我们感谢阿娇斯坦菲尔德对稿件的意见。 TAJ支持犹他州遗传学培训格兰特T32 GM007464从NIH NIGMS大学中。

1。马赛克代<ol><li> 遗传交叉 瓦特FRT 19A浴缸：多巴胺FLP 122 / Y; BTL-GAL4 UAS-GFP（男性）X * FRT 19A / BAL（女），其中*表示该突变体进行检查。注：建立交叉实验前至少一天，因为这使动物交配适当。 </li><li> 预产转让交叉飞食品用小药瓶放在媒体上的鲜酵母膏涂抹。允许打下1小时，在25°C </li><li> 居士：从预产小瓶传送动物用小鲜酵母膏涂抹新鲜小瓶。允许打下6小时，在25°C </li><li>传输成人预先打好小瓶在随后的实验中储存和使用。立即热休克裁员小​​瓶含0-6小时的胚胎在38°C，45分钟一个循环水浴。注：食物表面应低于水位，以验证所有胚胎适当加热震惊。 </li><li>孵育4-5天，直到三龄幼虫开始徘徊在25°C热休克小瓶。 </li></ol> 2。屏幕马赛克动物<ol><li>用细镊子轻轻一挑游荡第三龄幼虫从小瓶的两侧，并将它们放置在一个小塑料板进入冷冻100％的甘油。 </li><li>使用高倍率解剖显微镜配备荧光光学检查幼虫。识别幼虫与镶嵌表达荧光标记的气管细胞。通常情况下，这是可以做到在10-20X的放大倍率。 </li></ol> 3。热固<ol><li>用细镊子仔细挑选马赛克动物的菜和地方上75x25x1毫米白玻璃显微镜幻灯片一滴新鲜的100％的甘油。注意：多个动物可以被放置在下拉式菜单（ 图1A）。 动物也可直接到盖玻片上，放置在甘油下降但必须谨慎采取固定期间（步骤3.2），动物将加热速度非常快，可以成为超过固定。 </li><li> 70°C加热块，直到动物放置玻片上停止移动，盖玻片的幻灯片和10秒（ 图1B）不超过20秒。动物会蠕动，但最初停止，并变得僵硬和拉长一旦死了。注：热暴露过长会损坏终端分支机构和流明以及GFP信号暗淡和弥漫。为了获得最佳的效率，建议有作为显微镜在同一房间内的热块。 </li><li>用细镊子小心地定位在一个单一方向的下降，所有的幼虫在所有的幼虫在幻灯片上方向平行（ 图1C）。 </li><li>轻轻这里的18X18MM微玻璃盖之上幼虫（或翻转到载玻片盖玻片），避免形成气泡（ 图1D）。注：盖玻片可能不在完全相同平超过幼虫，这是没有问题的。 </li><li>图片幼虫在30分钟内。热量固定端子细胞会降低的GFP信号会变得非常漫反射。 </li></ol> 4，气管终端细胞活体成像<ol><li>将舞台上的一个复合体视显微镜幻灯片。使用5X的目标定位运行从前侧到后侧的动物（ 图1F）的背侧表面上的两个平行的背中继线。找到两个背终末细胞，这两者之间直接背树干。这有助于确定适当的分部分析。 </li><li>一旦为本，旋转图像所需的终端细胞动物。要做到这一点，小心地推盖玻片的边缘钳垂直于长轴的幼虫，，慢慢滚动幼虫盖玻片下方（ 图1E和1G）。 </li><li>一致的结果，选择适当的终端细胞是关键和突变体之间的比较。肥胖的身体支（FB）终端细胞很容易被发现背躯干，正中线旁开相邻。横向G终端细胞（LG），很容易被发现，且位于腹中线外侧（ 图1H）。注：终端单元安排在最前（TR1）和后段（TR10）是发散的，从其他分部。我们只分析终末段TR2-TR9。背终末细胞比其他气管终端细胞有千篇一律的分枝格局。这可能依赖于额外的非细胞自主的信号，并决定是否将它们包含在分析时，应考虑。 </li><li>一旦一个终端单元利息已被确定使用所需的放大倍率，拍摄图像，通常10X或20X是最好的（ 图2A）。注：因变量的分支模式，试图捕捉到的图像，包括尽可能为许多分支和分支提示。 </li><li>没有移动舞台，关闭打开的透射光荧光和明场图像捕捉相同的细胞和焦平面（ 图2B）。管腔内将可视化对比黑暗的终端内的细胞空间。如果规划量化终端细胞分支机构和流明，在多个焦平面每个终端单元采集多个图像。 </li></ol> 5。终端细胞形态分析和定量使用ImageJ <ol><li>打开使用插件NeuronJ（ <a href="http://www.imagescience.org/meijering/software/neuronj/" target="_blank">http://www.imagescience.org/meijering/software/neuronj/</a> ） ImageJ的（ <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ）的荧光灯和明图像。 </li><li>使用“添加描”工具中，手动跟踪整个终端单元的树枝和管腔。 </li><li>使用“标签描”工具，重命名和recolor在跟踪一个特定的指定给每个行。 </li><li>使用“设置参数”工具，从6-10像素（512×512得到的图像由数码相机连接到的化合物显微镜调整线宽，更高或更低的分辨率的相机，应适当缩放的像素宽度），使用的所有其他默认设置，并选择“确定”。然后使用“快照”工具拍摄影像。选择“画踪”的跟踪可以显示并排侧与原快照。 </li><li>选择“测量描”工具并选择跟踪类型（ 即分支机构来衡量的基础上分配的具体名称，在步骤5.3）。接着，选择“显示追查测量”和“清除以前的测量”，然后点击“运行”。这将产生一个电子表格中包含的名称，标签的长度（以像素为单位），和其它数据，用于图像。然后，这些数据可以被保存为Mic​​rosoft Excel电子表格，进一步的统计的分析。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+for+the+PAR+complex+in+subcellular+morphogenesis+of+tracheal+terminal+cells+in+Drosophila+melanogaster.">A novel function for the PAR complex in subcellular morphogenesis of tracheal terminal cells in Drosophila melanogaster.</a> Genetics. 189 (1), 153-164 (2011).</li><li>Gervais, L., Casanova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+coupling+of+cell+elongation+and+lumen+formation+in+a+single+cell.">In vivo coupling of cell elongation and lumen formation in a single cell.</a> Curr. 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作者宣称，他们有没有竞争的财务权益。

这里描述的热固定技术是一种快速，方便的工具，成像果蝇幼虫气管终末细胞。在这里，我们使用这种技术来研究野生型细胞分支和流明模式。气管细胞表达GFP，气管特异性启动子气喘吁吁驱动，可以很容易地可视化，通过热固定后的幼虫角质层。的具体的分支型态的单独终端细胞，以及那些充满空气的腔，可快速可视化，并使用这个方法测量。这种技术也可以进行第一和第二龄幼?...

细胞的形状为单个细胞内的生物体中的功能是至关重要的，以及细胞，组织或器官的一部分功能。我们使用果蝇气管终末细胞，昆虫呼吸系统的一个组成部分，调查的分子机制，参与在控制两个保守类型的细胞形态：分支和管形成（lumenogenesis）。网络支管的前端位于终端细胞的功能来输送氧气的内部组织1，有一个复杂的支链的形态，取决于所控制的局部氧水平于靶组织2 FGF信号通路。端细胞分支细管，与一个充满气体的贯穿每个分支的亚细胞管腔。不同的蜂窝结构的终端单元，以及由在果蝇中，可以进行遗传分析的简易性，使这些细胞的优异的F型或调查机制的细胞生长，分支，和细胞内管形成。终末细胞的信号转导通路，导致支细胞分化的生长，成熟2-4了解一些已经证明一个有用的模型。使用该系统的公正，正向遗传筛选的细胞形态突变已被执行，控制细胞的形状5,6机制产生的见解。例如，这些屏幕显示，特定RabGAP的所需的细胞骨架的极性和管腔形成囊泡运输和定位7，整合素介导的粘附所需的分支稳定性8，该上皮细胞的PAR极性蛋白调节所需的偏振膜运输分支和管腔形成9。终末细胞的其他研究表明，非对称的肌动蛋白的积累和微管组织需要细胞伸长和lumenogenesis的<s高达&gt; 10。因此，多样化的，保守的细胞生物学机制有助于终端细胞形态。 这里，我们描述的方法迅速修复分析端细胞分支和管腔形成完好的第三龄果蝇幼虫。此协议也可以进行第一和第二龄期的动物。这种技术的关键是客户终端可视化细胞的基因标记的荧光蛋白表达直接通过完整的动物的幼虫角质层。由于此过程不需要任何固定后的处理，如抗体染色，观察细胞，它是非常适合高通量分析，包括基因或药物筛选。荧光蛋白的表达揭示了细胞质充满分支的结构。管形成，可以并行使用明视场显微术，以确定充有气体的腔，它与周围的流体填充对比监测ED组织。 包括在此协议中的方法，用于产生根据MARCM系统11，以产生纯合突变终端另有未标记动物与荧光蛋白标记细胞的遗传马赛克。这是必要的，因为只有终端细胞阐述其复杂的结构发展相对较晚，要求在其他组织早在发展基因遗传马赛克允许旁路。要产生MARCM克隆，气管被标记为使用这里描述的气管专用驱动气喘吁吁（BTL）12。果蝇 X染色体的协议;其他染色体的相似的方法可以被使用，适当的染色体与遗传试剂审查。在此，气管的细胞质本地化GFP标记的表达，但该过程同样适用与其他荧光蛋白，红色荧光蛋白等的表达。 此外，我们还包括量化分支模式和管腔形成终末细胞的基础上发展为特征的神经元分支图案13的方法，一种方法。这种量化的数据可以识别基因的确切作用在的分支或lumenogenesis过程中，以及允许不同的突变体9之间的直接比较中是至关重要的。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Student Dumont #5 forceps</td>
			<td>Fine Scientific Tools</td>
			<td>91150-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leica MZ16 Dissecting microscope (or equivalent)</td>
			<td>Leica</td>
			<td>MZ16</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AxioImager M1 compound microscope (or equivalent)</td>
			<td>Carl Zeiss</td>
			<td>&nbsp;</td>
			<td>Equiped with AxioCam MRm, A-PLAN 10X/0.25, EC PLAN-NEOFLUAR20X/0.5, Filter Set 38HE</td>
		</tr>
		<tr>
			<td>100% Glycerol</td>
			<td>BioExpress</td>
			<td>M152-4L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fly stock</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Genotype: w FRT19A tub:GAL80 FLP122; Btl-GAL4, UAS-GFP y w FRT19A</td>
		</tr>
		<tr>
			<td>Fly stock</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Genotype: y w FRT19A</td>
		</tr>
	</tbody>
</table>

examination of drosophila larval tracheal terminal cells by light microscopy

细胞的形状是细胞功能的关键。然而，尽管细胞形态的重要性，很少有人了解单个细胞如何产生特定的形状。 果蝇气管码头细胞已经成为一个强大的遗传模型生成细胞形态识别和阐明基因的角色。终端细胞是支链的管状网络的一个组成部分，气管系统，内部组织提供氧气的功能。终端细胞是细胞形态的调查问题，因为他们拥有两个不同的蜂窝结构的一个很好的模型。首先，终端细胞有一个复杂的支链的形态，类似的复杂的神经元;第二端细胞分支形成为细管，并包含一种膜结合的细胞内管腔。定量分析终端小区内支路数，枝条组织和个人分支形状，可以用来提供特定的基因机甲的角色信息anisms在决策支电池。在这些细胞中的小管形成的分析可以揭示保守机制的体外小管到其他管状网络，如脊椎动物的血管。这里，我们描述的技术，可以用来迅速修复，图像，并分析两个分支模式和管在终端细胞内形成果蝇幼虫。这些技术可以用于分析终端细胞中野生型和突变型的动物或遗传马赛克。因为此协议的高效率，它也适用于遗传，RNAi为基础，或药物屏幕在果蝇气管系统。

结果示于图2。一个单一的横向组（LG）终端细胞显示了广泛的亚分支（可视化通过GFP，A）和一个充满气体的亚细胞腔贯穿每个分支（明视场显微镜可视化; B）。收集这些图像的马赛克L3幼虫，使用的MARCM第1＆2中所述的系统，产生固定的热成像，第3及4中所描述的。 C及D面板显示E及F一个NeuronJ生成的跟踪，如在第5条，树枝和管腔，分别为A和B的面板显示的图像显示的位置在幼虫的脂肪?...

Watch this Scientific Journal Video about Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy at JoVE.com

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

在这里，我们提出了一个方法气管终端细胞在光镜下分析<em&gt;果蝇</em&gt;幼虫。这种方法允许分支和管腔形态在整个动物的快速检查和分析个别突变体或突变影响终端细胞发育的屏幕将是有益的。

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

examination-drosophila-larval-tracheal-terminal-cells-light

Research

JoVE Journal

Biology

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

10.2K 次观看.  University of Utah. 在这里，我们提出了一个方法气管终端细胞在光镜下分析果蝇幼虫。这种方法允许分支和管腔形态在整个动物的快速检查和分析个别突变体或突变影响终端细胞发育的屏幕将是有益的。

视频: Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

光镜研究沉默突触的突触前

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...