作者要感谢博士。郑和平，惠良张和斯蒂芬Kolwicz的宝贵意见，并在开发这种方法的技术支持。这项研究是由美国国立卫生研究院资助和科学家发展资助从美国心脏协会WW支持。

1。实验准备<ol><li>制备含有等渗平衡盐溶液（50毫升）:140 mM氯化钠，5mM的氯化钾，2.5mM的氯化钙 ，2毫摩尔MgCl 2和10mM HEPES（pH 7.2）中， 在体内骨骼肌成像。 </li><li>制备1升含的Krebs-Henseleit缓冲液（KHB）:118 mM氯化钠，5.3 mM的氯化钾，1.2mM的MgSO 4干燥，0.5mM的EDTA和25mM的NaHCO 3。 </li><li>气泡的KHB与含氧气体95％O 2/5％CO 2 10-15分钟之前，加入2mM的CaCl 2。添加代谢底物（ 如 10毫米的葡萄糖和0.5 mM的丙酮酸）。 </li><li>通过灭菌在70％乙醇钳，剪刀，镊子和然后漂洗于灭菌双蒸2 O制备的外科器械</li><li>打开心脏灌注系统上，调整在水浴和环行器的温度，设置蠕动泵的速度到6毫升/分钟。 </li><li>泡沫高雄港务局与95％的O 2/ 5％CO 2气体，并监测流出物的流率和温度（37℃，通过一个光纤温度探头）。该系统需要大约20分钟的气体和温度平衡。 </li></ol> 2。骨骼肌在体内的共聚焦成像<ol><li>麻醉小鼠用戊巴比妥（80毫克/千克，IP）。动物将在10-15分钟达到手术麻醉（以脚趾捏无响应），并保持在此状态为1-1.5小时，足以使骨骼肌的体内成像。 </li><li>在后肢之一除去毛发和消毒用70％乙醇在皮肤上。 </li><li>使上沿肢体，以暴露腓肠肌外侧皮肤上的切口。 </li><li>轻轻拿起肌外膜用锋利的钳子和做一个切口，通过用剪刀。进一步剖析，除去肌外膜和揭露肌纤维下方。 </li><li>用于体内装载TMRM进入骨骼肌肉，包括TMRM（500纳米）的等渗平衡盐溶液浸渍肌肉30分钟，然后洗出由指示器无解。 </li><li>把鼠标在其一侧上的共聚焦显微镜（Zeiss LSM 510）的阶段，抑制后肢在该露出骨骼肌面临对形成所述腔室的底部的盖玻片的位置上。盖玻片是在肌肉组织和反相物镜（40X油）之间。 </li><li>按腿轻轻放下，使组织和盖玻片之间的紧密接触。沉浸在等渗平衡盐溶液暴露的肌肉。 </li><li>记录二维（2D，xy）的共焦图象以每帧一秒的采样率。每个像素的强度被数字化，以8位深度。通常，一个串行扫描包含100帧。 </li><li>在&gt; 505nm处收集顺序图像由第1激励在405nm处，并收集，然后在488nm处&gt; 505 nm的收集，同时对双波长激励下MT-cpYFP n个影像。使用顺序激发在405，488和543 nm，而在505-545收集排放，505-545，和&gt; 560 nm处，分别为MT-cpYFP和TMRM的三波长激发成像。 </li></ol> 3。的灌注小鼠心脏共聚焦成像<ol><li>骨骼肌的体内成像后立即用200单位肝素钠（IP）的注射的小鼠。十几分钟后，安乐死鼠标开胸并迅速连接到它的肺和胸腺取出心脏。 </li><li>在冰冷的缓冲液快速清除肺部。识别胸腺瓣，轻轻剥开，露出升主动脉。 </li><li>取出胸腺和仔细去除任何周围组织分离主动脉。 </li><li>主动脉弓的第一分支之前做一个切口在升主动脉的上端。 </li><li>用两个微缝合镊持有的主动脉壁轻轻地以暴露腔和主动脉小心放置到大约nnula（PE50管）。主动脉被保持在适当位置用一个微型钳容器而缝合线围绕主动脉迅速栓。 </li><li>取出钳，仔细检查套管钳，以确保套管的尖端高于主动脉根部。其他关系都根据需要添加，以固定的心脏。 </li><li>打开蠕动泵并在1毫升/分钟灌注心脏。心脏会恢复灌注后跳动。 </li><li>调整灌注系统的位置，把心脏上的显微镜载物台。该阶段有一个适配器，允许保存心脏的腔室加热。 </li><li>加入1 ml的KHB灌注液在室部分淹没的心脏。监视室的温度在37℃。通过使用蠕动泵从腔室中移除流出物。 </li><li>提高蠕动泵的速度逐渐以提供足够的流量（大约2毫升/分钟）至心脏。 </li><li>后稳定10分钟，灌注老天室温下用10μMII型肌球蛋白和100-500纳米TMRM。 10分钟后，心脏跳动会减慢。 </li><li>适用于心脏温和的压力，以确保心脏的紧密接触盖玻片在腔室的底部，并进一步抑制心跳。 </li><li>按照相同的程序，如上面的步骤2.8所描述完整的心肌的共聚焦成像。仔细调整焦平面显露最清晰的图像成为可能。 </li></ol> 4。图像处理和数据分析<ol><li>使用由共聚焦软件来分析单个线粒体闪烁和膜电位变化的"生理学分析"模块提供的工具。该模块通常被包括在共聚焦系统的图像获取软件，并允许确定的特定区域的图像中，以及荧光强度的输出与时间标签。 </li><li>打开数据库并进行分析，然后串行2D图像文件。 </li><li>点击"区域权益回报率（ROI）的意思是"切换到"平均投资回报率的"模式。点击"感兴趣区域（ROI）的意思是"切换到"平均投资回报率的"模式。 </li><li>关闭其它信道的不同之处cpYFP 488nm的用于选择闪烁的信道的显示。 </li><li>放大图像并​​手动移动滑动条玩串行2D图像。 </li><li>通过查找网站，荧光信号增加瞬时识别单个线粒体超氧化物闪烁。使用适当的投资回报率的工具，以纪念闪烁。跟踪显示每个ROI的时间依赖性荧光的变化将出现在图像旁。 </li><li>选择所有闪烁后，打开其他渠道的显示屏上。选择单元格的背景信号减法之外在图像上的投资回报率。输出的每个ROI的平均荧光连同时间标记。 </li><li>与扫描时间和面积闪烁记录在每个串行扫描的图像文件的数目一起的细胞。使用Excel来计算闪光频率为每100秒/ 1,000μm2的电池面积闪烁的数字。 </li><li>使用写在交互式数据语言（IDL，ResearchSystems）来计算每个闪存（振幅，达峰时间和衰减时间）的参数定制开发的程序。 IDL软件是市售和闪光灯参数分析的定制开发的程序可以从作者要求索取。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

成像在活的动物或器官灌注线粒体单事件具有比传统方法的线粒体功能评价17,19,21,22,24,25显著优势。这里描述的技术可以实现实时的在一个真实的生理状态在亚细胞分辨率线粒体功能的原位测定。这是特别有用的，当与其它测量值相结合，系统地研究线粒体在体内特定器官或细胞类型的正常功能的作用。这是一个复杂的技术，它结合了共焦显微术和复杂的灌注系统的各...

线粒体在细胞生物能量核心作用，自由基信号，氧化还原平衡，离子调控和细胞命运决定1,2。线粒体功能障碍常伴和的基础疾病3-6的发病机制。尤其是在肌肉系统，如心脏和骨骼肌线粒体呼吸提供了广大的ATP以支持及时调控细胞内钙离子和强大的开发力量7,8。这些肌肉具有大量线粒体经常占据高达总细胞体积的20-40％，并在肌丝2之间被"固定"的。 尽管大量的研究，我们对线粒体功能的调节，特别是在体内和在生理学相关条件的理解，是有限的。其中一个原因是，多数的评价线粒体功能开发的方法依赖于在VITRo或体外的方法，如监测分离线粒体辅以人工基质的耗氧量，并通过形态学（ 如电子显微镜），酶的活性（ 如乌头酸酶活性）的间接测定线粒体功能，或细胞内ATP水平9-11 。 最近，小分子荧光指示剂与线粒体相对富集已应用于提供一瞥线粒体信号，包括膜电位，钙和活性氧簇（ROS），在完整细胞中11-13。此外，一些绿色荧光蛋白（GFP）的氧化还原和ROS指标，已经开发了实现了条块细胞内的氧化还原或ROS信号14-16的更具体的评价。这中间，我们开发了一种基因编码的超指标，圆形排列的黄色荧光蛋白，并targeteD它进入线粒体（MT-cpYFP）17。 MT-cpYFP可以在405或488 nm的激发与在515 nm的两个发射峰。在488nm激发的发光是专门响应于在体外和体内的校准17,18所示由以前超氧。在405nm处激发的发光被用作内部控制（请参考图1号17的对MT-cpYFP的发射光谱和激发光谱的详细信息在各种条件下）。随着时间推移共焦成像，这一指标检测爆棚超生产活动，名为超氧化物闪烁，在单个线粒体完整细胞。超闪作为线粒体呼吸，伴随的瞬态线粒体膜去极化和活性氧的产生17-20的复合函数。最近，我们已经生成使用PUC-CAGGS-MT-cpYFP矢量17,19上C57/BL6背景的泛组织MT-cpYFP转基因小鼠和验证了强烈的表达锡永这一指标在心脏，骨骼肌等组织中（ 图2）。根据要求和MTA批准由华盛顿大学的转基因小鼠将供感兴趣的学术研究。 在这项研究中，我们描述了在超闪烁的Langendorff灌流心脏原位成像以及体内成像的麻醉MT-cpYFP转基因小鼠17,19骨骼肌Flash事件。此技术允许在生理相关的条件下或在体内21,22单个线粒体ROS产生事件的实时监控。它也是可行使用该系统来监测其它单线粒体参数如膜电位和钙与适当的荧光指示剂。与细胞内事件（ 如钙瞬变）或心脏功能（线粒体功能进一步，同步或并行的评价如。射血分数）就可以实现。病理扰动，如局部缺血和再灌注，可以被应用到灌注心脏评估应力对未受影响的心肌单线粒体功能的影响。

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			<td>REAGENTS</td>
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			<td>CaCl2</td>
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			<td>Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including &#34;Physiological Analysis&#34; module.</td>
		</tr>
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confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo

线粒体是负责能源生产和细胞内信号在真核系统中的关键细胞内的细胞器。线粒体功能障碍常伴随并有助于人类疾病。已开发，以评估线粒体功能和功能障碍的方法的大多数都是在体外或离体测量的基础上。这些实验的结果具有有限的在确定体内的线粒体功能的能力。在这里，我们描述了一种新的方法，利用激光共聚焦扫描显微镜对完整的组织的活缩醛胺的成像，其允许单个线粒体功能的评价在一个实时的方式在体内 。首先，我们生成表达线粒体超氧针对性的指标，循环置换黄色荧光蛋白（MT-cpYFP）转基因小鼠。麻醉MT-cpYFP鼠标固定在一个特制的舞台适配器和时间推移图像拍摄fROM中的后肢暴露的骨骼肌。鼠标随后被处死，心脏被设置为Langendorff灌流与生理溶液在37°C灌注心脏被定位在一个特殊的腔上的共聚焦显微镜阶段，温柔的压力施加到固定的心脏，抑制心脏的跳动引起的运动伪影。超氧化物闪烁是由实时二维共焦成像，每秒1帧的频率检测到。该灌注溶液可修改成包含不同呼吸底物或其它荧光指示剂。灌注也可以进行调整，以产生疾病模型，如缺血再灌注损伤。这种技术是用于确定单个线粒体在完整的组织和体内的功能的独特的方法。

原位成像灌注心脏（ 图1），根据该协议，单个线粒体事件的体内成像可以在麻醉小鼠的骨骼肌完成之后。的成像条件的最佳设置将确保清晰的图像的完整肌肉组织中，并与单一的线粒体的分辨率（ 图2）。 TMRM经常被用来验证的mt-cpYFP的位置和应显示一个完整的重叠图案与MT-cpYFP信号（ 图2）。 TMRM对于在完整细胞中23线粒体膜电位的测定...

Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

共聚焦扫描显微镜是适用于在成像灌注心脏或骨骼肌线粒体单事件活的动物。单个线粒体过程，如超氧化物闪烁和膜电位的波动进行实时监控，使线粒体功能的评价在生理相关的背景和病理过程中的扰动。

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

confocal-imaging-single-mitochondrial-superoxide-flashes-intact-heart

Research

JoVE Journal

Biology

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

14.5K 次观看.  University of Washington. 共聚焦扫描显微镜是适用于在成像灌注心脏或骨骼肌线粒体单事件活的动物。单个线粒体过程，如超氧化物闪烁和膜电位的波动进行实时监控，使线粒体功能的评价在生理相关的背景和病理过程中的扰动。 

视频: Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

科研

生物学

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.