Inyecciones de vena de la cola de los tintes fluorescentes dextranos <ol><li> Con el fin de la vasculatura cerebral imagen, los animales deben ser inyectadas por vía intravenosa con un tinte fluorescente. Usamos dextrano conjugado con tintes porque la fracción de dextrano impide que el tinte de cruzar la barrera sangre-cerebro y la fuga de los vasos sanguíneos. </li><li> Los ratones son anestesiados con isoflurano (4% para la inducción, 1,5-2% durante la inyección). </li><li> La cola es desinfectada con alcohol al 70%. </li><li> Con una aguja de calibre 26, 75-100 l de un 5% v / v de rodamina dextrano disuelto en solución salina se inyecta a través de la vena de la cola, a medio camino a lo largo del eje de la cola. </li><li> Los animales pueden ser reflejados inmediatamente después de la inyección vena de la cola hasta que el colorante se excreta en la orina, que se vuelven de color rosa si se utiliza un colorante rodamina en alrededor de 2 horas. </li></ol> Imágenes del flujo sanguíneo utilizando microscopía de dos fotones (la duración total de 30-60 minutos por sesión, dependiendo del número de buques de imágenes) <ol><li> Después de la inyección de dextrano tinte fluorescente, el ratón es anestesiados con isoflurano (4% para la inducción, 1-1.5% de imagen). </li><li> Ratón está bien conectado con la barra de titanio a la platina del microscopio, que contiene una almohadilla térmica termo-regulado para mantener al animal caliente. Algunos ungüento oftálmico se aplica para mantener los ojos húmedos. </li><li> La cubierta de vidrio de la ventana del cráneo se limpia con alcohol al 70%. </li><li> La ventana se coloca paralela al plano focal y se centra en el campo de visión en el objetivo de 4X. </li><li> Lo mejor es tomar una fotografía de los vasos cerebrales superficie con una cámara digital. Esta imagen se utilizará como imagen de referencia en las siguientes sesiones de formación de imágenes para encontrar la zona visualizada en repetidas ocasiones el día a día. </li><li> El objetivo de 4X se reemplaza con el objetivo de inmersión en agua 40 veces sin mover el escenario. Una fotografía digital del campo de visión es tomada de nuevo. Las coordenadas en el manipulador de la etapa se fija en cero. </li><li> Usamos scanimage como el software de adquisición de imágenes. Esto fue escrito en Matlab Tom Pologruto y Bernardo Sabatini en el laboratorio de Karel Svoboda (Pologruto et al., 2003). Pasamos a continuación a todos los otros equipos: el láser, el medidor de potencia, los tubos foto multiplicadores, los amplificadores, etc </li><li> El área de la ventana adecuada para obtener imágenes digitalizadas es brevemente a bajo aumento para encontrar las mejores regiones. Una vez que estos se identifican, una pila de bajo aumento de la región elegida para la imagen está tomada, y sus coordenadas XY se anotan. </li><li> Para grabar la dinámica del flujo sanguíneo en los vasos pequeños o capilares que usamos líneas de barrido a lo largo de por lo menos 40 m de la embarcación de interés. Estos solo &quot;barrido&quot; que dura un segundo se realizan utilizando la potencia del láser como poco como sea posible y las coordenadas y el ángulo de exploración están escritas. </li><li> Una vez que la sesión de imágenes es más, el animal se mueve a una cámara de calentamiento en los que puede recuperarse de la anestesia. </li></ol>

<ol>
	<li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proc Nat Acad Sci. 95, 15741-15746 (1998).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed. Eng. Online. 17, 2-13 (2003).</li><li>Zhang, S., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+impact+of+cortical+microcirculation+on+synaptic+structure+and+sensory-evoked+hemodynamic+responses+in+vivo.">Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo.</a> 5, (2007).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proc Nat Acad Sci. 104, 365-370 (2007).</li></ol>

Cortical dinámica del flujo de sangre se pueden estudiar en vivo por la proyección de imagen fluorescente tintes dextrano se inyecta en la vena de la cola de los roedores con microscopia de dos fotones. Este video muestra un método para saber cómo la dinámica de la imagen del flujo sanguíneo en la corteza cerebral de los ratones a través de una cubierta de vidrio preparación ventana craneal.

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a method for 2-photon imaging of blood flow in the neocortex through a cranial window

La capacidad de la imagen de la vasculatura cerebral (desde los grandes buques a los capilares) y la dinámica de registro de flujo sanguíneo en el cerebro intacto de los roedores que viven es una poderosa técnica. El uso en vivo de 2 fotones microscopía través de una ventana craneal es posible tintes fluorescentes imagen inyecta por vía intravenosa. Esto le permite a uno la imagen de la vasculatura cortical y también para obtener mediciones del flujo sanguíneo. Esta técnica fue desarrollada originalmente por David Kleinfeld y Winfried Denk. El método puede ser usado para estudiar la dinámica del flujo sanguíneo durante o después de la isquemia cerebral, en las enfermedades neurodegenerativas, en los tumores de cerebro, o en la fisiología normal del cerebro. Por ejemplo, se ha utilizado para estudiar cómo se produce movimiento en la dirección de los cambios del flujo sanguíneo y cambios en la velocidad de los glóbulos rojos o de flujo en y alrededor del infarto. Aquí se demuestra cómo utilizar dos fotones microscopía a la dinámica de la imagen del flujo sanguíneo en la corteza cerebral de ratones vivos utilizando colorantes fluorescentes se inyecta en la vena de la cola.

Watch this Scientific Journal Video about A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window at JoVE.com

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

Cortical dinámica del flujo de sangre se pueden estudiar en vivo por la proyección de imagen fluorescente tintes dextrano se inyecta en la vena de la cola de los roedores con dos fotones microscopía. Este vídeo muestra cómo la dinámica de la imagen del flujo sanguíneo en la corteza cerebral de los ratones a través de una cubierta de vidrio preparación ventana craneal.

Un método para el 2-Photon imágenes del flujo sanguíneo en la neocorteza a través de una ventana craneal

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

a-method-for-2-photon-imaging-blood-flow-neocortex-through-cranial

Research

JoVE Journal

Biology

18.7K vistas.  University of California, Los Angeles. Cortical dinámica del flujo de sangre se pueden estudiar en vivo por la proyección de imagen fluorescente tintes dextrano se inyecta en la vena de la cola de los roedores con dos fotones microscopía. Este vídeo muestra cómo la dinámica de la imagen del flujo sanguíneo en la corteza cerebral de los ratones a través de una cubierta de vidrio preparación ventana craneal.

Video: A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

La disección y 2-Photon imagen de los ganglios linfáticos periféricos en ratones

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Investigación

Biología

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

En imágenes in situ del timo de ratón utilizando 2-Photon Microscopía

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

En Vivo 2-Photon imágenes de calcio en la capa 2 / 3 de los ratones

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Generación de médula ósea procedentes de células murinas dendríticas para su uso en dos fotones de imágenes

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

El uso de Rotorod como un método para el análisis cualitativo de la Ruta de la rata

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

Dendra2 Photoswitching a través de la ventana de imágenes mamarias

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Un método simple para imágenes Arabidopsis Uso de hojas perfluorodecalina como un medio de imágenes infiltrativas

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un ensayo de explante para evaluar el comportamiento celular del mesénquima craneal

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Identificación de un murino eritroblasto subpoblación Enriquecido en enucleación Eventos Multi-espectral Imaging Citometría de Flujo

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...