Kuyruk ven enjeksiyonları floresan dextrans boyalar <ol><li> Görüntü serebral vasküler amacıyla, hayvanların bir floresan boya ile intravenöz olarak enjekte edilmelidir. Dekstran benzer parçaları beyin kan bariyerini geçen ve kan damarlarının dışarı sızdıran boya engeller çünkü dekstran-konjuge boyaları kullanabilirsiniz. </li><li> Fareler izofluran (% 4, indüksiyon için enjeksiyon sırasında% 1.5-2) ile anestezi. </li><li> Kuyruk% 70 alkol ile dezenfekte edilir. </li><li> 26 gauge iğne ile, 75-100 ul, tuzlu su içinde çözünmüş rodamin dekstran% 5 v / v çözümü, kuyruk mili boyunca kuyruk ven yarıda ile enjekte edilir. </li><li> Boya, yaklaşık 2 saat içinde rodamin boya kullanılarak, pembe dönecek idrar atılır kadar hayvanların hemen kuyruk ven enjeksiyondan sonra görüntülü olabilir. </li></ol> Iki foton mikroskopi kullanılarak kan akışı Görüntüleme (görüntülü damarların sayısına bağlı olarak toplam süresi 30-60 dakika, seans başına) <ol><li> Floresan dekstran boya enjeksiyonu takiben, fare (indüksiyon için% 4,% 1-1.5 görüntüleme) izofluran anestezisi. </li><li> Fare sıkıca hayvan sıcak tutmak için bir termo-ayarlı ısıtma yastığı içeren mikroskop sahne, titanyum çubuğunu kullanarak eklenir. Bazı göz merhemi, gözleri nemli tutmak için uygulanır. </li><li> Kraniyal pencere cam kapak,% 70 alkol ile temizlenir. </li><li> Pencere odak düzlemli paralel olarak konumlandırılmış ve 4X objektif altında görüş alanı merkezli. </li><li> Beyin yüzey damarlarının dijital bir kamera ile bir fotoğraf çekmek için en iyisidir. Bu resim görüntüleme oturumları her geçen gün art arda görüntülü bölgeyi bulmak için aşağıdaki referans görüntü olarak kullanılır. </li><li> 4X objektif sahne hareket ettirmeden suya daldırma 40X hedefi ile değiştirilir. Görüş alanında dijital resim tekrar alınır. Sahne manipülatör koordinatları sıfır ayarlanır. </li><li> Biz görüntü elde etme yazılımı olarak ScanImage kullanın. Bu laboratuvar Karel Svoboda (Pologruto ve ark, 2003) Tom Pologruto ve Bernardo Sabatini tarafından MatLab yazılmıştır. Lazer, güç ölçer, fotoğraf çarpanları tüpler, yükselteçler, vb: Önümüzdeki tüm diğer ekipman açmak </li><li> Yansıması uygun pencere alanı kısa bir süre için en iyi bölgeyi bulmak için, düşük büyütmede taranır. Bir kere bu, düşük bir büyütme yığını görüntünün çekildiği için seçilen bölgenin tespit ve onun XY koordinatlarını açıklamalı. </li><li> Küçük damarlar ve kılcal kan akış dinamikleri kaydetmek için ilgi geminin en az 40 mikron hat boyunca taraması kullanın. Bu tek bir &quot;tarama&quot; kalıcı bir saniye, mümkün olduğunca az lazer güç ve tarama koordinatları ve açı yazılı olarak kullanarak yapılır. </li><li> Görüntüleme oturumu bittikten sonra, hayvanın anestezi kurtarmak sıcak bir odaya taşınır. </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>

Kortikal kan akımı dinamikleri görüntüleme floresan dekstran boyalar iki foton mikroskobu ile kemirgen kuyruk damar içine enjekte in vivo olarak incelenebilir. Bu video görüntü kan akışını dinamikleri ile farelerin bir cam kaplı kafatası pencere hazırlama neokorteks nasıl bir yöntem gösterdi.

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

Yaşayan kemirgenler bozulmamış beyin beyin damarsal (kılcal büyük damarları) ve kan akışını dinamikleri kaydetmek için resim yeteneği güçlü bir tekniktir. Kafatası bir pencereden in vivo olarak 2 foton mikroskopi kullanılarak damar içine enjekte görüntü floresan boyalar mümkün. Bu kan akışı ölçümleri elde etmek için de görüntü kortikal damarsal bir izin ve. Bu teknik aslında David Kleinfeld ve Winfried Denk tarafından geliştirilmiştir. Bu yöntem, nörodejeneratif hastalıklar, beyin tümörleri, ya da normal beyin fizyolojisi, serebral iskemi sırasında veya sonrasında kan akış dinamiklerini incelemek için kullanılabilir. Örneğin, kırmızı kan hücre hız veya akı infarkt ve çevresinde ne kadar inme nedenleri kan akış yönü vardiya ve değişiklikleri incelemek için kullanılır olmuştur. Burada kuyruk damar içine enjekte floresan boyalar kullanarak yaşayan farelerin neokorteks görüntü kan akışını dinamikleri 2-foton mikroskopi nasıl kullanılacağını göstermektedir.

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

Kortikal kan akımı dinamikleri 2-foton mikroskobu ile kemirgen kuyruk damar içine enjekte görüntüleme floresan dekstran boyalar in vivo olarak incelenebilir. Bu video görüntü kan akışını dinamikleri ile farelerin bir cam kaplı kafatası pencere hazırlama neokorteks nasıl gösterir.

Kranial Pencere üzerinden neokorteks Kan Akış 2-Foton Görüntüleme Bir Yöntem

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.7K Görüntüleme.  University of California, Los Angeles. Kortikal kan akımı dinamikleri 2-foton mikroskobu ile kemirgen kuyruk damar içine enjekte görüntüleme floresan dekstran boyalar in vivo olarak incelenebilir. Bu video görüntü kan akışını dinamikleri ile farelerin bir cam kaplı kafatası pencere hazırlama neokorteks nasıl gösterir.

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.

Farelerde Periferik lenf nodu diseksiyonu ve 2-Foton Görüntüleme

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

Biyoloji

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.

2-Foton Mikroskop kullanarak Fare Thymus in situ Görüntüleme

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 .

Katman In Vivo 2-Foton Kalsiyum Görüntüleme Fare 2 / 3

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.

Kemik İliği Üretimi 2-foton Görüntüleme Kullanım murin Dendritik Hücreler Türetilmiş

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.

Rotorod Rat Yürüyüş Kalitatif Analiz için bir yöntem olarak kullanımı

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.

Meme Görüntüleme Penceresi yoluyla Dendra2 Photoswitching

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.

Görüntüleme için Basit Bir Yöntem Arabidopsis Infiltratif Görüntüleme Orta Perfluorodecalin kullanarak bırakır.

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.

Kranial mesenchyme Hücresel Davranış Değerlendirme için bir Eksplantasyon Assay

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.

Multi-spektral Görüntüleme Sitometrisi ile Enucleating Olaylar bir Kemirgen erythroblast ICSI&#39;nin Zenginleştir tanımlanması

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

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Kinaz İnhibitörleri Karşı Dayanıklı Mutasyonlarının Tarama ve Doğrulama için Bir Yöntem

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...