja muchos parámetros de estímulo no controlado. Se describe aquí un sistema controlado por ordenador estímulo táctil, lo táctil automático pasiva dedo estimulador (TAPS), que se aplica espacialmente los estímulos estructurados para la piel, el control de la velocidad de inicio, la fuerza de contacto, y la duración del contacto. TAPS es un sistema versátil y programable, capaz de llevar a cabo de manera eficiente una gran variedad de procedimientos psicofísicos. Se describen los componentes de grifería, y mostrar cómo GRIFOS se utiliza para administrar un intervalo de dos pruebas de elección forzada táctil orientación rejilla. Correspondientes Autor: Daniel Goldreich

Watch this Scientific Journal Video about A Tactile Automated Passive-Finger Stimulator TAPS at JoVE.com

A Tactile Automated Passive-Finger Stimulator TAPS

Se describe un dispositivo controlado por ordenador para investigar el sentido del tacto: la táctil automático pasiva dedo estimulador (TAPS). Se describen los componentes de grifería, y mostrar cómo GRIFOS se utiliza para administrar un intervalo de dos pruebas de elección forzada táctil orientación rejilla.

A táctil automático pasivo-Finger Stimulator (TAPS)

Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering ...

a-tactile-automated-passive-finger-stimulator-taps

Research

JoVE Journal

Biology

A Tactile Automated Passive-Finger Stimulator (TAPS)

13.6K vistas.  Duquesne University. Se describe un dispositivo controlado por ordenador para investigar el sentido del tacto: la táctil automático pasiva dedo estimulador (TAPS). Se describen los componentes de grifería, y mostrar cómo GRIFOS se utiliza para administrar un intervalo de dos pruebas de elección forzada táctil orientación rejilla.

Video: A Tactile Automated Passive-Finger Stimulator TAPS

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 ...

Investigación

Biología

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Microfluídica digitales para el procesamiento automatizado de Proteómica

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

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.

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 ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Imaging semi-automatizado de fluorescencia específica de tejido en embriones de pez cebra

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

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+ ...

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 ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Open Source de análisis de contenidos de alta Utilizando microscopía de fluorescencia automatizado de imágenes por vida

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Un Protocolo Automatizado Rápido de Fibra Análisis Población en músculo de rata Secciones representativas Uso de la cadena pesada de miosina inmunohistoquímica

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Medición de la confluencia de iPSCs utilizando un sistema automatizado de imágenes.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...