Name: photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells
Uploaded: 2010-06-26T23:00:00
Description: Watch this Scientific Journal Video about Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells at JoVE.com

<p class="jove_content">我们感谢迈克W.戴维森（佛罗里达州立大学）提供荧光蛋白的质粒DNA编码。这项工作是由国家卫生授予GM72048（DWP）研究院的支持。 </p>

<p class="jove_title"> 1。荧光蛋白滴样品制备</p><p class="jove_content">一种荧光蛋白液滴样本包括与居住在水相中的荧光蛋白质1-octanol/water乳液。这个乳液是夹在显微镜幻灯片和一个22平方毫米的玻璃盖显微镜应用。 </p><ol><li>需要清理，并具有疏水性剂涂在荧光蛋白液滴样本的显微镜载玻片和盖玻片。 </li><li> 5分钟，用丙酮洗涤清洁的玻璃器皿和离开干燥空气。 （可选，清洗玻璃器皿后，可以被视为在等离子体清洁30秒，...

<ol>
	<li>Kremers, G. J., Hazelwood, K. L., Murphy, C. S., Davidson, M. W., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+in+orange+and+red+fluorescent+proteins.">Photoconversion in orange and red fluorescent proteins.</a> <em style='font-style: italic'>Nature Methods</em>. <b>6</b>, 355-358 (2009).</li></ol>

<p class="jove_content">纯化的荧光蛋白液滴样本的荧光蛋白的光物理特性是非常方便的样本格式，例如漂白动力学和光转化动力学研究。液滴体积非常小（〜20皮升）促进漂白和光转化实验，它可以比色皿的系统难以执行。此外，如图所示，这里液滴样本非常适合设立了光转化应用共聚焦显微镜。疏水性涂层和BSA的存在是重要的，获得均匀的液滴。无涂层的液滴往往是对玻璃表面的挤压和缺乏对BSA的荧光蛋白容易聚积?...

<!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>Microsope slides</td>
<td>&nbsp;</td>
<td>VWR Scientific</td>
<td>48312-003</td>
<td>&nbsp;</td>
<tr>
<td>22 mm cover glass</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940-245</td>
<td>&nbsp;</td>
<tr>
<td>1-octanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4500</td>
<td>&nbsp;</td>
<tr>
<td>methyltrimethoxysilane</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6420</td>
<td>&nbsp;</td>
<tr>
<td>MatTek dishes</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G-1.5-14-C</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

photoconversion of purified fluorescent proteins and dual-probe optical highlighting in live cells

<p class="jove_content"> Photoconvertible荧光蛋白（PC - FPS）是一种荧光蛋白类的“光学荧光笔”的能力，这意味着暴露于一个特定波长的光可以通过改变颜色的荧光。光突出允许非侵入性标记的荧光分子亚群，因此跟踪单个细胞或细胞器的理想选择。 </p><p class="jove_content">高效光转化的关键参数的光转化光的强度和曝光时间。如果强度太低，将光转化缓慢或不发生在所有。另一方面，强度太大或太长时间曝光可以光漂白的蛋白质，从而减少了光转化效率。 </p><p class="jove_content">本协议描述了如何设置PC - FP光转化应用激光共聚焦扫描显微镜的一般方法。首先，我们描述准备纯化蛋白液滴样本的一个过程。此样本格式是非常方便的在显微镜下研究荧光蛋白的光物理行为。其次，我们将使用蛋白质液滴示例显示如何配置的光转化显微镜。最后，我们将展示如何执行在活细胞，包括mOrange2和Dronpa双探头光学突出中的光学突出。 </p>

Watch this Scientific Journal Video about Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells at JoVE.com

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

<p class="jove_content">这个协议描述的一般方法执行在激光共聚焦显微镜，荧光蛋白的光转化。我们描述puried蛋白质样品的光转化，以及与mOrange2和Dronpa活细胞的双探头光学突出的程序。</p>

在活细胞荧光蛋白的双探头光学突出纯净的光转化的

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Research

JoVE Journal

Biology

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Proper Care and Cleaning of the Microscope

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase Contrast and Differential Interference Contrast DIC Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Polarized Translocation of Fluorescent Proteins in <em>Xenopus</em> Ectoderm in Response to Wnt Signaling

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

偏光易位荧光蛋白<em>爪蟾</em>外胚层响应Wnt信号

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Live Imaging of GFP-labeled Proteins in <em>Drosophila</em> Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

<em>In vivo</em> Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...