这项工作是由东风集团。

蜜蜂的制备及回填<ol><li>赶上在蜂房和关于固定冰的寒意蜜蜂觅食。 </li><li>安装在有机玻璃录音商会3。低熔点硬蜡录音室的墙壁修复眼睛和胸部。 </li><li>拉玻璃毛细管，俗称为电，突破，并在尖端获得了CA的尖端直径。 10μm的。盖毛细管尖端的染料粘贴。染料粘贴由一个10:1混合FURA - 2葡聚糖和赖氨酸可修复四甲右旋糖酐。 </li><li>对于染色过程中，固定天线与细枝末节或正二十烷。取出一块上面的大脑角质层和腺体和气管推到一边允许访问的蘑菇体。 </li><li>注入到蘑菇体神经元的胞体或轴突地区的毛细血管。这是可以做到放开手脚或使用微机器人。然后恢复角质层的一块头胶囊和松开天线。 </li><li> 30％的蔗糖溶液蜜蜂饲料存储在加湿的情况下，至少有四个小时前，或在夜间20 ° C。 </li></ol>活体成像<ol><li>防止蜜蜂，例如运动的海绵小块，压腹部，并用一个夹子或磁带录音室固定。 </li><li>为了防止大脑造成食道抽水的运动，角质层以上唇切成一小切口，小心地拉出食管和其周围的固体结构，把张力下，在不损害食道4。封面有两个组分硅酮。 </li><li>用针的孔，并插入M17录音电极丝。 </li><li>删除角质层片，气管和腺体以上的大脑。吸里面的一张纸头胶囊heamolymph。 </li><li>填充双组分硅酮头囊。重要的是，大脑是完全覆盖。 </li><li>蜜蜂在显微镜阶段和地方的水的硅沉浸到液滴显微镜浸目标表面上的下降。专注于染色的神经元。 </li></ol>气味的刺激和记录信号 嗅觉：我们使用计算机控制的，定制的刺激装置或Galizia和Vetter 3前面所述的“嗅觉” 。气味是稀释针对触角到一个恒定的空气流。任何刺激包括一个3S0.2毫升气味饱和空气的气味脉冲。可以设置在录制软件TILLVision刺激协议。 显微镜和影像设置：我们使用了蔡司荧光显微镜。在25 ° 5 Hz的采样率使用一个免耕光电成像设备安装在显微镜的彗星图像记录。钙信号记录通过一个X60的0.9 W，奥林巴斯DIP与意象CCD相机（640x480像素，160x120像素芯片分级4X）的目标。 FURA - 2是兴奋与340和380nm的比例测量波长的单色光。荧光检测到通过410nm分色镜和一个440nm的长通滤波器。录音软件录制的参数设置。每次测量持续10S和两个波长的曝光时间可以进行调整，以准备在每个染色强度。 M17录音：阴唇（M17）量角器肌肉细胞外记录到监控与学习的行为反应，即每五，六 。注入口部位的肌肉铜线。接地电极注入眼。肌肉潜力与自定义生成预放大器放大，数字化，并存储在计算机上。刺激呈现所引发的嗅觉。 数据分析<ol><li>在实验过程中钙信号的录音送入一台计算机和存储。录音软件TILLVision允许信号的初步检查，如之间的波长（340nm/380nm）和deltaF（基准加减）计算的比例计算。 </li><li>自定义编写的IDL程序进行图像处理。比例计算的Ca 2 +信号从340nm的测量和380nm的每个像素。确定背景荧光（F）和刺激前的平均帧减去比例的信号（deltaF）。 </li><li>要定义区域利息率（ROI）：计算平均帧期间的刺激，适用于低通滤波器（3X3px）。灰度转换伪彩色规模。确定假彩色图像中的活动点的神经细胞结构的活动，并定义为投资回报率。 </li><li>计算平均无任何过滤或更正的投资回报率超过所有帧像素的时空动态的活跃的地区。 </li></ol>连同FURA - 2右旋糖酐填写与可修复染料罗丹明右旋糖酐（“小红宝石”）的神经元。这两种染料具有相同的分子量;他们应在神经元合署办公。宽视场成像只允许非常有限的空间分辨率，因此建议使用共焦扫描显微镜的实验调查后染色结构。 <ol><li>生理测量后，解剖大脑和注视。 </li><li>冲洗PBS和乙醇和甲基salycilate明确逐步脱水。 </li><li>共聚焦显微镜嵌入甲基salycilate大脑。 </li><li>我们使用了Leica TCS SP2的显微镜。激发波长为543nm。 </li><li>我们用空气或油浸物镜扫描大脑成像数据进行比较的染色结构。 </li></ol>代表性的成果： 如果染色和准备是成功的，假色标信号，例如蘑菇体阿尔法叶外在神经元，可能看起来像图1。 行为反应（PER），可以计算出在刺激减去前刺激（图2）尖峰频率尖峰频率。 染色结构，可在更大的使用共聚焦显微镜（图3）的详细调查。 <img src="/files/ftp_upload/1353/figure1_new.png" alt="图1" /> 图1：代表钙成像的信号（刺激与庚） 。答：蜜蜂的大脑成像区域。乙：通过激发波长在380nm的一个X20 DIP目标的原始荧光图像，蘑菇体的α-叶概述紫色。注入α-叶腹外侧轮辋染料染色外在的神经元。选定的ROI：deltaF（三百八分之三百四十零），平均信号，在第二轮刺激派生的假彩色图像;α-叶紫色概述，概述了黑色。 D：钙信号的时空动态响应气味的刺激，从感兴趣的区域的平均信号C.电子表示：钙信号的时空动态响应蔗糖刺激IPSI和对侧天线的投资回报率分别表示在C <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> 图2：代表M17的反应。蜜蜂的嗅觉调节后延伸时所发出的气味，没有奖励的长鼻。红线表示刺激气味。插图：蜜蜂人均。 <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> 图3：形象代表栈。成像实验后的大脑解剖。从图1中的神经元是使用一个X20的油浸目标，在紫色概述α-瓣共聚焦显微镜扫描。

<ol>
	<li>Joerges, J., K&#252;ttner, A., Galizia, C. G., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representations+of+odors+and+odor+mixtures+visualized+in+the+honeybee+brain.">Representations of odors and odor mixtures visualized in the honeybee brain.</a> Nature. 387 (6630), 285-288 (1997).</li><li>Rehder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+honeybee's+proboscis+reflex+by+electromyiographic+recordings.">Quantification of the honeybee's proboscis reflex by electromyiographic recordings.</a> J. Insect Physiol. 33, 501-507 (1987).</li><li>Galizia, C. G., Vetter, R. S., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Analyzing+Odor-Evoked+Activity+in+the+Insect+Brain.">Optical Methods for Analyzing Odor-Evoked Activity in the Insect Brain.</a> Methods in Insect Sensory Neuroscience. , 345-388 (2004).</li><li>Mauelshagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+olfactory+learning+paradigms+in+an+identified+neuron+in+the+honeybee+brain.">Neural correlates of olfactory learning paradigms in an identified neuron in the honeybee brain.</a> J Neurophysiol. 69 (2), 609-625 (1993).</li><li>Kuwabara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bildung+des+bedingten+Reflexes+von+Pavlovs+Typus+bei+der+Honigbiene+Apis+mellifica,+Hokaido+Univ.">Bildung des bedingten Reflexes von Pavlovs Typus bei der Honigbiene Apis mellifica, Hokaido Univ.</a> Zool. J. Sci. 13, 458-464 (1957).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera.">Classical conditioning of proboscis extension in honeybees (Apis mellifera.</a> Journal of Comparative Psychology. 97 (2), 107-119 (1983).</li></ol>

在此演示中，我们经历过的所有步骤 ，以调查在蜜蜂体内的钙信号。我们应用这种技术的蘑菇体神经元，但可能对神经元的染色技术可以建立完成成像。我们一直专注于蜜蜂的嗅觉通路中的神经元。实验都是在暗室进行。设立是长波光线照射位于蜜蜂的光学感知范围，并没有激发染料。重要的是要避免任何荧光文物，例如气泡在硅。 这个系统的主要优点之一是样品在空间和时间分辨率的神经元活动的信息的可能性。同时，我们可以观察蜜蜂的相关行为进行调查时，如神经元在学习相关的可塑性。然而，在空间和时间分辨率是有限的。 体内钙成像技术的使用，实验时间是有限的。同时，随着外的电生理方法，录音，可以持续数小时或数天，我们的实验是有限的90分钟左右。信号仍然相当不错，当我们执行一个5Hz的采样率，每次测量持续10秒，20ms和380nm的曝光持久5ms的340nm的曝光，分别由两个波长85测量与激励。之后染料漂白可能会成为一个问题。

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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

在体内和半体内钙成像准备在我们的实验室已开发Joerges，库特纳和Galizia十几年前，来衡量在触角叶1的气味诱发的活动。从那时起，它一直不断完善和应用在蜜蜂大脑中的不同neuropiles。在这里，我们描述了目前在实验室中用来测量使用葡聚糖加上钙敏感染料（FURA - 2）在蘑菇体神经元活动的准备。我们逆行染色注入其轴突的染料或SOMA地区的蘑菇体神经元。我们专注于减少的侵袭，达到了准备，它仍然是可能培养的蜜蜂，每空调使用的。我们是能够监测和量化的行为反应录音电myograms肌肉控制的PER（M17）2。 后生理实验成像结构更大的使用共焦扫描显微镜的详细地址的神经元的身份进行调查。

Watch this Scientific Journal Video about In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee at JoVE.com

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

蜜蜂可以调节食欲嗅觉学习范式（每空调）。作为刺激的气味，我们建立了一个行为记录的同时，钙成像是用来衡量蘑菇体神经元的气味诱发活动的方法<em&gt;在体内</em&gt;。

在体内的Ca 2 + -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

in-vivo-ca2-imaging-mushroom-body-neurons-during-olfactory-learning

Nanyang Technological University

Research

JoVE Journal

Biology

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

13.9K 次观看.  Freie Universität Berlin. 蜜蜂可以调节食欲嗅觉学习范式（每空调）。作为刺激的气味，我们建立了一个行为记录的同时，钙成像是用来衡量蘑菇体神经元的气味诱发活动的方法在体内。

视频: In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Olfactory Behavioral Testing in the Adult Mouse

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible circuitry. In addition, this area's unique ability to generate new neurons throughout an organism's lifetime makes it an attractive system for developmental and regenerative biologists alike. Such interest necessitates a means for a quick, yet reliable assessment of olfactory function. Many tests of olfactory ability are complex, variable or not specifically designed for mice. Also, some tests are sensitive to memory deficits as well as defects in olfactory abilities, confounding obtained results.
Here, we describe a simple battery of tests designed to identify defects in olfactory sensitivity and preference. First, an initial general health assessment allows for the identification of animals suitable for further testing. Second, mice are exposed to various dilutions of scents to ascertain whether there is a threshold difference. Third, mice are presented with various scents, both attractive and aversive, that allow for the assessment of olfactory preference. These simple studies should make the initial characterization of olfactory behavior accessible for labs of varied resources and expertise.

Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference.

在成年小鼠的嗅觉行为测试

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

科研

生物学

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性的微型驱动器阵列在体内录音：驱动器的制造

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential amplitude of action potential signatures over the channels of a tetrode allows for the isolation of single-unit activity from multi-unit signals.  The ability to precisely control the stereotaxic location and depth of the tetrode is critical for studying coordinated neural activity across brain regions.  In combination with a micro-drive array, it is possible to achieve precise placement and stable control of many tetrodes over the course of days to weeks.  In this protocol, we demonstrate how to fabricate and condition tetrodes using basic tools and materials, install the tetrodes into a multi-drive tetrode array for chronic in-vivo recording in the rat, make ground wire connections to the micro-drive array, and attach a protective cone onto the micro-drive array in order to protect the tetrodes from physical contact with the environment.



Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly

In this protocol we demonstrate how to fabricate and condition tetrodes for use with a micro-drive array, which was designed for chronic electrophysiological recordings in rats. In addition, we illustrate the final stages of micro-drive array construction, which includes installing ground wires and a protective cone.

慢性的微型驱动器阵列在体内录音：四极大会

The tetrode, a bundle of four electrodes, has proven to be a valuable tool for the simultaneous recording of multiple neurons in-vivo.  The differential ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

获取标本减慢，加速和反老化的蜜蜂型号

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

对于在比较营养Beebread由非洲化和欧洲蜜蜂的方法和淋巴蛋白质滴度影响

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

在体内的Ca ^{2 +} -蜜蜂的嗅觉学习期间的蘑菇体神经元的成像

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