Prueba de calor del dolor mediante la Thermostimulator TSA II 1. Introducción <ol><li> Este procedimiento utiliza la TSA II, disponible en el mercado un termo-sensorial analizador que ofrece estímulos de calor a la piel con una thermode (Medoc Advanced Medical Systems, Durham, NC). Una unidad central genera y transmite la señal a la thermode. Esta señal es controlada por el software propietario normalmente se almacena en un ordenador portátil conectado a la unidad central. También se adjunta a la unidad central es un control de mano que permite a un objeto de estudio para detener el calentamiento de la thermode. </li><li> Siete estímulos de calor y secuencial idéntico se entregan, con intervalos entre estímulos de 30 años. La temperatura basal del thermode se fija en un cómodo 35 º C. La velocidad a la que se calienta thermode se fija en 1 º C / s, mientras que la velocidad a la que se enfría es de 10 º C / s. La temperatura máxima se fija en thermode 52 º C. </li></ol> 2. Preparación y capacitación de los sujetos <ol><li> Para preparar los temas para el asiento de Ensayo térmico dolor ellos cómodamente en una silla reclinable que provee soporte para la cabeza, brazos y piernas. La silla también debe permitir un fácil acceso a los sitios del cuerpo examinado. </li><li> La instalación de prueba debe estar a una temperatura ambiente confortable, y proporcionar un ambiente tranquilo y neutral, sin distracciones. </li><li> Antes de comenzar el experimento, los participantes deberán estar bien entrenados en todos los aspectos de las pruebas de dolor por calor. La formación puede tener lugar en cualquier momento antes del experimento. </li><li> Las interacciones entre los sujetos de estudio y los investigadores deben limitarse a las necesarias para la recogida de datos. Bromas y charlas se desaniman porque puede comprometer la coherencia de los datos. Los sujetos del estudio se informó que las interacciones intencionalmente será un poco monótona y neutral durante los experimentos. </li><li> Mantenga las instrucciones claras, simples y consistentes. Haga hincapié en que el dolor es subjetivo, y no hay respuestas correctas o incorrectas. Lo más importante es ser consistente. Definir el umbral del dolor de calor como el punto entre &quot;estar a punto de ser doloroso&quot; y &quot;sólo se convirtió en dolor&quot;. </li><li> Es muy importante que un sujeto sabe con precisión qué esperar durante una sesión de pruebas. Cualquier &quot;sorpresas&quot; pueden resultar en menor cantidad de datos consistente, ya que los factores de confusión como la ansiedad puede ser generada. Por ejemplo, si siete estímulos dolorosos calor se utilizan para determinar el umbral del dolor de calor, el sujeto debe saber esto. </li><li> En cualquier momento, el sujeto puede detener el thermode de conseguir más caliente pulsando el botón de un control de mano. Tener el control se reduce la ansiedad del sujeto y que los datos sean más fiables. </li><li> La sesión de entrenamiento ofrece una oportunidad para evaluar el rendimiento de un sujeto del estudio. Puede ser mejor para entrenar a los sujetos durante dos a cuatro ciclos de prueba para sus umbrales de dolor individuales varían en no más de 0,8 ° C, e idealmente no más de 0,6 ° C entre los ciclos de prueba los dos últimos. Los sujetos que no cumplan consistentemente suelen ser excluidos del estudio. </li></ol> 3. Determinar el umbral del dolor <ol><li> Para comenzar los ciclos de pruebas de umbral de dolor marca la ubicación de la thermode en la piel del sujeto. En este experimento la misma lesión inflamación de la piel utilizada para microdiálisis se pondrá a prueba para el dolor para aprender acerca de bioquímicos libera en respuesta a las pruebas de dolor. </li><li> Luego, comenzar cada ciclo mediante la colocación de la thermode en el área de la piel marcada. Coinciden con la posición de la thermode con las marcas en la mayor medida posible. Deje que la temperatura entre la piel y thermode la que se equilibre a 35 ° C - una temperatura que con seguridad no causará dolor. Esto tomará alrededor de 5 segundos y se indicará con una lectura de 35 ° C en la pantalla del ordenador. </li><li> Antes de comenzar, dar el control de mano para el tema. </li><li> Use una presión constante y suave para mantener toda la superficie de la thermode en pleno contacto con la piel del sujeto durante la prueba. </li><li> Ahora inicie el algoritmo de prueba pre-programados. El thermode comenzará a calentar hasta a 1 ° C / s. Tan pronto como el sujeto experimenta el dolor, van a presionar el dispositivo de mano. </li><li> Después de pulsar el botón en el dispositivo de mano, el dolor que causa la temperatura thermode se registrará automáticamente y comenzará a disminuir a un ritmo de 10 ° C / s hasta su temperatura de referencia de 35 ° C. </li><li> La sonda de temperatura de forma automática se mantendrá a 35 ° C durante 30 s antes de que comienza a elevarse de nuevo en 1 º C / s, hasta un tema nuevo pulsa el botón del dispositivo de mano. Justo antes de que la temperatura thermode está a punto de aumentar de nuevo, recordar el tema que está a punto de suceder. Esto evitará que el objeto enfocado. </li><li> Esta secuencia se repite hasta siete registros de temperatura se han registrado. Calcular el promedio de estas temperatura lecturas, lo que es el umbral del dolor de calor. </li></ol> Prueba de dolor mecánico con sondas de presión puntuado 1. Introducción <ol><li> Usamos encargo calibrado puntuado sondas de presión, ya que proporcionan resultados más consistentes que las tradicionalmente utilizadas y disponibles en el mercado de von Frey pelos. Las sondas están hechas de cables idénticos cilíndrica de acero inoxidable (240 m de diámetro) montados sobre varillas de cobre de diferentes pesos (1, 2, 4, 8, 16, 32, 49, 64 y 81 gramos) que se mueven libremente dentro de una amplia mano lugar del tubo de aluminio. </li><li> Las mismas condiciones experimentales generales descritos para las pruebas de calor el dolor se aplican a las pruebas de dolor mecánico. Los temas deben estar bien preparados y entrenados. Es especialmente importante para tranquilizar a los sujetos que las sondas son contundentes y no se perfora la piel. Esto ayudará a los sujetos para relajarse y proporcionar datos coherentes. </li></ol> 2. Determinar el umbral de dolor mecánico <ol><li> Para determinar el umbral de dolor mecánico, aplicar primero la punta del alambre de acero de la más ligera de la sonda perpendicular a la piel en el centro de la lesión experimental inflamada. </li><li> El tubo de aluminio de mano se hace descender hacia la piel, pero sin tocarlo. De esta manera, todo el peso de la varilla de cobre en el interior del tubo de aluminio se apoya en la punta del alambre de acero. </li><li> El objeto de estudio se indica si la sonda produce dolor al decir &quot;sí&quot; o &quot;no&quot;. </li><li> Si el sujeto responde &quot;no&quot;, ya sea la misma sonda o la sonda más pesada que viene será utilizado siguiente. Este algoritmo se llama una secuencia de estimulación ascendente. Al azar determinar si la próxima sonda es más pesado o del mismo peso. Esto evitará que un sujeto ciego con el algoritmo. </li><li> Aplicar las sondas en orden ascendente hasta que un sujeto responde &quot;sí.&quot; El peso promedio de los últimos dos sondas de la serie se registra como el punto de deflexión de primera. </li><li> Después de que el sujeto responde &quot;sí&quot;, empiece a usar una secuencia de estimulación descendente, y utilizar una sonda o la sonda más ligero que antes. Al azar determinar si la próxima sonda es más claro o más del mismo peso. Esto evitará que un sujeto ciego con el algoritmo. </li><li> Aplicar las sondas en una secuencia descendente hasta que el sujeto responde &quot;no&quot;. El peso promedio de los últimos dos sondeos en esta secuencia constituye el punto de deflexión de segundo. </li><li> Cuando el sujeto responde &quot;no&quot;, volver a una secuencia de estimulación ascendente. Alternar entre ascendientes y descendientes hasta las siete secuencias de puntos de desviación se registran. El promedio de estos siete puntos es el umbral de dolor mecánico. </li></ol>

<ol>
	<li>Angst, M. S., Clark, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+Koltzenburg+et+al.:+Differential+sensitivity+of+three+experimental+pain+models+in+detecting+the+analgesic+effects+of+transdermal+fentanyl+and+buprenorphine.">Comment on Koltzenburg et al.: Differential sensitivity of three experimental pain models in detecting the analgesic effects of transdermal fentanyl and buprenorphine.</a> Pain. 126, 165-174 (2006).</li><li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation,+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: A microdialysis study.</a> Pain. , (2008).</li><li>Angst, M. S., Koppert, W., Pahl, I., Clark, J. D., Schmelz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+infusion+of+the+mu-opioid+agonist+remifentanil+in+humans+causes+hyperalgesia+during+withdrawal.">Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal.</a> Pain. 106, 49-57 (2003).</li><li>Lotsch, J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mu-opioid+agonist+remifentanil+attenuates+hyperalgesia+evoked+by+blunt+and+punctuated+stimuli+with+different+potency:+a+pharmacological+evaluation+of+the+freeze+lesion+in+humans.">The mu-opioid agonist remifentanil attenuates hyperalgesia evoked by blunt and punctuated stimuli with different potency: a pharmacological evaluation of the freeze lesion in humans.</a> Pain. 102, 151-161 (2003).</li></ol>

Este video muestra los algoritmos para evaluar el calor y el umbral de dolor mecánico en la piel inflamada de voluntarios humanos. La percepción del dolor es subjetiva. Recogida de datos adecuado del dolor requiere una instalación de prueba adecuados (por ejemplo, tranquilo y confortable), la formación completa de los participantes mediante la incorporación de medidas de control de calidad de datos (test-retest), la estricta adhesión a un protocolo de estudio estandarizado, y la evitación de factores de confusión (por ejemplo, estrés, ansiedad). Fiables los datos subjetivos de la sensibilidad al dolor, junto con intervenciones como la administración de un fármaco analgésico y la recolección de datos de mecanismo como la liberación local de mediadores nociceptivos proporcionan información valiosa sobre los mecanismos del dolor, y los medios para prevenir o atenuar la misma.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>TSA-II Neuro Sensory Analyzer</td>
<td>Tool</td>
<td>Medoc Advanced Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Puntuated Pressure Probes</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>Alternatively, commercially available von Frey Hairs can be used.</td>
</tr>		</tbody>
		</table>
		</div>

determining heat and mechanical pain threshold in inflamed skin of human subjects

En un artículo anterior en el Diario de los experimentos visualizados hemos demostrado las técnicas de la piel de microdiálisis para la recogida de tejidos específicos bioquímicos nociceptivas e inflamatorias en los seres humanos. En este artículo vamos a mostrar las pruebas de dolor paradigmas que se utilizan a menudo en combinación con procedimientos de microdiálisis. La combinación de pruebas de dolor con microdiálisis proporciona el vínculo crítico entre los datos de comportamiento y la bioquímica que permite la identificación de sustancias bioquímicas clave responsables de la generación y propagación de dolor. Dos modelos de dolor que evocan en la piel inflamada de los participantes humanos se muestran. El primer modelo provoca dolor con la ayuda de estímulos de calor. El dolor evoca el calor como se describe aquí es predominantemente mediada por pequeñas, no mielinizadas fibras nerviosas periféricas nociceptivas (fibras C). El segundo modelo provoca dolor a través de estímulos de presión marcada. Dolor marcada presión evocados es predominantemente mediada por pequeñas fibras mielínicas de los nervios periféricos nociceptivos (fibras A-delta). Los dos modelos son mecánicamente distinta e independiente examine el procesamiento nociceptivo por las dos principales poblaciones periféricas de las fibras nerviosas implicadas en la señalización del dolor. El dolor se evoca el calor con la ayuda de la TSA II, disponible en el mercado un termo-sensorial analizador (Medoc Advanced Medical Systems, Durham, NC). Configuración del estímulo y la entrega se maneja con la ayuda de software específico. Thermodes varían en tamaño y forma, pero en principio, por una placa de metal que pueden ser calentados o enfriados a distintas velocidades y por diferentes períodos de tiempo. Algoritmos de evaluación de calor provocado dolor son múltiples. En los experimentos se muestra aquí, los participantes del estudio se les pidió que indicaran en qué momento comienza a experimentar dolor mientras que la thermode en contacto con la piel se calienta a una velocidad predeterminada a partir de una temperatura que no provoca dolor. La temperatura thermode en la que un sujeto comienza a experimentar el dolor constituye el umbral del dolor de calor. Dolor mecánico se evoca con sondas puntuado. Estas sondas están disponibles en el mercado de varios fabricantes (filamentos de von Frey). Sin embargo, la precisión de von Frey pelos ha sido criticado y muchos investigadores utilizan encargo marcado sondas de presión. En los experimentos que se muestran aquí ocho sondas de medida marcada de diferentes pesos se aplican en orden consecutivo, un procedimiento llamado algoritmo de arriba-abajo, para identificar los puntos de percepción de desviación, es decir, un cambio de no sentir dolor para sentir dolor, o viceversa. El peso medio causando una deformación perceptiva constituye el umbral de dolor mecánico.

Watch this Scientific Journal Video about Determining heat and mechanical pain threshold in inflamed skin of human subjects at JoVE.com

Determining heat and mechanical pain threshold in inflamed skin of human subjects

Algoritmos de evaluación de los umbrales de dolor mecánico y el calor en la piel inflamada forma experimental los sujetos de estudio humanos se muestran. Los dos paradigmas dolor prueba independiente examine el procesamiento nociceptivo por las dos principales poblaciones periféricas de fibras nerviosas de la transmisión del dolor, es decir, no mielinizadas fibras C y pequeñas mielinizadas fibras A-delta.

Determinar el calor y el umbral de dolor mecánico en la piel inflamada de los sujetos humanos

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

determining-heat-mechanical-pain-threshold-inflamed-skin-human

Research

JoVE Journal

Biology

20.7K vistas.  Stanford University School of Medicine. Algoritmos de evaluación de los umbrales de dolor mecánico y el calor en la piel inflamada forma experimental los sujetos de estudio humanos se muestran. Los dos paradigmas dolor prueba independiente examine el procesamiento nociceptivo por las dos principales poblaciones periféricas de fibras nerviosas de la transmisión del dolor, es decir, no mielinizadas fibras C y pequeñas mielinizadas fibras A-delta.

Video: Determining heat and mechanical pain threshold in inflamed skin of human subjects

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Utilizando Micro-Electro-Mechanical Systems (MEMS) para desarrollar herramientas de diagnóstico

Investigación

Biología

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Trasplante de piel murina

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantación de la ingeniería tisular en el corazón de rata

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

Demostración de La alodinia cutánea En asociación con el dolor pélvico crónico

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

Digital Inline holográfica Microscopía (MHDL) de sujetos débilmente dispersión de

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagen confocal de Soltero mitocondriales superóxido Parpadea en corazón intacto o<em&gt; En Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex vivo Cultura de embriones de ratón con la piel y en vivo de imágenes de melanoblasto Migración

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Brecha ocasiones intercelular de comunicación: un biomarcador funcional para evaluar los efectos adversos de las sustancias tóxicas y toxinas, y de Salud Beneficios de Productos Naturales

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

Un método basado en la flotabilidad de la determinación de los niveles de grasa en<em&gt; Drosophila</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...