格兰特支持，国家科学基金会，美国国立卫生研究院NIH CA165263 CA150197：NIH CA150197S1（HJ功夫），：NIH CA150197S1（CA Changou）PHY-0120999 中心生物光子学科学与技术 （DL欧胜，庄花园街），国防部PC073420（RJ大胆），研究挪威理事会，：儿子里夫埃埃里克森旅游格兰特209286/F11（BS阿鲁瓦莉）。 HJ功夫也承认奥本社区癌症基金的支持。 RJ大胆也承认J.麦当劳养老的支持。 我们感谢珍妮博士伟仁西贡博士和吴柏文DesigneRx慷慨供给ADI。

1。第1部分：细胞培养，免疫荧光标记<ol><li> CWR22 RV1人前列腺肿瘤细胞生长在盖玻片（＃1.5或170微米厚）置于6孔培养板中，含有10％胎牛血清（FBS）和1％青霉素/链霉素/谷氨酰胺的RPMI（敏达，VA）。 </li><li>引起细胞自噬与精氨酸脱亚胺酶（ADI，0.3微克/毫升）的磷酸盐缓冲盐水（PBS）处理选定的样品。 </li><li>固定细胞，用4％多聚甲醛（PFA，Fisher Scientific公司，NH）在PBS中稀释，将100μl每盖玻片，在RT下10分钟。 </li><li>用1毫升HBS / BSA洗涤细胞3次。 </li><li>通透细胞用0.05％皂甙（Sigma公司），HEPES（HBS）/ BSA，加入100μl每盖玻片，在RT下的15分钟。 </li><li>免疫标记，块样品与2.5％的酪蛋白（Sigma公司），每盖玻片100微升，HBS / BSA 1小时RT之前。 </li><li>孵育固定细胞主要抗体稀释在2.5％酪蛋白/ HBS / BSA，每coversl 100微升IP，O / N在4°C。 </li><li>用1毫升HBS / BSA洗涤细胞3次。 </li><li>固定细胞孵育二次抗体稀释在2.5％酪蛋白/ HBS / BSA，每100微升盖玻片，在室温下1小时。 </li><li>用1毫升HBS / BSA洗涤细胞3次。 </li><li> SlowFade金制备的盖玻片，在显微镜玻片安装或延长Gold抗淬灭试剂（Invitrogen，CA），含有4&#39;，6 - 二脒-2 - 苯基吲哚（DAPI）。 </li><li>指甲油密封盖玻片边缘。为了获得最佳的成像效果，让时间（理想的O / N）为防褪色媒体的彻底渗透细胞。 </li><li>清洁盖玻片的表面与甲醇和/或氯仿。 </li><li>浸油（折射率1.520成像时，在37°C或1.516成像时，在RT）60X 1.42 NA物镜（奥林巴斯，日本）。 </li><li>滑动感兴趣的细胞在显微镜舞台上，并建立重点。 </li></ol> 2。第2部分：准备细胞实时成像<ol><li>长大无缝线路22 RV1细胞表达绿色荧光蛋白偶联型轻链3（LC3-GFP）的RPMI培养基中，在35 mm的聚-D-赖氨酸涂覆的玻璃底培养皿（MatTek，MA）中含有10％FBS和1％抗生素。应接种于细胞足够的密度，方便快速扩散，但没有那么多细胞覆盖，成群成像时。 </li><li>治疗选择与ADI（0.3微克/毫升）的PBS的细胞样本。 </li><li>约1小时，在成像之前，稀释红色LysoTracker DND-99（Invitrogen公司，CA），用20毫升的RPMI 1.5微升。含有10％FBS和1％抗生素。使用的解决方案，包含ADI选定样本。预热到37°C前加入培养皿所有媒体。 </li><li>与红的RPMI含有LysoTracker的DND-99 15-45分钟，在37°C孵育</li><li>约30分钟，在成像之前，打开的WeatherStation环境围封，并允许平衡到37℃和5％CO 2的（humidif与IED的空气）。 </li><li>用PBS洗涤细胞，并与标准的RPMI只含有10％FBS和1％抗生素更换介质。 ADI表示样品。 </li><li>安装35毫米盖玻片底培养皿定制适配器（应用精密公司，WA），并在显微镜舞台上的位置。 60X 1.42 NA物镜用浸油（折射率1.520），并在舞台上的位置安装的培养皿。 </li></ol> 3。第3部分：反卷积显微镜和分析该协议在本节中假定DeltaVision的个人DV卷积显微镜和相关的应用程序套件（应用精密公司，WA）SoftWorX使用。 <ol><li>打开显微镜系统，包括采集工作站和仪器控制器。打开Resolve3D应用初始化显微镜阶段，并开启氙灯光源。允许光源的热身和达到稳定的条件下为10分钟。 </li><li>添加滴浸油（折射率1.520活样品在37℃或固定的样品在RT 1.516）60X 1.42 NA物镜，在物镜的中心滑动试样（盖玻片正面朝下）。 </li><li>无论是使用明视场或外部照明和粗调调节旋钮，慢慢地提高胎圈浸油物镜，直到与倒置的玻璃盖接触。 （从这点来说，应该成为焦点的细胞层内几圈微调对焦调节旋钮）。当使用固定的样品在幻灯片上，它被建议，以避免细胞内或附近有气泡的任何领域。当观看直播样品上的玻璃底菜，避免移动物镜玻璃盖玻片的边界之外。 </li><li>自噬体可以识别eGFP的信号（绿色）。溶酶体确定由LysoTracker红（现场采样）或反灯泡1荧光抗体（固定样本）。细胞的细胞核用DAPI圣发现癌宁（固定样本）。 </li><li>选择理想的视野。理想的情况下，细胞应具有良好的荧光信号在所有适当的渠道，并充分地附着盖玻片表面上摊开，使细胞内的内容是很容易想象的。侧的x，y和z聚焦在小的调整，可以控制由计算机使用Acquire3D接口。设置分级1X1或2x2的，这取决于所需的视场（更多的细胞中可以看出分级设置为2x2的领域）。 </li><li>对于一个给定的图像通过一个电池的中部，设置最大像素强度不超过3000个计数的曝光参数（ 例如 ％的发送功率和曝光时间）。一般情况下，％透光率应尽可能低的设置，相应的曝光时间在1秒内没有提高。重复此过程，每个荧光色进行成像，并为每个单独的视野，以获得最高质量的图像。 </li><li>设置在u稍稍调整焦点分别在顶部和底部的细胞样品，Z堆栈PPER和下限。 Z堆栈中的图像的总数，从而来确定这些限制和层与层之间的间距（默认值：0.2微米）。 </li><li>为了最大限度地减少运动伪影，图像采集过程中，我们建议设置图像“波长，那么Z-Stack的”用于固定试样，“Z-Stack的波长”现场样品采集模式。 </li><li>荧光图像反褶积使用SoftWorX（应用精密，WA）以后分析使用VoloCITY（即兴，现在上Perkin Elmer公司，MA）。 </li></ol> 4。第4部分：超高分辨率，结构照明显微镜（OMX） 本节中的协议适用于使用结构照明显微镜OMX（应用精密，WA）。 <ol><li>接通主电源和所需的激光器（410纳米，488纳米，和/或532纳米）。等待20英里n为激光热稳定。 </li><li>浸油（用于固定试样在室温下的折射率1.516）60X 1.42 NA物镜。如果气泡出现在油滴，清洁的目的，并再次添加机油。 </li><li>如将样品幻灯片的舞台上，并在必要时，允许10分钟的细胞在盖玻片上定居。 </li><li>初始化收购方案。使用荧光照明，直到锋利的细胞轮廓可以看出，调整的重点。 （在任何时候都应该小心，以尽量减少暴露的细胞荧光激发，直到实际图像采集）。 </li><li>螺旋拼接扫描可以获取的样品预览较大的地区，选择单元格或地区的利益。建议使用短的曝光时间（1-10毫秒），低激磁功率（0.1-1％传输），同时扫描样品或寻找目标。 </li><li> GFP-LC3荧光识别自噬。识别溶酶体的Alexa Fluor 555抗LAMP（溶酶体相关膜蛋白）和细胞核用DAPI（固定的样品）。 </li><li>选择的实验条件，包括激发波长，图像区域（ 例如，512×512），堆栈的厚度，曝光时间，激光透射百分率。对于超分辨图像，确保结构照明选项启用。在OMX卷积显微镜，选择传统的照明选项。 </li><li>为了获得最佳的重建结果中，选择的实验条件下，例如，在采集过程中在10,000和15,000之间的最大强度计数。漂白如果是一个问题，也可以使用在一个较低的曝光（5000和10000之间的数）的成像。理想情况下，不应该减少计数超过整个采集过程中的两个因素，应避免超过四个因素的减少。如果有必要，减少组的厚度，以确保持续的强度计数。相机饱和，所以在64,000计数最大张力不应该超过此值。为了获得良好的样本，我们发现1％的激光传输的实验条件下，10〜50毫秒的曝光时间。可以反复成像明亮的和耐光样品的首选。需要一个稳定的污渍时间推移，超分辨图像。 </li><li>为了减少噪音，95MHz读出速度（中等速度选项）和采集时间为2毫秒或以上推荐。增加文物得到重建成像，当样品在收购移动。由于这个影响，短期风险，建议非贴壁或快速移动的细胞。 </li><li>多通道成像可以同时或依次进行。顺序模式产量减少串扰和建议。 </li><li>为了减少光漂白，一般建议图像较长的激发波长（532纳米，488纳米和410纳米）。然而，对于在图3所示的实验结果，该顺序是反向。（ 即 48纳米，532纳米和410纳米）由于这个特定样品的光稳定性。 </li><li>设置上限/下限的Z堆栈的顶部/底部的细胞移动显微镜阶段，直到稍微失焦。在0.125微米的超分辨率成像所需的每幅图像之间的距离应保持不变，重建软件借此为默认值。 </li><li>图像重建栈使用SoftWorX（应用精密，WA）和分析使用的6.0 VoloCITY（Perkin Elmer公司，MA）。 </li></ol>

<ol>
	<li>Kim, R. H., Coates, J. M., Bowles, T. L., McNerney, G. P., Sutcliffe, J., Jung, J. U., Gandour-Edwards, R., Chuang, F. Y., Bold, R. J., Kung, H. 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没有利益冲突的声明。

虽然被广泛接受为一个标准的方法来确认自噬反应6相同的系统（因为我们已经做了），定量三维成像提供了前所未有的信息和细节的复杂过程，细胞自噬对LC3荧光探针标记细胞直接观察。特别是，我们观察到自噬诱导后80分钟内，数百（如果不是数千）在活细胞中的自噬体形成。同样，我们观察到在自噬诱导溶酶体舱室的分布非常有趣的形态学变化。在一个给定的单元格，在自噬体的数?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Arginine Deiminase (ADI)</td>
			<td>DesigneRx</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C5890</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>4042</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S4521</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa anti-mouse 555</td>
			<td>Invitrogen</td>
			<td>A21422</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa anti-rabbit 647</td>
			<td>Invitrogen</td>
			<td>A21244</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LysoTracker Red DND-99</td>
			<td>Invitrogen</td>
			<td>L7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-Lamp1</td>
			<td>DSHB</td>
			<td>H4A3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-Cadherin</td>
			<td>Cell Signaling</td>
			<td>#3195</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlowFade Gold</td>
			<td>Invitrogen</td>
			<td>S36936</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-d-lysine coated glass bottom plate</td>
			<td>MatTek</td>
			<td>P35GC-1.5-1.4-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>No.1, 22 mm coverslip</td>
			<td>Corning</td>
			<td>#2865-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Globe Scientific</td>
			<td>1324G</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

quantitative analysis of autophagy using advanced 3d fluorescence microscopy

手术虽然带有明显的阳痿和尿失禁的风险，前列腺癌是美国男性恶性肿瘤之间的主要形式，传统的化疗方法已基本成功。激素治疗是有效的，在早期阶段，但往往失败，并最终发展为激素难治性肿瘤。我们一直有兴趣在开发针对特定的肿瘤细胞代谢缺陷的疗法。我们最近发现，前列腺肿瘤细胞的特异性缺乏一种酶（精氨琥珀酸合成酶，或ASS）的合成中所涉及的氨基酸精氨酸1。这种情况会导致肿瘤细胞变得依赖于外生精氨酸，他们经过代谢应激游离精氨酸耗尽时由精氨酸脱亚胺酶（ADI）1,10。事实上，我们已经表明，人类前列腺癌细胞CWR22 RV1有效地杀死ADI依赖caspase的细胞凋亡和积极autophaGY（或macroautophagy）1,2,3。细胞自噬是一种进化上保守的过程，使细胞代谢多余的蛋白质在营养饥饿4,5溶酶体破裂。虽然这一途径的重要组成部分，良好的特点6,7,8,9，很多方面的分子机制仍不清楚-特别是，什么是自噬作用的前列腺癌细胞死亡响应ADI治疗后？为了解决这个问题，我们需要的实验方法来衡量的水平和程度在细胞中的自噬反应 - 因为没有已知的分子标记物，可以准确地跟踪这个过程中，我们选择了开发基于成像的方法，使用定量三维荧光显微镜11,12的 。 使用专门CWR22Rv1细胞自噬体和溶酶体的荧光探针标记，我们证明了收购3D图像栈或者无线defield卷积显微镜（以及后来的超高分辨率，结构照明显微镜），可以清晰地捕捉到自噬诱导的早期阶段。市售数字图像分析的应用程序，我们可以很容易地获得统计信息自噬体和溶酶体的数量，规模，分布和程度从任何成像细胞共存。这个信息让我们能够精确地跟踪在活细胞自噬的进展，使我们继续调查自噬作用在癌症化疗。

图1中所示的图象序列显示在第一个80分钟的自噬诱导的在CWR22细胞发生的物理变化。在这方面和其他的研究（图中未示出）中，我们持续观察到：（1）核内的位移相差的小区中心;（2）减少了粘着点，以及（3）朝向中心的自噬体和溶酶体一般易位细胞。此外，我们还观察到自噬体和溶酶体（绿色）（红色）之间的共存小幅增加（黄色），在稍后的时间点。 基于?...

Watch this Scientific Journal Video about Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy at JoVE.com

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

细胞自噬是一个无处不在的进程，使细胞蛋白质和细胞器的降解和回收利用。我们采用先进的荧光显微镜可视化和量化小，但必要的，与诱导自噬相关的物理变化，包括自噬体和溶酶体的形成和分布，以及他们融合成auto​​lysosomes。

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, ...

quantitative-analysis-autophagy-using-advanced-3d-fluorescence

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Cell Death

SCIENTISTS IN ACTION

17.8K 次观看.  University of California, Davis. 细胞自噬是一个无处不在的进程，使细胞蛋白质和细胞器的降解和回收利用。我们采用先进的荧光显微镜可视化和量化小，但必要的，与诱导自噬相关的物理变化，包括自噬体和溶酶体的形成和分布，以及他们融合成auto​​lysosomes。

视频: Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

科研

生物学

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 ...

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 ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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 ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

自噬的分析<em&gt;产黄青霉</em&gt;通过结合使用饥饿垫荧光显微镜

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

使用荧光显微镜驱动蛋白-1的货物的识别

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...