Name: quantitative analysis of aspergillus nidulans growth rate using live microscopy and open-source software
Uploaded: 2021-07-24T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software at JoVE.com

这项工作部分得到了"希腊可视化和监测基本生物过程研究基础设施"项目（MIS 5002755）的支持，该项目是在"加强研究和创新基础设施"行动下实施的，该项目由"竞争力、创业和创新"业务方案（NSRF 2014-2020）资助，由希腊和欧盟共同资助。

1. 接种准备
注：所有步骤应在层压流柜下执行。
<ol><li>摆脱感兴趣的真菌菌株， 从使用无菌接种环的甘油库存 （-80 °C） 到最小介质 （MM） 板，辅以与所检查的菌株相关的适当营养要求 [MM： 10.0 g/L 葡萄糖，20 mL/L 盐溶液（盐溶液：26 克/升 KCl，26 克/升 MgSO4+7H2O， 76 克/升 KH2PO4， 2.0 mL/L 氯仿） 和 1 mL/L 微量元素 （微量元素： 40 毫克/L Na2...

<ol>
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通过延时显微镜监测真菌细胞生长和表型是实时、定量和准确地评估细胞行为的有力方法，它能够确定特定的药物治疗和/或基因干预是否导致可检测到的细胞生长或表型差异。
在这项研究中，描述了一个可靠的活细胞成像方法来测量和定量分析真菌的发展，包括细菌管的动态和直肠尖生长在 A.尼杜兰。使用传输的光显微镜技术监测随着时间推移的形态变化，对于识别?...

丝状真菌具有重要的社会经济和生态重要性，作为工业/农业工具，酶和抗生素生产1，2和作物植物的病原体3，害虫昆虫4和人类3。此外，像阿斯珀吉卢斯尼杜兰这样的丝状真菌被广泛用作基础研究的模型生物体，如遗传学、细胞和进化生物学的研究，以及连字符延伸5的研究。纤维真菌是高度两极分化的生物体，通过膜脂质/蛋白质的连续供应和细胞壁在延伸尖端6的脱光合成而拉长。在直写尖端生长和极性维护中的核心作用是名为"斯皮琴科珀"（SPK）的专门结构，这是一种高度有序的结构，主要由细胞骨骼成分和Golgi6、7、8的极化分布组成。
环境刺激/信号，如水-空气接口，光，CO2 浓度和营养状况负责这些模具9的发展决定。在水下（液体）培养物中 ，A.尼杜兰的 分化被抑制，生长通过连字符尖拉长6发生。在植物生长过程中，无性孢子（conidia）通过顶点延伸发芽，形成一个由相互关联的连字符细胞（mycelium）组成的无差别网络，只要有营养和空间，这种细胞就可能无限期地生长。另一方面，在固体介质连字符提示拉长和经过一个明确的植物生长期（发育能力），无性繁殖开始和空中锥形鼻从细胞的专门足细胞延伸。这些产生专门的发展多细胞结构称为针叶，产生长链的黄体针叶10， 可以在有利的环境条件下重新启动生长。
测量丝状真菌生长的一种广泛使用的方法是在培养皿中含有的营养阿加上接种孢子，并在几天后11日宏观测量菌落的直径。殖民地的直径/面积，最依赖于天体生长速度的变化，而较少依赖于圆锥体密度12，然后用作生长值。虽然测量在固体表面生长的真菌种群（科洛尼）大小是相当充分的，但这绝不是最准确的生长量。与种群水平平均值（真菌群大小的平均值）相比，单细胞测量可以捕获细胞群的异质性，并允许识别细胞的新型亚种群，状态13，动力学，通路以及细胞对内源和环境变化作出反应的生物机制14，15。通过延时显微镜监测真菌细胞生长和表型可以说是最广泛采用的定量单细胞观察方法。
在此，我们详细介绍了一个无标签的实时成像协议，使用传输的光显微镜技术（如相对比度、差分干扰对比度 （DIC） 和极化显微镜）来捕捉图像，这些图像独立于荧光显微镜的组合使用，可用于分析和量化水下文化和固体介质中 A. nidulans 菌株的极性生长。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Slide 8 Well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>Imaging slides</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic acid</td>
			<td>Merck</td>
			<td>A9878</td>
			<td></td>
		</tr>
		<tr>
			<td>azhA&#916; ngnA&#916;</td>
			<td></td>
			<td></td>
			<td>Genotype: zhA&#916;::pyrGAf; ngnA&#916;::pyrGAf; pyroA4 pantoB100 / References:Laboratory collection, Athanasopoulos et al., 2013</td>
		</tr>
		<tr>
			<td>Bacto Casamino Acids</td>
			<td>Gibco</td>
			<td>223030</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Merck</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate</td>
			<td>Merck</td>
			<td>C8027</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Statistical Software</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image processing and analysis software</td>
		</tr>
		<tr>
			<td>Inoculating Loop</td>
			<td>Merck</td>
			<td>I8263-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) phosphate</td>
			<td>Merck</td>
			<td>1.03935</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Microscope software</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Merck</td>
			<td>63138</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese(II) sulfate monohydrate</td>
			<td>Merck</td>
			<td>M7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Leica TCS SP8</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide (Niacinamide)</td>
			<td>Supelco</td>
			<td>47865-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Millipore</td>
			<td>68971</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes for Microbiology Culture</td>
			<td>KISKER</td>
			<td>G090</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Merck</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxine hydrochloride</td>
			<td>Merck</td>
			<td>P6280</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Microcentrifuge Tubes, 1,7 mL, DNase-, RNase and pyrogen free, sterile</td>
			<td>KISKER</td>
			<td>G052-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Microcentrifuge Tubes, 2.0 mL, sterile</td>
			<td>KISKER</td>
			<td>G053-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Standard Tips, Bevelled, 100-1000 &#181;L</td>
			<td>KISKER</td>
			<td>VL004G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali - Standard Tips, Bevelled, 1-200 &#181;L</td>
			<td>KISKER</td>
			<td>VL700G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quali Microvolume Tips, DNase-, RNase free, 0,1-10 &#181;L/clear</td>
			<td>KISKER</td>
			<td>GC.TIPS.B</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin (B2)</td>
			<td>Supelco</td>
			<td>47861</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades NO. 11</td>
			<td>OdontoMed2011</td>
			<td>S2771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate decahydrate</td>
			<td>Merck</td>
			<td>S9640</td>
			<td></td>
		</tr>
		<tr>
			<td>VS151 (PilA-GFP and H1-mRFP)</td>
			<td></td>
			<td></td>
			<td>Genotype: pyrG89; pilA::sgfp::AfpyrG+ argB2 nkuA&#916;::argB+&#160; pyroA4 hhoA::mrfp::Afribo+ riboB2 / References:Laboratory collection, Biratsi et al., 2021</td>
		</tr>
		<tr>
			<td>WT</td>
			<td></td>
			<td></td>
			<td>Genotype: nkuA&#916;::argB; pyrG89; pyroA4;pyrG89 / References: TN02A3 -FGSC A1149</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Millipore</td>
			<td>70161</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of aspergillus nidulans growth rate using live microscopy and open-source software

众所周知，通过比较菌落大小，在凝固介质上对长丝真菌的菌落生长进行宏观估计，主要取决于海眠/肌皮生长速度的变化。然而，在不同的环境/生长条件下（pH值、温度、碳和氮源、抗生素等）定量测量基因不同真菌株或菌株的生长速度是具有挑战性的。因此，为了更好地了解真菌细胞的生长，必须采用补充方法来量化生长动力学。此外，众所周知，丝状真菌，包括阿斯珀吉卢斯花，在固体介质或水下文化的亚空中条件下具有独特的生长和分化模式。在这里，我们详细介绍了一个定量的微观方法，用于分析模型真菌 阿斯珀吉卢斯尼杜兰的生长动力学，使用实时成像在水下文化和固体介质。我们使用开放源、免费的生物图像软件（例如斐济）以可重复和可靠的方式捕获不同真菌菌株的增长率，分析和量化其增长率，无需用户提供任何事先的图像分析专业知识。

按照这个协议，我们捕获并分析了各种图像对应于不同生长/发育阶段的长丝真菌 A.尼杜兰。本研究中提供的数据是使用斐济软件进行处理和分析的。测量保存为 csv 文件，使用商业统计软件进行统计分析并准备为图形和/或 Python 编程语言使用软件库，如熊猫、笨重、统计模型、马图利布和海出生。更多详情可在引用的原始出版物中找到。
为了解释人口的反应，重要的?...

Watch this Scientific Journal Video about Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software at JoVE.com

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

我们使用传输的光显微镜技术呈现一个无标签的实时成像协议，以捕获图像，分析和量化在水下培养物和固体介质中长丝真菌 A.尼杜兰的 生长动力学。此协议可与荧光显微镜配合使用。

使用实时显微镜和开源软件对 阿斯珀吉卢斯尼杜兰 生长速度的定量分析

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...

在这段视频中，我们描述了一个标签免费的实时成像协议，使用传输的光显微镜技术捕捉模型真菌阿斯珀吉卢斯尼杜兰的图像。我们使用此图像分析和量化液体和固体介质中阿斯珀吉卢斯尼杜兰的生长动力学。此协议的视觉表示允许用户熟悉图像捕获和显微镜图像处理的重要步骤。用于测量长丝真菌生长的常见方法是，在含有营养阿加的培养皿中接种两个接种真菌孢子，并在几天后测量发育中的菌落的直径。然而，这种方法不够敏感，无法量化精细的增长差异。因此，利用延时显微镜进行了单细胞测量。这种测量不仅能够提供准确和定量的结果，而且还能够反映人群中不同细胞的生长动力学。此外，这种方法旨在更好地了解真菌生长对内源性和环境信号的反应所涉及的分子机制。首先将15毫升的液体营养阿加倒入培养皿中。将盖子放在顶部，让它冷却。使用紫外线辐射对板进行消毒。在从股票中划出感兴趣的真菌菌株之前，加热邦森燃烧器中的点头环，直到它变热，通过刺入阿加来冷却环。漩涡管和条纹环穿过表面的agar最小的介质补充了适当的营养要求。在37摄氏度下孵育板两到三天。使用无菌牙签，通过轻轻触摸单个菌落将少量针叶转移到完整的介质板上。在37摄氏度下孵育板三到四天。阿斯珀吉卢斯尼杜兰的Conidia，在无菌的1.5毫升漩涡管中收获，1.0毫升的高压灭菌蒸馏水每卷含有0.05%的体积，Tween 80，用于减少针叶团的数量。倒置阿加方法的修改版本用于在阿加中等表面成像丝状真菌。最初发现10微升的大力涡旋锥体悬浮物，大约2倍于每毫升104细胞到15毫升的Petri菜最小介质，每卷一个重量的阿加。根据研究的发展阶段孵化实验文化。在这里，我们在30摄氏度下孵育三天。之后，使用无菌手术刀切出一块含有菌落的阿加。在这里，我们在殖民地边缘切出一块美洲狮楔子，倒置，将阿加片放入八个井滑梯中。转移10微升的大力涡旋锥体悬浮约2倍10在井滑梯的井中，包含200微升的最小介质与适当的补充。每次检查的时间和温度孵育。显微镜的选择取决于可用的设备。在任何情况下，设置的显微镜应包括倒置舞台、环境室或至少具有精确空气温度控制的房间。最初预热恒温器和显微镜室，以稳定所需的温度。在这里，我们把温度定在30摄氏度。打开显微镜、扫描仪电源、激光电源和计算机，并加载成像软件。将先前准备好的滑动放在显微镜阶段并集中注意力。查找包含隔离、不重叠的单元格或至少不过度拥挤的视图字段，以便在图像分析期间促进生长测量。选择要使用的传输光显微镜技术，并在必要时激活传输的光探测器以及荧光探测器。设置显微镜，以所需的时间间隔获取图像，并开始时间系列采集。图像的加载、可视化和处理是通过开源图像J斐济软件完成的。首先，使用插件格式将图像导入斐济。选择默认颜色模式和自动刻度。为了可视化时间戳和刻度栏作为叠加去分析，工具，刻度栏。设置有关秤杆宽度、高度、颜色和位置的预期参数。去图像，属性，以显示图像属性在图像J斐济软件。观察帧间隔信息。在这里，我们使用大约15分钟的时间间隔，并按好。然后转到图像、堆叠、标记，并将正确的信息设置为间隔。通过修改 X 和 Y 设置现场文本中的测量单元并按预览设置标签的位置。使用直方图匹配，通过选择图像、调整、漂白校正、直方图匹配，在不同帧之间进行照明校正。出现了一个带有校正照明的新窗口。使用 MtrackJ 插件插件插件插件 MtrackJ 来跟踪不断增长的连字符提示要添加轨道，请选择工具栏中的添加按钮，然后使用鼠标的左键将第一点放在连字符提示处。时间系列将自动移动到下一帧。要完成跟踪过程，请双击鼠标到最后一点，或按下逃生键。滚动输出表，计算连字符尖端增长的最重要列，是任何给定帧的速度。安全跟踪测量，添加文件，保存为您选择的文件格式，例如CSV，将保存的文件导入您的电子表格程序，并计算可视化和进一步测试。按下电影按钮生成电影 RGB 颜色类型，其中包含画入其中的轨道的帧，可在任何标准多媒体播放器中可视化。按照这个协议，我们捕获并分析了与长丝真菌阿斯珀吉卢斯尼杜兰的不同生长发育阶段相对应的各种图像。图为野生类型与阿扎德三角洲 ngnA 三角洲增长率测量的比较。azhAD 德尔塔 ngnA 三角洲是两个基因中的双重删除菌株，与两种卡西酸中添加的有毒战斗机产品 L 的排毒和同化有关。与水下液体培养物中的野生类型菌株相比，其统计学上显著降低增长率测量值。通过用固体最小介质测量这两个菌株的菌落面积，无法检测到这种生长差异。单细胞、长期活成像对于获取细胞蛋白动力学的时空信息具有重要的价值。此协议也适用于进行长期活细胞成像。在这里，我们看到科尼迪亚共同表达GFP标记的核心艾索马尔蛋白PilA和mRFP组织H1的发芽。在这里，我们提出了一个协议，以可重复和可靠的方式分析真菌生长动力学，而无需任何来自用户的任何以前的图像分析经验。此协议允许对真菌生长进行客观准确的量化。

Research

JoVE Journal

Biology

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

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

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

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 ...

科研

生物学

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

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

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 ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

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, ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

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 ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

使用实时显微镜和开源软件对 阿斯珀吉卢斯尼杜兰 生长速度的定量分析

使用实时显微镜和开源软件对阿斯珀吉卢斯尼杜兰生长速度的定量分析