我们感谢塔夫茨大学神经科学研究中心，P30的NS047243（杰克逊），线粒体亲和性研究协作（mtARC）埃文斯在波士顿大学医学校区，链接医药公司的跨学科的生物医学研究中心的支持，并支持这项工作的蔡司。

1。图像板的制备<ol><li>文化INS1细胞在RPMI媒体含有10％的标准胎牛血清血清，1％青霉素，链霉素，2毫米L-谷氨酰胺，50微米2 -巯基乙醇， 碳酸氢钠 5毫米，2毫米的肝素钠，2毫米丙酮酸，和11毫米葡萄糖80％汇合。 </li><li> Trypsinize INS1细胞培养与0.05％胰蛋白酶和板到聚-D-赖氨酸涂布盖玻片底成像板（30-40％汇合）。 </li><li>允许板达到60-80％汇合（〜2天），并添加有针对性的线粒体基质（COXVIII）的PA-GFP腺病毒为24小时（MOI = 5）。交换媒介，使细胞生长成像前2天。 </li><li>对成像的日子，添加7-15纳米TMRE成像板，平衡至少45分钟。 </li><li>在此期间对孵化器的导通时间（舞台顶部孵化器已用在这里），并允许在显微镜平衡至37℃，约1小时。打开的5％的CO 2。 </李&gt; </ol> 2。蔡司LSM的710共聚焦显微镜成像参数<ol><li>显微镜具有平衡后，根据收购"选项卡上，单击"秀手工工具"，打开成像设立面板，并选择"通道模式"切换跟踪每一个"框架"。 </li><li>在光路径面板，选择LSM和渠道模式。从标本图标向上的工作，选择"后方"，MBS的690 +和MBS 488。在面板的底部，选择"T型光电倍增管"，使亮场或DIC通道的可视化。 </li><li>完成调整光路参数设置范围490-540纳米到580-700纳米的TMRE染料的绿色荧光染料。 </li><li>在采集模式，使用512×512像素的扫描场，平均4倍，并选择一个缩放因子（你可能要保持校准的目的相一致）。 </li></ol> 3。成像参数优化<ol><li>使用计划复消色差透镜100X（1.4 NA）的物镜，FOC我们对你的细胞，卤素灯，荧光灯，光毒性，以保护线粒体。 </li><li> PA-GFP的表达，使用最大开放针孔扫描发现的细胞，是明亮的绿色屏幕。 </li><li>找到需要激活的PA-GFP和优化成像参数，确保信号不饱和探测器的双光子激光功率最低。检查PA-GFP信号与几个Z系列TMRE信号的共定位。损失的TMRE信号的指示线粒体去极化或光毒性，细胞表现出这些特点，不应该被用于分析。 </li><li>找到一个PA-GFP表达细胞，并指定一个缩放因子。 </li><li>设置Z系列范围内收集6片（此范围内，将需要满足所有10个细胞，除非特定的Z-焦点设置在每个位置）。 </li><li>保存为每个单元格（单元1，单元2等）的成像方法。 </li></ol> 4。调整的"自动"ð部分计划和指定的10个细胞，你将遵循为1小时线粒体融合含量的<ol><li>多时间窗口的左侧面板上，选择"节能"面板，文件将被保存在指定位置。 </li><li>在"收购"面板，载入保存扫描配置1细胞的采集方法和检查Z堆栈框。也可以选择"标记的Z-z的栈中间"。 </li><li>在"块"面板中，选择"在每个位置的单块"。点击"添加模块"每次要测量的时间间隔。 </li><li>在"计时"面板，选择"只在第一的位置块前的等待时间间隔""等待的时间间隔"中键入"0"。块2-4将有"15分钟"这一节。 </li><li>在位置面板中，选择"移动焦点加载位置之间的位置"和"负载扫描配置时，"禄"或"下一步禄"点击在"编辑位置列表"选择"全部清除"，然后选择"多地点机动的阶段"。 </li><li> &gt;ER"死神"面板，单击"漂白"框中，指定在"配置"配置文件下拉菜单，在主要软件的窗口中指定。然后，在"漂白"窗口，保存相应的光敏方法。在该地区面板，选择这个特定的细胞的投资回报率，并选择"添加当前区域的投资回报率清单"窗口添加了多时的投资回报率。务必选择相同的位置下拉菜单中的"投资回报率"。 </li></ol>正常工作的时机，每个单元有一个15分钟的时间间隔，这两种方法都需要被保存。在块列表中，第一个将"真正的"光敏配置，其余的将有一个"模拟"的配置，不使用双光子激光。首块也将是唯一的块，不会有延迟（BKIntv = 0）。因此，该方法开始一个由光敏扫描基线扫描，。其余的区块有900秒BkIntv的，和在每个时间点有两个扫描一样在时间0秒，保持时间的一致性。 <ol start="7"><li>对于每个10细胞随着时间的推移，执行此序列: 查找PAGFP表达细胞保存其成像方法 位置面板标记舞台上的地位，指定成像方法 主要的投资回报率面板选择感兴趣的区域，是光活化漂白剂面板保存特定的投资回报率也加载在投资回报率框中: 擦除的投资回报率和重置扫描放大到1 寻找下一个单元格，并设置变焦 重复的过程，在未来的9细胞</li></ol>检查每个阶段的位置和所有块有适当的成像方法（扫描配置）。还要确保在每个位置的第一个块对应到相应的光敏方法，而其余包含一个"模拟"的方法。最后，选择"运行"。 5。分析PA-GFP的信号强度<ol><l我&gt;对于细胞在photoactivatable-GFP信号用红色TMRE信号的共定位，减去背景在PAGFP图像（在这里我们使用metamorph）。 </li><li>然后将数据导出到Excel计算Z-堆叠在每个时间点的平均强度。丢弃有没有信号的光学部分。 </li><li>原光活化信号的百分比计算，测量信号在每个Z-Stack的稀释。 </li></ol>每个数据点扫描和记录了两次，导致重复的Z-Stack的每个时间点的信息。检查Z-Stack的值一致。如果不是，这是一个问题（如焦点，细胞运动）的指标。 6。代表结果激活和非激活PA-GFP的线粒体之间的融合事件发生时，在线粒体基质PAGFP混合矩阵的非标记和稀释成为了一个更大的区域，降低了信号强度（ 图1A）。在INS1细胞，发生在信号强度显着下降，每15分钟，直到已达到平衡线粒体融合（约1小时）。请注意，在图1B细胞呈现近完整的PA-GFP的和TMRE信号的共存。在这些化验TMRE的浓度非常低（15 nm）的使用，以帮助目标的光敏PAGFP，并同时监测细胞的健康。与去极化线粒体丰富的细胞将有PAGFP和TMRE不完整的共存和不应该加以分析，因为这表明无论是光毒性，或在一个垂死状态的细胞。 线粒体融合的平衡时间通常为1小时INS1细胞，当〜线粒体体积的15％被激活。有时，即使小面积被照亮，大多数线粒体成为由于高度网络化的进一步融合，在这种情况下是难以察觉的光活化。其他类型的细胞可能会出现不同的平衡时间，并应在较短的时间间隔，并在一个较长时期的测试表征线粒体的动态。为了抑制线粒体融合，细胞可以放置内lipotoxic环境。先前已被证明有0.4毫米棕榈片段线粒体，抑制线粒体融合9。这种效果可以看出，在图2，其中线粒体是支离破碎，但信号强度的mtPAGFP不会改变尽可能在正常条件下（ 图1）。因此，稀释的PA-GFP信号可能是由于线粒体融合，而不是核裂变。在其他细胞类型，我们建议使用其他方式诱导线粒体碎片，如沉默OPA1这是必要的线粒体融合14。 <img alt="图1" src="/files/ftp_upload/3991/3991fig1.jpg" /> 图1。 </stroNG&gt;典型稀释的PA-GFP在线粒体融合实验后光敏信号。的PA-GFP逐渐变得黯淡由于线粒体融合的事件，导致蛋白质的稀释，增加面积，可以看到这些投影图像6光学部分量化每15分钟。与PA-GFP TMRE表明，线粒体活跃，而不是去极化。 <a href="http://www.jove.com/files/ftp_upload/3991/3991fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3991/3991fig2.jpg" /> 图2。线粒体融合棕榈抑制0.4毫米减少了稀释的PA-GFP 6光学部分，显示短不变的信号强度随着时间的推移，线粒体A.预测B.共存的PA-GFP与TMRE显示，线粒体不德极化。 <a href="http://www.jove.com/files/ftp_upload/3991/3991fig2large.jpg" target="_blank">点击这里查看大图</a> 。

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这种方法允许约10细胞成像的时间，如果收购发生光敏后每隔15分钟。细胞的确切数目将取决于如何迅速，是能够找到和标记mtPAGFP表达细胞在培养皿中，如何快速，可以设置所有软件参数。使自动化运行顺利，甚至细胞层应使用指定的Z-Stack的利润率，因为将适用于所有细胞。 这一初步的光敏面积的大小，将治理的平衡时间。为了能够测量线粒体融合，这是重要的，以photoactivate只有10-20％的网络，例如，可随着时间的推移监测网络的其余部分的信号传播。如果太多的网络是光活化，这是完全融合可能会出现太快，而不会被捕获的事件。 必须采取非常谨慎调整激光功率的双光子激光以及TMRE浓度，以避免光毒性，从而导致线粒体去极化。确保该mtPAGFP信号TMRE信号的共定位，可以帮助评估光毒性和一般细胞的健康8,15。 epifluoerescent光照明应避免。而细胞表达mtPAGFP，针孔，应该最大限度地开放，而低功率488nm激发扫描。调整的双光子激光的权力photoactivate足够的PA-GFP来衡量1小时以上的信号，但不oversaturate细胞可能会非常棘手8。然而，时间应在这个优化步骤花了，因为一次自动启动程序，它是乏味停止，选择更多的细胞，并恢复。 对于质量控制，图像采集的微分干涉对比（DIC）（或透射光）重点监察细胞可以很乐于助人，也是一个很好的方法来检测扫描过程中形成的泡沫在浸油，这有时会发生从舞台的动作。 虽然使用此mtPAGFP方法收集数据上的光活化线粒体那些没有标记的线粒体基质蛋白的单向流动，可以想象的是，利用这种技术来研究其他进程。例如，特定的荧光可以连接到膜蛋白，观察其特定的运动融合事件，已显示为ABC，我从15水溶性基质蛋白的混合不同的时间尺度上发生融合。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> COXIII-PA-GFP的腺病毒</td><td>利平科特 - 施瓦茨博士</td><td></td></tr><tr><td> TMRE </td><td> Invitrogen公司</td><td> T669 </td></tr><tr><td>蔡司LSM的710聚焦</td><td>蔡司</td><td></td></tr></tbody></table>

a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

线粒体融合是衡量跟踪矩阵针对性photoconverted整个网络随着时间的推移线粒体的GFP平衡的。到目前为止，只有一个细胞可能受到的时间在一个小时长的动力学分析。我们提出了一个方法，同时测量多个细胞，从而加快了数据采集过程。

速度更快，高分辨率，MTPA-GFP基于线粒体融合含量的收购多个单元格的动力学数据，在并行使用共聚焦显微镜

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

a-faster-high-resolution-mtpa-gfp-based-mitochondrial-fusion-assay

Research

JoVE Journal

Biology

16.5K 次观看.  Tufts School of Medicine. 线粒体融合是衡量跟踪矩阵针对性photoconverted整个网络随着时间的推移线粒体的GFP平衡的。到目前为止，只有一个细胞可能受到的时间在一个小时长的动力学分析。我们提出了一个方法，同时测量多个细胞，从而加快了数据采集过程。

视频: A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

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 ...

科研

生物学

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

同时进行pH测量的内吞和胞车厢中使用共焦显微镜活细胞

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

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 temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

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

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...