Name: preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging
Uploaded: 2011-06-22T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

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1。 Sylgard盖菜，试验室，和刀片的制备<ol><li> 35毫米猎鹰的Petri Sylgard弹性体涂层的菜是一个孤立的视网膜上获得单的感光细胞需要适当切碎。根据供应商的说明和少量浇到每道菜，其底部覆盖一层弹性体准备。更换的菜后，涂层覆盖，并将其储存。在几天的时间弹性体变硬的菜都准备好了。 </li><li>隔离光感受器需要坚持的实验室的底部，让他们固定在成像实验的过程中。这是通过涂层与聚- L -?...

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	<li>Burns, M. E., Arshavsky, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Counting+Photons:+Trials+and+Trends+in+Vertebrate+Visual+Transduction.">Beyond Counting Photons: Trials and Trends in Vertebrate Visual Transduction.</a> Neuron. 48, 387-401 (2005).</li><li>Ebrey, T., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+Photoreceptors.">Vertebrate Photoreceptors.</a> Prog Retin Eye Res. 20, 49-94 (2001).</li><li>Fain, G. L., Matthews, H. R., Cornwall, M. C., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+in+Vertebrate+Photoreceptors.">Adaptation in Vertebrate Photoreceptors.</a> Physiol Rev. 81, 117-151 (2001).</li><li>Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptor+Rhodopsin.">G Protein-Coupled Receptor Rhodopsin.</a> Annu Rev Biochem. 75, 743-767 (2006).</li><li>Saari, 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=Biochemistry+of+Visual+Pigment+Regeneration:+The+Friedenwald+Lecture.">Biochemistry of Visual Pigment Regeneration: The Friedenwald Lecture.</a> Invest Ophthalmol Vis Sci. 41, 337-348 (2000).</li><li>Lamb, T. D., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dark+Adaptation+and+the+Retinoid+Cycle+of+Vision.">Dark Adaptation and the Retinoid Cycle of Vision.</a> Prog Retin Eye Res. 23, 307-380 (2004).</li><li>Imanishi, Y., Lodowski, K. H., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Microscopy:+Shedding+Light+on+the+Chemistry+of+Vision.">Two-Photon Microscopy: Shedding Light on the Chemistry of Vision.</a> Biochemistry. 46, 9674-9684 (2007).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleached+Pigment+Produces+a+Maintained+Decrease+in+Outer+Segment+Ca2++in+Salamander+Rods.">Bleached Pigment Produces a Maintained Decrease in Outer Segment Ca2+ in Salamander Rods.</a> J Gen Physiol. 111, 53-64 (1998).</li><li>Sampath, A. P., Matthews, H. R., Cornwall, M. C., Bandarchi, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Dependent+Changes+in+Outer+Segment+Free-Ca2++Concentration+in+Salamander+Cone+Photoreceptors.">Light-Dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors.</a> J Gen Physiol. 113, 267-277 (1999).</li><li>Woodruff, M. L., Sampath, A. P., Matthews, H. R., Krasnoperova, N. V., Lem, J., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Cytoplasmic+Calcium+Concentration+in+the+Rods+of+Wild-Type+and+Transducin+Knock-out+Mice.">Measurement of Cytoplasmic Calcium Concentration in the Rods of Wild-Type and Transducin Knock-out Mice.</a> J Physiol. 542, 843-854 (2002).</li><li>Leung, Y. T., Fain, G. L., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Measurement+of+Current+and+Calcium+in+the+Ultraviolet-Sensitive+Cones+of+Zebrafish.">Simultaneous Measurement of Current and Calcium in the Ultraviolet-Sensitive Cones of Zebrafish.</a> J Physiol. 579, 15-27 (2007).</li><li>Matthews, H. R., Fain, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Spot+Confocal+Technique+to+Measure+Cytoplasmic+Calcium+Concentration+in+Photoreceptors.">Laser Spot Confocal Technique to Measure Cytoplasmic Calcium Concentration in Photoreceptors.</a> Methods Enzymol. 316, 146-163 (2000).</li><li>Szikra, T., Cusato, K., Thoreson, W. B., Barabas, P., Bartoletti, T. M., Krizaj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Calcium+Stores+Regulates+Calcium+Influx+and+Signal+Transmission+in+Rod+Photoreceptors.">Depletion of Calcium Stores Regulates Calcium Influx and Signal Transmission in Rod Photoreceptors.</a> J Physiol. 586, 4859-4875 (2008).</li><li>Krizaj, D., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+Calcium+Extrusion+Mechanisms+in+the+Outer+and+Inner+Segments+of+Photoreceptors.">Compartmentalization of Calcium Extrusion Mechanisms in the Outer and Inner Segments of Photoreceptors.</a> Neuron. 21, 249-256 (1998).</li><li>Chen, C., Nakatani, K., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+Magnesium+Concentration+in+Salamander+Photoreceptor+Outer+Segments.">Free Magnesium Concentration in Salamander Photoreceptor Outer Segments.</a> J Physiol. 553, 125-135 (2003).</li><li>Chen, C., Jiang, Y., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Behavior+of+Rod+Photoreceptor+Disks.">Dynamic Behavior of Rod Photoreceptor Disks.</a> Biophys J. 83, 1403-1412 (2002).</li><li>Tsina, E., Chen, C., Koutalos, Y., Ala-Laurila, P., Tsacopoulos, M., Wiggert, B., Crouch, R. K., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+Microfluorometric+Studies+of+Reduction+and+Clearance+of+Retinal+in+Bleached+Rod+Photoreceptors.">Physiological and Microfluorometric Studies of Reduction and Clearance of Retinal in Bleached Rod Photoreceptors.</a> J Gen Physiol. 124, 429-443 (2004).</li><li>Ala-Laurila, P., Kolesnikov, A. V., Crouch, R. K., Tsina, E., Shukolyukov, S. A., Govardovskii, V. I., Koutalos, Y., Wiggert, B., Estevez, M. E., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle:+Dependence+of+Retinol+Production+and+Removal+on+Photoproduct+Decay+and+Cell+Morphology.">Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology.</a> J Gen Physiol. 128, 153-169 (2006).</li><li>Koutalos, Y., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluorometric+Measurement+of+the+Formation+of+All-Trans-Retinol+in+the+Outer+Segments+of+Single+Isolated+Vertebrate+Photoreceptors.">Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors.</a> Methods Mol Biol. 652, 129-147 (2010).</li><li>Wu, Q., Blakeley, L. R., Cornwall, M. C., Crouch, R. K., Wiggert, B. N., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphotoreceptor+Retinoid-Binding+Protein+Is+the+Physiologically+Relevant+Carrier+That+Removes+Retinol+from+Rod+Photoreceptor+Outer+Segments.">Interphotoreceptor Retinoid-Binding Protein Is the Physiologically Relevant Carrier That Removes Retinol from Rod Photoreceptor Outer Segments.</a> Biochemistry. 46, 8669-8679 (2007).</li><li>Kolesnikov, A. V., Ala-Laurila, P., Shukolyukov, S. A., Crouch, R. K., Wiggert, B., Estevez, M. E., Govardovskii, V. I., Cornwall, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Cycle+and+Its+Metabolic+Support+in+Gecko+Photoreceptors.">Visual Cycle and Its Metabolic Support in Gecko Photoreceptors.</a> Vision Res. 47, 363-374 (2007).</li><li>Chen, C., Blakeley, L. R., Koutalos, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+All-Trans+Retinol+after+Visual+Pigment+Bleaching+in+Mouse+Photoreceptors.">Formation of All-Trans Retinol after Visual Pigment Bleaching in Mouse Photoreceptors.</a> Invest Ophthalmol Vis Sci. 50, 3589-3595 (2009).</li></ol>

如果未取得健康孤立的细胞，但问题在于与隔离或视网膜的健康，或者其斩。通常情况下，取出后，眼睛的玻璃体前，视网膜随时升空色素上皮细胞。如果没有，尝试从外围眼罩开始剥离它关闭。如果它仍然是难以分开，一个可能性是，动物还没有足够的时间内，暗适应，或太亮了红灯。确保适当的暗适应的动物，和昏暗的红灯。在视网膜上的杆光感受器的存在，可以很容易地通过检查孤立的视?...

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>黑暗的房间（100-150英尺2） </td><td></td><td></td><td></td></tr><tr><td> 19个红灯</td><td></td><td></td><td>网上商店</td></tr><tr><td>红外光源和红外图像的观众</td><td> FJW光学系统，公司</td><td></td><td></td></tr><tr><td>解剖显微镜19 </td><td></td><td></td><td>配备红外观众</td></tr><tr><td>封闭在一个光紧笼19 Epifluorescence显微镜</td><td></td><td></td><td></td></tr><tr><td>剖析工具（剪刀，镊子，刀片持有） </td><td> Roboz或精细的科学工具</td><td></td><td></td></tr><tr><td> Sylgard弹性体</td><td>埃塞克斯郡（北卡罗莱纳州夏洛特） </td><td> Sylgard 184弹性体套件</td><td></td></tr><tr><td>聚- L -鸟氨酸（0.01％） </td><td> Sigma - Aldrich公司</td><td> P4957 </td><td></td></tr><tr><td>聚- L -赖氨酸（0.1％） </td><td> Sigma - Aldrich公司</td><td> P8920 </td><td>稀释至0.01％ </td></tr><tr><td>实验商会</td><td>华纳仪器（哈姆登，CT） </td><td> D3512P </td><td></td></tr><tr><td>培养皿，塑料移液器</td><td> Fisher Scientific则</td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

在脊椎动物视网膜，phototransduction，光转换为电信号，进行了由杆和锥感光细胞1-4。棒感光器负责在昏暗的光线，在明亮的光线锥的视野。 Phototransduction的感光细胞外，一个专门的车厢，其中包含一个视觉色素，主光探测器的高浓度段的地方。视色素是一个生色，11 - 顺视网膜附着的蛋白质，视蛋白组成。视觉色素吸收光子异构从11生色团- 独联体国家全反式 。这种光致异构化带来了一个启动一个级联反应，最终在膜电位的变化，为电信号的传导光刺激的视觉色素的构象变化。光刺激的细胞恢复涉及的失活，由光激活的中间体和膜电位的重建。的Ca 2 +调节几个phototransduction涉及的酶的活性，减少光刺激后，其浓度。在这样的Ca 2 +在细胞光刺激，其背景光的适应复苏起着重要作用。 恢复过程的另一个重要组成部分，已在光检测摧毁其11的光致异构化的视觉色素的再生-独联体生色全反式5-7。这种再生开始的全反式视网膜的光敏色素的释放，留下的APO -蛋白视蛋白。被释放的全反式视网膜利用NADPH的全反式视黄醇，视蛋白反应迅速减少，结合新鲜的11 -顺视网膜带来改革的视觉色素外段。全反式视黄醇，然后转移到邻近的细胞外段和专门载体Interphotoreceptor维甲酸结合蛋白（IRBP）。 单一的感光细胞的荧光成像可用于他们的生理和细胞生物学研究。的Ca 2 +敏感的荧光染料，可用于详细研究外段的Ca 2 +的变化和反应之间的相互作用 ，以光 8-12以及内心的Ca 2段中的作用的Ca 2 +存储+动态平衡13,14 。荧光染料，也可用于测量镁2 +浓度，pH值，以及水和膜车厢 16示踪剂。最后，全反式视黄醇（维生素A）的内在荧光可以用来监测其形成的动力学和17日至19日在单一的感光细胞去除。

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

一个方法是为单身的生活，从不同的脊椎动物物种的荧光成像的感光细胞的准备。该方法可用于图像的内源性荧光基团，如NADH或维生素A，的荧光，或外源性补充钙敏感的荧光染料，<sup&gt; 2 +</sup&gt;或其他因素。

生活荧光成像的隔离脊椎动物的感光细胞的制备

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

Research

JoVE Journal

Neuroscience

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

内吞体动力学在生活神经末梢与四维荧光成像可视化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

的vibratome切片小鼠视网膜准备感光文化

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

毫秒氢/氘交换质谱法用于生理条件下α-突触核蛋白结构动力学的研究

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...