我们要感谢在弗雷德哈钦森癌症研究中心的乔纳森·库珀实验室的许可使用低温恒温器。这项工作是由美国国立卫生研究院和WM凯克基金会的支持。 BM是一个戈登和贝蒂·摩尔的生命科学研究基金会的研究员。我们非常感谢丹尼尔Gottschling，与我们分享他的固定协议。

1。固定酵母社区<ol><li>酵母社区成长的膜过滤器（ 例如 ，Millipore公司的MF膜的过滤器， 图2A）。该协议对应于小于2×10 8个细胞的一个6毫米直径的膜过滤器上的一个典型的社区。 </li><li>使用一块的Whatman 541滤纸覆盖1厘米直径的（ 图2B）的底部。此Whatman滤纸将用作载体，以在以后的阶段转让社区。加入1毫升100％的甲醇井，离开以及-20°C的温度达到平衡。 </li><li>把在杜瓦瓶中的液N 2。社区浸渍在液体中的至少15秒N 2使用镊子（ 图2C），在液氮中冻结的社群。 </li><li>把冷冻社区以及-20°C甲醇（ 图2D）。确保社会保持水平时淹没在甲醇。等待20分钟。 MF膜过滤器开始在甲醇中崩解。 </li><li>社区，使用该载体的Whatman 541滤纸转移到冷的陪替氏培养皿保持在-20℃的甲醇蒸发（ 图2E）以及从甲醇。传送时，社会上运营商Whatman过滤器，引诱多余的甲醇在寒冷的培养皿中，然后再将使用另一块厚的滤纸置于滴。这将确保样品与样品蒸发的时间是一致的。 </li><li>等待4-6小时蒸发，直到运营商Whatman过滤器看起来干的。蒸发留下残余的不足，甲醇干扰切片。过度干燥产生裂纹和变形的社区。 </li><li>样品在-20℃的冰箱中。切掉的Whatman 541滤纸覆盖的社区。 </li><li>车型的矩形立方体铝箔容器，该容器足够大，该样本具有至少〜2毫米毛利从的每一侧（ 图2F）。将社会上的铝箔容器的底面;，立刻覆盖的样品，10月至3〜4毫米以上社会高度。如果重要的是，样品的取向，可以调整在此步骤中与关于所述容器的侧面。等待10分钟。 </li><li>将OCT-嵌入式样品干冰冻结的金属板。保存在-20℃或-80℃的冰箱中冷冻社区。 </li></ol> 2。用于荧光成像切片冷冻社区<ol><li>冰冻切片室的温度设置到-25℃，并离开样品在室中，直至其温度达到该室的温度。 </li><li>样品安装到冷冻安装应用下降了OCT的安装后，。均匀地传播出去OCT暴露Whatman滤纸（在容器的底部）上的样品的侧使OCT的厚度为2-3毫米。让OCT冻结本身低温恒温器内室前ctioning。确保OCT缘2-3毫米的样品各方面的帮助，以保持完整的部分。 </li><li>可以使用OCT取样块的两侧对齐样品。如果样品未对齐相对于刀片，切片将开始的样品从一个角落或侧面。调整的安装，以确保刀片切割一个完美的垂直（或水平）面的样品块。 </li><li> 〜14微米厚的典型酵母社区部分。切割后的部分中，使用在室温下保持在显微镜载玻片，收集部分中，慢慢接近滑动到切割部分。本节将融化，转移到显微镜幻灯片。多达18个部分可以转移到一个幻灯片。 </li><li>在寒冷的温度，最好是-20°C，在成像之前，存储的幻灯片。是最小的，在4℃下保持一个星期时的信号损失为了获得最大的荧光信号，成像，优选立即进行后切片。 </li></ol>

<ol>
	<li>Tolker-Nielsen, T., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Organization+of+Microbial+Biofilm+Communities.">Spatial Organization of Microbial Biofilm Communities.</a> Microbial Ecology. 40 (2), 75-84 (2000).</li><li>Moller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Gene+Expression+in+Mixed-Culture+Biofilms:+Evidence+of+Metabolic+Interactions+between+Community+Members."> In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members.</a> Appl. Environ. Microbiol. 64 (2), 721-732 (1998).</li><li>Lepp, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methanogenic+Archaea+and+human+periodontal+disease.">Methanogenic Archaea and human periodontal disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6176-6181 (2004).</li><li>V&#225;chov&#225;, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environmental Microbiology. 11 (7), 1866-1877 (2009).</li><li>Min&#225;rikov&#225;, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+Gene+Expression+in+Cells+within+Yeast+Colonies.">Differentiated Gene Expression in Cells within Yeast Colonies.</a> Experimental Cell Research. 271 (2), 296-304 (2001).</li><li>Shou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+cooperation+in+engineered+yeast+populations.">Synthetic cooperation in engineered yeast populations.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1877-1882 (2007).</li><li>Momeni, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+generates+a+unique+spatial+signature+in+microbial+communities.">Cooperation generates a unique spatial signature in microbial communities.</a> , (2012).</li><li>Kiernan, J., ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histological and Histochemical Methods: Theory and Practice. , 4 (2008).</li><li>Piccirillo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+Pathway+and+Alkaline+pH+Regulate+Pattern+Formation+in+Yeast+Colonies.">The Rim101p/PacC Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies.</a> Genetics. 184 (3), 707-716 (2010).</li></ol>

没有利益冲突的声明。

这里介绍的过程提供了一种方法来检查的内部结构的酵母社区。甲醇定影步骤使得酵母菌落刚性8并保留的菌落的完整性，但是，它也导致收缩8，应考虑在解释结果。我们通常会看到30％左右的收缩高的酵母菌菌落，菌落直接嵌入到OCT无固定9。 为了避免收缩为cryosectioning，另一种方法是直接嵌入在华侨城社区，并冻结他们。荧光是保存在方法基于直接?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 100％甲醇</td><td> JT贝克</td><td> 9070-01 </td><td></td></tr><tr><td>组织康OCT化合物</td><td> Andwin科学</td><td> 4583 </td><td></td></tr><tr><td> Whatman公司级541号定量滤纸</td><td> Whatman公司</td><td> 1541-047 </td><td></td></tr><tr><td>微孔-MF膜过滤器</td><td>密理博</td><td> HAWP04700 </td><td></td></tr><tr><td>低温恒温器</td><td>徕卡</td><td> CM 1510小号</td><td></td></tr></tbody></table>

cryosectioning yeast communities for examining fluorescence patterns

微生物通常生活在社区。社区空间组织的细胞内被认为是影响社区1的生存和功能。光学切片技术，包括共聚焦和双光子显微镜，已被证明可用于观察细菌和古细菌的社区空间组织的2,3。共聚焦成像和物理切片的酵母菌菌落的组合显示内部组织单元4。然而，直接用激光共聚焦或双光子显微镜的光学切片只能达到几个细胞层深入酵母菌菌落。这种限制可能是因为强散射光从酵母细胞中4。 在这里，我们提出了一种基于固定和cryosectioning获得的荧光细胞内的空间分布酿酒酵母（Saccharomyces cerevisiae）社区。我们使用甲醇作为定香剂保存细胞的空间分布。固定社区的渗透与OCT化合物，冷冻，和cryosectioned在低温恒温器。荧光成像的部分揭示了内部组织社区内的荧光细胞。 酵母表达红色和绿色荧光蛋白的菌株组成的社区的例子演示的cryosectioning方法来揭示的空间分布的荧光细胞，以及酵母菌落内的基因表达2,3的电位。虽然我们的重点是在酿酒酵母（Saccharomyces cerevisiae）社区，同样的方法也可能适用于研究其他的微生物群落。

在图3中示出的荧光图像的垂直截面包含红色和绿色标记的竞争种群6酵母社区。这些社区包括两个原养型菌株的共享资源，包括空间的酵母和其竞争，导致，显示在它们的垂直的横截面的柱状图形7。分辨率下降到一个单一的单元，可以解决在横截面， 图3比较复制社区获得的（底部）和无（顶部）甲醇-定影后，在图1中的协议的横截面。不固?...

Watch this Scientific Journal Video about Cryosectioning Yeast Communities for Examining Fluorescence Patterns at JoVE.com

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

我们提出了一个协议冻结和cryosectioning的酵母菌社区观察荧光细胞的内部模式。该方法依赖于甲醇固定，OCT-嵌入社区内的荧光蛋白失活的细胞没有保留的空间分布。

检查荧光模式Cryosectioning酵母社区

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

cryosectioning-yeast-communities-for-examining-fluorescence-patterns

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.8K 次观看.  Fred Hutchinson Cancer Research Center. 我们提出了一个协议冻结和cryosectioning的酵母菌社区观察荧光细胞的内部模式。该方法依赖于甲醇固定，OCT-嵌入社区内的荧光蛋白失活的细胞没有保留的空间分布。

视频: Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

闪存冻结和Cryosectioning E12.5小鼠脑

科研

生物学

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

Biology of Microbial Communities  - Interview

微生物群落的生物 - 面试

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

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 ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

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 ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...