D.萨麦斯感谢G.哈罗德和莱拉Y。马瑟斯慈善基金会和哥伦比亚大学科学与工程学院的倡议。 D.萨麦斯和D。苏尔寿感谢麦克奈特技术创新McKnight基金会在神经科学奖 D.苏尔寿感谢NIDA的，镍氢电池，和Picower和帕金森氏病的基础。 H.张感谢NARSAD。 相对湿度爱德华兹感谢迈克尔J.福克斯基金会，全国帕金森基金会，奈达和镍氢。 我们感谢罗伯特伯克技术支持与TIRF显微镜设置为6 - OHDA注射和建议，有益的讨论，Merek成像分析编程的小马克Sonders，和扬Schmoranzer。

使用这种方法在研究中报道的<a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator 等人，科学 324（5933）。 1441-1444（2009） 。</a> 1。急性纹状体切片准备<ol><li>准备急性纹状体片之前，必须氧气的人造脑脊液（ASCF）和冷却用冰前至少15分钟，大脑提取。保持学联冰冷和含氧在解剖。学联（毫米）：125氯化钠，2.5氯化钾，碳酸氢钠3月 26日，氯化钙2.4，硫酸镁1.3，KH 2 PO 4 0.3，葡萄糖10，HEPES 5，pH值7.3-7.4，290-295 mOsm。 </li><li>杀头无需麻醉雄性小鼠。 </li><li>从小鼠中提取的全脑，并安装使用Krazy ®胶水vibratome上vibratome托盘。脑提取和安装，必须在2分钟内完成。在这段时间内，一定要保持学联冰冷和含氧保持组织的健康。 </li><li>切割厚度在250微米之间囟1.54 0.62 1冠状纹状体的脑切片。这样就可以削减到6个半片，用针将获得整整3片。 </li><li>含氧学联在室温孵育探头负载前至少1小时的脑切片。片显示良好FFN511加载，并保持积极成像长达4小时后切片准备。 </li></ol> 2。载入FFN511 <ol><li>准备FFN511装载解决方案（10μMFFN511学联），还准备在学联100μm的ADVASEP - 7。所有的解决方案应准备新鲜和含氧加载到片之前至少15分钟。 </li><li>单独孵育FFN511 30分钟，在室温下加载溶液片。 </li><li>然后删除约束孵化100μm的含氧ADVASEP - 7学联为30分钟，在室温下 2装入片外组织的染料。现在切片成像准备。 </li></ol> 3。在大脑切片成像FFN511 FFN511成像切片是使用多光子激光扫描显微镜进行。我们使用的是一个草原终极多光子激光扫描显微镜（以前我们使用的Zeiss LSM 510 NLO多光子激光扫描显微镜和取得了一定的成效，与蔡司显微镜）。 <ol><li>将在录音室FFN511装片（RC - 27L，华纳）在1-2毫升，每分钟的流量和含氧学联superfuse。然后将切片上的尼龙绳铂金竖琴在实验过程中，尽量减少运动。为了进一步减少运动，让片，影像稳定之前至少10分钟，在会议厅内。 </li><li>可视化和定位的10倍水浸泡的目标下的明亮的领域发光的背侧纹状体。 </li><li>在这一地区的地点和位置的两极扭曲钨电极，如果执行刺激实验。理想的成像区域位于内300微米之间的刺激双极电极的两个小技巧。放置在视野的中心区域。 </li><li>图片FFN511标记下一个63X（0.9 NA）的水浸泡紫外线目标纹状体终端。 FFN511是兴奋与麦大激光光谱物理和纹状体终端的最佳荧光，在760纳米，是通过一个带通滤波器（480-520纳米）。 75 × 75微米地区的利益，在512 × 512像素分辨率与12位格式拍摄的图像。为刺激脱色实验，以弥补Z轴转移，5-7图像的Z系列，相隔1微米在z平面，获得每个时间段。 </li></ol> 4。 FFN511脱色 destained FFN511标签可以通过高浓度氯化钾（我们使用70毫米氯化钾），（+），安非他明类兴奋剂的硫酸盐（AMPH，20微米），或电刺激。在氯化钾脱色实验，当高的钾学联解决方案适用于脑片，FFN511在2分钟内标签destains。记录XYZ - T图像随着时间的推移跟踪FFN511脱色。下面介绍了电刺激依赖多巴胺终端脱色： <ol><li>随着时间的推移跟踪FFN511脱色，XYZ - T的图像记录。为了确保最小的片运动（Z轴少于4微米）并没有漂白Z - Stack的激光，控制时间序列图像，应取得至少5分钟前刺激。否则，调整激光功率。 </li><li>这些控制图像之后，继续XYZ - T成像，同时启动的刺激频率1，4或20赫兹。 1，4或20 Hz（300微秒× 1毫安）的刺激，适用于本地纹状体通过ISO - Flex的刺激，引发主8脉冲发生器使用双极电极（AMPI，耶路撒冷，以色列）隔离。为了尽量减少在发布网站的去极化的变化，我们已经使用了刺激协议应用循环伏安法确定的最大刺激强度的150％。 </li><li>使用适当的软件成像分析。在我们的实验室中，我们使用图像J（韦恩Rosband，美国国立卫生研究院，洛克维尔，MD）和自定义编写的软件在IDL（研究系统，博尔德，CO）的量化与时间3 puncta荧光的变化。 </li></ol> Represenative结果图1显示FFN511加载到纹状体切片，现在已经完成。代表使用10倍和60倍的目标的图像如图1所示。多巴胺终端的选择性标签可以destained 20μM的安非他明。 图2显示了FFN511高氯化钾标签脱色。 图3所示的FFN511标签由当地的电刺激脱色。 <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="图1" /> 图1 FFN511标签多巴胺终端，在现场的皮质-纹状体急性片（一）标记FFN511急性现场皮质纹状体切片。丰富的标签在纹状体（STR）的，稀疏的皮质标签（CTX），并没有标签胼胝体（CC）的。比例尺：100微米（二）由安非他明纹状体FFN511脱色。左侧面板：前安非他明类兴奋剂;右图：20μM的安非他明20分钟后。比例尺：10微米。 <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="图2" /> 图2。FFN511高氯化钾标签脱色。 FFN511标签destained在2分钟70毫米氯化钾在学联的应用。比例尺：10μm的。 <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="图3" /> 图3。FFN511纹状体标签的频率依赖性脱色。（一 ）在4赫兹的局部刺激导致脱色从终端。在t = 0开始的刺激。比例尺：5微米（二）FFN511脱色4赫兹的Ca 2 + -和频率依赖性。控制收到无刺激（153 puncta从3片）。氯化镉（200μM）的脱色未刺激控制（475 puncta从5片）相同。由一个单一的指数衰减函数和半衰期（T 1 / 2）每个刺激频率的脱色曲线拟合值计算τx 0.693（1赫兹：765 puncta从9片，4赫兹：410 puncta从7片， 20赫兹：6片）416 puncta。请参阅期间在纹状体脑片制备的胞外分泌FFN511脱色的双光子显微镜视频显微镜。

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

在这段视频中，我们展示了一种方法，使用荧光多巴胺类似物光学可视化的神经传递。 FFN511是我们已经开发出第一代的FFNs。虽然它的目的是针对神经元囊泡单胺转运体（VMAT2），携带从细胞质进入突触囊泡单胺类神经递质，并专门标签在皮质纹状体和推定儿茶酚胺和/或羟色胺终端的多巴胺终端（在科学所示纸），适当的装载期间是关键的特殊性，因为FFN511是相对的疏水性。我们发现，孵化的时间超过40分钟，将导致在纹状体切片广泛的非特异性染色。脱色使用高浓度的氯化钾，应引起FFN释放功能的突触囊泡内的特异性，可以直截了当地确定。不到15分钟的片FFN511暴露将导致在微弱的荧光信号，由于染料在码头的不足装载。在这个协议中，我们使用100微米ADVASEP - 7删除绑定到细胞外组织的染料。这一步是没有必要的，但如果它被省略，在学联的冲洗时间需要延长。总之，取决于您选择的准备，您将需要修改的浓度和装载期间，以确定最佳的标签。

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<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
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<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
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<td>&nbsp;</td>
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<td>Ted Pella, Inc.</td>
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<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
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dopamine release at individual presynaptic terminals visualized with ffns

中枢神经系统神经元之间通过突触囊泡融合过程中的神经递质释放的信号传输。观察神经递质的摄取和释放个人直接突触前终端，我们设计了荧光假神经递质的突触囊泡单胺转运体的基板。我们使用这些探头形象在纹状体多巴胺的释放，突触可塑性相关的若干意见。我们发现，每刺激释放神经递质的突触小泡的一小部分是依赖于刺激频率。这些实验条件下未观察到一个动力学鲜明的“储备”突触囊泡人口。一个依赖于频率的异质性突触前终端显示，依赖D2多巴胺受体的一部分，表明了突触前的选择依赖于频率的编码机制。 张辉和尼科G. Gubernator的贡献同样对这项工作。

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

一种新的手段使用荧光多巴胺类似物的光学测量神经递质。

多巴胺的释放突触前货箱码头在个别的可视化与FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Research

JoVE Journal

Biology

24.7K 次观看.  Columbia University. 一种新的手段使用荧光多巴胺类似物的光学测量神经递质。

视频: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

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

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 labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

荧光显微镜下细胞电融合的可视化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...