我们感谢我们株教授平冈。我们承认从戈兰Gustafssons基金会和瑞典癌症协会（九百三十九分之二千零八）的支持。

1。裂殖酵母的培养<ol><li>准备爱丁堡最小媒体（EMM）没有ammoniumchloride和EMM（EMM - N）的8。为了减少自体荧光的葡萄糖溶液不应进行高压灭菌，但而不是使用一个0.2微米的过滤器和随后添加到蒸压媒体的过滤消毒。 </li><li>接种裂殖酵母细胞新鲜琼脂平板上生长，具有丰富的媒体， 雅 8，3毫升液体介质与过滤消毒葡萄糖EMM 。使用13mL管轻轻推第一个的细胞，以确保良好的通风。让细胞增长225 RPM摇晃30 ° C。其次是适当稀释，每天早晨和晚上保持细胞生长在2天的日志阶段（1 × 10 6 - 2 × 10 7细胞/毫升）计数使用Burker室。 </li><li>在实验当天确保细胞在早期的日志阶段，5 × 10 6 - 1 × 10 7细胞/ ml 。 </li><li>要饿死吨他对氮细胞在20分钟，从电解金属锰媒体交换机EMM - N的媒体。这是在1.5 mL Eppendorf管在板凳最大1.5 RCF的离心机，以更快的速度离心的收获3毫升的细胞可能诱发应激反应在裂殖酵母9。使用双纺丝技术，这意味着第一自旋为2分钟，然后打开Eppendorf管180 °和自旋为1.5 RCF 2分10。这有助于之一颗粒收集所有的细胞在试管底部。洗一次用EMM - N，然后溶解在EMM - N的沉淀和15分钟的细胞孵育30 ° C晃动在225转，然后继续第2点。样品制备。 </li></ol> 2。样品制备<ol><li>在1.5毫升的Eppendorf管在一条长凳最大1.5 RCF离心机，以更快的速度离心收获1.5毫升的细胞可能诱发应激反应在裂殖酵母9。使用双纺丝技术，meani吴;自旋为2分钟，然后打开Eppendorf管180 °，再次自旋为2分10 1.5 RCF。这有助于之一颗粒收集所有的细胞在试管底部。 </li><li>去除上清，但离开10-15μL重悬细胞，在余下的媒体。另外添加10μL新鲜的媒体重悬细胞沉淀。 </li><li>确保有明确的载玻片和盖玻片（1.5）。通常情况下，你没有清理，但确保他们不满是灰尘。 </li><li>从冰箱已过滤消毒的1mg/ml的外源凝集素原液等分。凝集素的解决方案可以再冷冻几次。凝集素是用来固定在玻璃盖的酵母细胞。 </li><li>放置5μL电解金属锰与客观玻璃过滤消毒葡萄糖。 </li><li>将5μL在角落的玻璃盖的外源凝集素的解决方案。随后放置在同一个角落5μL细胞培养，即在凝集素下降。混合吹打几次，然后蔓延整个玻璃盖的细胞文化凝集素混合使用枪头长边。根据细胞混合物的密度，放弃一切，或吸在玻璃盖的对面角落里多余的液体。 </li><li>将玻璃盖，顶升，客观玻璃和对方在板凳上的一面。让玻璃盖干一点几分钟。它绝对不应该被完全干燥，但它不应该过多的液体在玻璃上。 </li><li>玻璃盖放置一个近似70 °从客观的玻璃天使电解金属锰下降。让玻璃盖去，使其将下降最电解金属锰下降下来。 </li><li>为了密封硅脂的边缘，准备2毫升注射器。削减200μL提示结束，并将它附加到注射器。约1毫升硅脂填充注射器。罚款的硅线适用于玻璃盖的边缘轻轻脓兴活塞的注射器。现在你有一个小S 的生长室裂殖酵母细胞。 </li></ol> 3。显微镜<ol><li>打开汞/氙灯，显微镜和计算机初始化的荧光显微镜。放置在荧光显微镜下的酵母生长室。使用一个63 X目标或与NA 100 × = 1.3或更高的目标。如果使用石油的目标是，添油。 </li><li>使用明场细胞，并获得清晰的图像。 </li><li>荧光显微镜的设置取决于用来标记的酵母细胞和显微镜的荧光。我们用共聚焦显微镜蔡司LSM 700激光扫描显微镜计划- 63X油复消色差透镜物镜（NA = 1.4）与16线的平均平面扫描设置。针孔应设置为1-1.1艾里单位，给出了一个0.8毫米的光学切片。我们检测GFP在音轨1，使用Alexa的488分束器在582纳米过滤器的设置，从而ðetecting 488和582 nm之间的波长。轨道2中，我们使用一个过滤器使用mCherry在578 nm处的分束器的最佳设置，从而检测波长为578和600 nm之间。这意味着，在第2轨将被检测的MRFP（SPB）和新墨西哥州（mCherry）。 </li><li>以许多图片需要能够来衡量，每个菌株在60不同的细胞。通常，15个独立的显微镜领域照片是不够的。这是建议，如果您的显微镜时间超过60分钟，是一个新的增长与新鲜细胞室。 </li></ol> 4。定量测定细胞内的距离<ol><li>蔡司禅光版程序中打开图片。使用的测量工具测量距离在微米之间的所有信号在同一焦平面的所有细胞的不同荧光。调整光的强度和对比度，以确定荧光信号的中心。这种简化的协议仅使用两个不同的Colours，因此国家邮政局和NM都在同一个通道。国家邮政局，是其在新墨西哥州的一轮结构大单挑。转移到一个记事本表的距离。衡量在至少60个细胞。其他节目，如ImageJ也可以用来测量距离，但蔡司禅灯是首选，由于其更高分辨率的图片，并轻松地放大和使用该程序。在ImageJ打开LSM格式的图片，将有两个通道彼此顶部。为了使两个通道的照片，第一次分裂的两个通道，并在其后将它们合并。此后行工具可以用来衡量如上所述的距离。 </li><li>比较使用统计程序，例如t检验或Mann - Whitney秩和检验，例如，通过使用SigmaStat 3.5软件之间的不同菌株和治疗的平均数或中位数的亚细胞距离。频繁的数据不是正态分布自会有一个较短的细胞选择荧光信号，因为所有的信号之间的距离，需要在同一焦平面来衡量。 T -测试只能使用数据时有一个正常的分布，而采用Mann - Whitney秩和检验允许比较缺乏正常分布的数据集，的 ​​。 在 T -测试两个不同的数据集的平均值相比，中位数，而在采用Mann - Whitney秩和检验比较。 </li></ol> 5。代表性的成果应变PJ1185：（H + his7 +：dis1placR - 绿色荧光蛋白 Chr1 [：ura4 + hphMX6 LACO] sid4 - MRFP：kanMX6 cut11 mCherry：natMX6 ura4 - D18 leu1 - 32 ADE6 - DN / N）的成长中的EMM 。一个样品被撤回，装在一个小的生长室和照片拍摄（图1A + N）。随后取代EMM的W / O ammoniumchlo​​ride（EMM - N）的生长介质，细胞克15分钟rown一阵晃动。氮饥饿细胞，然后安装在与EMM - N和图片的生长室（图1A - N）。的测量工具是用来测量轨迹（GFP）和国家邮政局（图1B和表1）之间的距离。此外，测量轨迹（GFP）和网管之间的距离（图1B和表2）。前后氮枯竭的亚细胞的距离中位数相比，使用的SigmaStat 3.5软件（见表3）。有一个统计学上的重大转变，在本土化的基因远离对收缩压的网管集群。 GFP和深圳湾口岸之间的距离测量的数据正态分布，因此平均距离（1.777微米+ N和1587微米- N），可以比较采用t检验（表1和3 ）。是两者之间的平均值（P = 0.008，t检验）有显着性差异。 GFP和NM之间的距离测量的数据没有一个正常的直接投资stribution，因此中位数的距离（0微米+ N和0.390微米氮）相比，使用Mann - Whitney秩和检验（表2和3）。有一个两者之间的中间值（P &lt;0.01，Mann - Whitney秩和检验）的显著区别。 <img alt="图1" src="/files/ftp_upload/3454/3454fig1.jpg" /> 图1。命名Chr1基因组的国产化，绿色荧光蛋白标记，改变后的氮饥饿。 （一）左列+ N，A代表从一个细胞生长电解金属锰右列，- N，代表生长在EMM - N一个细胞的细胞核的细胞核。绿色标签的Chr1集群是GFP信号，红色是MRFP和mCherry标签国家邮政局和NM，分别。 （一）（二）相同的细胞核，但现在测得的亚核的距离;黄色：细胞核的直径，蓝色：SPB和GFP信号和粉红色之间的距离：GFP信​​号和核膜之间的距离。 <pCLASS =“jove_content”&gt; <img alt="表1" src="/files/ftp_upload/3454/3454table1a.jpg" /> <img alt="表1" src="/files/ftp_upload/3454/3454table1b.jpg" /> <img alt="表1" src="/files/ftp_upload/3454/3454table1c.jpg" /> 表1。电解金属锰增长PJ1185应变微米的亚核的距离。第一排，直径细胞（D），第二行，GFP和国家邮政局之间的距离，第三排，GFP和新墨西哥州之间的距离。 <img alt="表2" src="/files/ftp_upload/3454/3454table2a.jpg" /> <img alt="表2" src="/files/ftp_upload/3454/3454table2b.jpg" /> <img alt="表2" src="/files/ftp_upload/3454/3454table2c.jpg" /> 表2亚核生长在EMM - N PJ1185应变微米的测量距离。第一排，直径细胞（D），第二行，GFP和国家邮政局之间的距离，第三排，GFP和新墨西哥州之间的距离。 <imgALT =“表3”SRC =“/ files/ftp_upload/3454/3454table3.jpg”/&gt; 表3。应变PJ1185观 ​​察亚核距离的描述性统计（+ N）和（- N）的氮饥饿。第一行：细胞的数量来衡量，第二行：平均直径（D），第三行：平均直径（D），来回行：GFP和国家邮政局，第五行之间的平均距离：GFP和网管之间的中位数的距离。

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我们什么都没有透露。

在过去十年中的活细胞成像的使用，监测细胞活动已成为越来越受欢迎。它开始从维多利亚水母水母的绿色荧光蛋白的使用，现在都可以通过许多不同的荧光发光的荧光从青色的宽光谱（475纳米）远红光（648 nm）的11。对免疫活细胞成像的主要优点之一是细胞是不固定的甲醛或乙醇/丙酮处理之前显微镜，从而避免可能的固定过程的文物。此外，活细胞成像，提供了可能，按照单个细胞，并采取频繁的照片在小时或天的潜伏期，从而有可能获得12个细胞的活动电影。哺乳动物细胞的尺寸较大的优势，直径大约100微米，直径约3-4相比较小的酵母细胞mu的M.的另一方面，与酵母的优点是很容易被操纵的基因组。同源重组发生在酵母和非常有效地用于保险丝利益的3个不同的荧光蛋白质。此外，使用有针对性的法律咨询及田土转易处的序列和随后LacR - GFP蛋白表达的一体化，使细胞核内的基因组中的一个特定部分的检测。使用S。裂殖酵母细胞在显微镜具有额外的优势，因为它们是自然生长快一代人的时间在低成本的细胞培养条件的单细胞生物。此外，S.酵母是一个很好的，因为它有后生动物同源基因的真核模式生物。 这项技术的主要限制之一是令人不安的真实信号的检测在酵母细胞自体荧光。这个问题是可以克服的，由最小的生长介质，而不是使用过滤消毒葡萄糖蒸压。在此外，酵母细胞应安装前增长数期为2天。这里介绍的协议提供了一个相对简单，但尚未定量的方法来确定的酵母细胞内蛋白质的亚细胞定位。此外，在不同时间点拍照，我们可以按照细胞的活动。

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>凝集素</td><td>西格玛</td><td> L1395 </td><td></td></tr><tr><td>硅脂</td><td>道康宁</td><td></td><td></td></tr><tr><td>激光扫描显微镜 LSM 700 </td><td>蔡司</td><td> LSM 700 </td><td>可用于其他共聚焦显微镜。 </td></tr><tr><td> SigmaStat 3.5 </td><td> Statcon </td><td> SigmaStat 3.5 </td><td>可用于任何软件进行统计分析。 </td></tr></tbody></table>

quantitative live cell fluorescence-microscopy analysis of fission yeast

一些显微技术，可以检测出一个特定的蛋白质在细胞内的今天。在过去的十年使用直接连接到蛋白的绿色荧光蛋白（GFP）的荧光活细胞成像已成为越来越受欢迎。使用GFP和类似荧光蛋白质的亚细胞本地化和运动可以在荧光显微镜检测。此外，亚核本地化的染色体中的某些区域也可以使用这种技术的研究。 GFP是融合到lac阻遏蛋白（LacR）和异位的法律咨询及田土转易处序列串联重复序列已被插入到2号染色体上的兴趣区域的细胞中表达。 LacR - GFP将绑定到的法律咨询及田土转易处重复和该区域的基因组将作为一个绿点在荧光显微镜可见。酵母是特别适合这种类型的操纵，因为同源重组是非常有效的，从而可以有针对性的统一的法律咨询及田土转易处重复和工程融合蛋白与GFP 3。在这里，我们描述了裂殖酵母活细胞分析的定量方法。裂殖酵母活细胞分析的附加 ​​议定书“，可以发现，例如，如何使电影减数分裂染色体行为4。在这个特殊的实验中，我们将集中在亚核组织，以及它是如何在基因诱导的影响。我们引进法律咨询及田土转易处结合位点的基因附近的一个基因簇，Chr1命名，标记。丰富，早在裂殖酵母5氮饥饿诱导基因的基因簇。应变核膜（nm）为标记由mCherry附件NM蛋白Cut11上升到一个红色荧光信号。 Sid4纺锤体极体（SPB）化合物是融合红色荧光蛋白（Sid4 MRFP）6 </s&gt;。在酵母细胞无性增长中的着丝粒是一贯重视的国家邮政局，是嵌入在NM 7 。被认定为国家邮政局在新墨西哥州的一家大型圆形结构。由成像前20分钟，氮源耗尽后，我们可以判断基因簇（GFP）和网管/国家邮政局之间的距离。氮耗尽之前和之后的均值或中位数的距离相比，因此，我们可以量化是否有一个氮枯竭后的基因簇的亚细胞定位的转变。

Watch this Scientific Journal Video about Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast at JoVE.com

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

裂殖酵母，<em&gt;裂殖酵母</em&gt;，是一个很好的模型系统研究的基本细胞过程。在这里，我们描述了一种方法来执行裂殖酵母活细胞定量分析。在这个特殊的实验中，我们专注于细胞核内的基因组的组织，但该方法也可用于研究细胞质的因素。

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

quantitative-live-cell-fluorescence-microscopy-analysis-fission

Research

JoVE Journal

Biology

20.1K 次观看.  University of Uppsala. 裂殖酵母，裂殖酵母，是一个很好的模型系统研究的基本细胞过程。在这里，我们描述了一种方法来执行裂殖酵母活细胞定量分析。在这个特殊的实验中，我们专注于细胞核内的基因组的组织，但该方法也可用于研究细胞质的因素。

视频: Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

科研

生物学

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Cell Electrofusion Visualized with Fluorescence Microscopy

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

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

荧光显微镜下细胞电融合的可视化

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

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...