Name: automated measurement of cryptococcal species polysaccharide capsule and cell body
Uploaded: 2018-01-11T15:00:00
Description: Watch this Scientific Journal Video about Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body at JoVE.com

我们要感谢安东尼. 鲍文, 他的幻灯片被作为第二个人并排比较, 以及他的幻灯片被用作第三人并排和第二次显微镜比较。

1. 印墨幻灯片的制作
<ol><li>吸管10µL 的隐球菌样本到幻灯片上。任何循环酵母菌株将工作, 但这个实验 H99 是唯一的应变使用。 注: 如果样品直接来自培养基, 用 PBS 或水稀释1:2 可以帮助防止墨汁结块。</li>
	<li>吸管2µL 印度墨水污点的样品和混合通过物理推动吸管尖端的样品和移动, 直到印度墨水出现均匀分布。</li>
	<li>通过将片的左边缘与幻灯片的表面片, 然后在样品上轻轻地、均匀地降低片?...

<ol>
	<li>Park, B. J., Wannemuehler, K. A., Marston, B. J., Govender, N., Pappas, P. G., Chiller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+current+global+burden+of+cryptococcal+meningitis+among+persons+living+with+HIV/AIDS.">Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS.</a> AIDS. 23 (4), 525-530 (2009).</li><li>Kwon-Chung, K. J., 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=Cryptococcus+neoformans+and+Cryptococcus+gattii,+the+etiologic+agents+of+cryptococcosis.">Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.</a> Cold Spring Harb Perspect Med. 4 (7), 019760 (2014).</li><li>Granger, D. L., Perfect, J. R., Durack, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Cryptococcus+neoformans.+Regulation+of+capsule+synthesis+by+carbon+dioxide.">Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide.</a> J Clin Invest. 76 (2), 508 (1985).</li><li>Rumbaugh, J., Pool, A., Gainey, L., Forrester, K., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Cryptococcal+Capsule+in+Pathogenesis+of+Cryptococcal+Meningitis.">The Role of Cryptococcal Capsule in Pathogenesis of Cryptococcal Meningitis.</a> Neurology. 80 (7), 007 (2013).</li><li>Bojarczuk, A., 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=Cryptococcus+neoformans+Intracellular+Proliferation+and+Capsule+Size+Determines+Early+Macrophage+Control+of+Infection.">Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection.</a> Sci Rep. 6, (2016).</li><li>Robertson, E. J., 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=Cryptococcus+neoformans+Ex+Vivo+Capsule+Size+Is+Associated+With+Intracranial+Pressure+and+Host+Immune+Response+in+HIV-associated+Cryptococcal+Meningitis.">Cryptococcus neoformans Ex Vivo Capsule Size Is Associated With Intracranial Pressure and Host Immune Response in HIV-associated Cryptococcal Meningitis.</a> J Infect Dis. 209 (1), 74-82 (2014).</li><li>Araujo, G. d. e. 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=Capsules+from+Pathogenic+and+Non-Pathogenic+Cryptococcus+spp.+Manifest+Significant+Differences+in+Structure+and+Ability+to+Protect+against+Phagocytic+Cells.">Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells.</a> PLoS One. 7 (1), 29561 (2012).</li><li>Garc&#237;a-Rivera, J., Chang, Y. C., Kwon-Chung, K. J., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+CAP59+(or+Cap59p)+Is+Involved+in+the+Extracellular+Trafficking+of+Capsular+Glucuronoxylomannan.">Cryptococcus neoformans CAP59 (or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan.</a> Eukaryot Cell. 3 (2), 385-392 (2004).</li><li>Guimar&#227;es, A. J., Frases, S., Cordero, R. J. B., Nimrichter, L., Casadevall, A., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+responds+to+mannitol+by+increasing+capsule+size+in+vitro+and+in+vivo:+Mannitol+impacts+the+structure+of+C.+neoformans+capsule.">Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo: Mannitol impacts the structure of C. neoformans capsule.</a> Cell Microbiol. 12 (6), 740-753 (2010).</li><li>Zaragoza, O., Fries, B. C., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Capsule+Growth+in+Cryptococcus+neoformans+by+Mammalian+Serum+and+CO2.">Induction of Capsule Growth in Cryptococcus neoformans by Mammalian Serum and CO2.</a> Infect and Immun. 71 (11), 6155-6164 (2003).</li><li>Rossi, S. A., 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=Impact+of+Resistance+to+Fluconazole+on+Virulence+and+Morphological+Aspects+of+Cryptococcus+neoformans+and+Cryptococcus+gattii+Isolates.">Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates.</a> Front Microbiol. 7, (2016).</li></ol>

这一技术的关键步骤是准备印度墨水幻灯片和获取显微镜图像。虽然该算法已成功地测试了各种幻灯片和图像技术的推荐协议在这篇手稿中描述。该多糖胶囊是基于印度墨水微粒从胶囊的领域排除, 因为这些粒子太大, 渗透到多糖纤维网络。印度油墨的排斥导致一个明亮的圆圈顶部的黑暗背景。该算法根据它们与背景的对比度来检测圆。因此, 大量印墨色斑会导致多糖胶囊与背景的对比度增高, 增?...

新生隐球菌是一种致病性酵母, 在全球范围内发现无所不在, 与人类疾病主要是免疫人群有关。C. 隐球菌最显著的原因是在撒哈拉以南非洲地区因传染性疾病而导致的全年死亡的一个重要因素1。隐球菌感染的主要临床表现是脑炎, 它是在受感染的巨噬细胞 (特洛伊木马方式) 或直接越过血脑屏障之后, 通过中枢神经系统的侵袭而发生的。C. 隐球菌表达了多种致病因子, 包括在人体温度、脲酶活性、黑和多糖胶囊的形成过程中复制的能力2。该多糖胶囊是由重复 glucuronoxylomannan 和 glucoronoxylomannangalactan 聚合物和功能作为保护屏障的因素, 如环境压力和宿主免疫应答2。
虽然隐球菌多糖胶囊大小的大小并没有持续与毒力相关, 但有证据表明它是致病因素2,3,4,5, 6,7。胶囊大小与脑膜炎病理相关6, 可影响巨噬细胞控制新生感染的能力5, 如果缺席则可能导致毒力丧失8。因此, 胶囊大小测量是常见的隐球菌研究, 但没有 fieldwide 标准的方法的胶囊测量。
目前, C. 隐球菌多糖胶囊的测量是基于手工测量的显微图像, 而且图像和测量的精确方法在实验室中各有不同9,10, 11。这种方法的一个直接的关注是, 一些研究需要获得数以千计的个人测量, 这使得保持准确性和可靠性的困难。此外, 即使在公布结果时, 对测量方法的描述也往往不够充分。许多出版物不解释如何获得他们的测量, 使用了什么焦平面, 他们如何确定胶囊的识别阈值, 无论他们使用的半径或直径, 无论是使用一个测量或平均数, 或其他详细.有些出版物仅说明使用的程序的方法, 例如, "Adobe Photoshop CS3 用于测量单元格"11。这种缺乏标准化和报告细节的情况可能会使重现性困难, 如果不是不可能的话。人的视力、电脑亮度、显微镜设置、滑动照明和其他因素之间的差异不仅在个体之间, 而且在样本之间也会有所不同, 而基于像素强度值的比值的计算将保持不变,适用于样品之间。这项技术是在提供一个标准化的, 准确的, 快速的, 简单的技术来测量胶囊大小的领域中, 没有以前。
如前所述, 海隧算法是长期的, 并且脚本自动地检测圈子以前被写了。此方法在其他脚本不足的两个方面有所改进。首先, 仅仅检测圆是不够的, 因为与隐球菌细胞的两个不同的圆圈必须相互关联检测。这种方法专门检测胶囊内的细胞体, 区分两者之间, 并只对相关的身体-胶囊对进行计算。第二, 即使遵循相同的协议, 不同的调查人员最终会得到不同的获取图像。通过允许调查人员控制每个算法参数, 可以调整该工具以匹配广泛的获取方法。不需要标准化的范围、目标、筛选等。
这种技术可以很容易地应用到任何情况下, 调查人员需要检测的圆圈内的图像, 与他们的背景形成对比。两个圆圈比他们的背景更亮, 更暗, 可以检测, 计数, 并用这种技术测量。

<table border="0" cellpadding="0" cellspacing="0" style="width:875px;" width="875">
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">India Ink</td>
			<td style="width:99px;">Becton, Dickinson and Co.</td>
			<td style="width:119px;">261194</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Fisherbrand Superfrost Microscope Slides</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-143</td>
			<td>25x75x1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Fisherfinest Premium Cover Glass</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:119px;">12-548-B</td>
			<td>22x22-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Sally Hansen HardasNails Xtreme Wear Nail Polish</td>
			<td style="width:99px;">Sally Hansen</td>
			<td style="width:119px;">N/A</td>
			<td>109 invisible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">SAB Media</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">S3306</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:213px;">Cryptotoccus neoformans</td>
			<td style="width:99px;">ATCC</td>
			<td style="width:119px;">208821</td>
			<td>H99 strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Olympus AX70 Microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:119px;">AX70TRF</td>
			<td>Discontinued ; Bright Field Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Qimaging Retiga 1300</td>
			<td style="width:99px;">Qimaging</td>
			<td style="width:119px;">N/A</td>
			<td>Discontinued ; Camera Microscope Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">MATLAB</td>
			<td style="width:99px;">MathWorks</td>
			<td style="width:119px;">N/A</td>
			<td>Most recent version recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Python Programming Language</td>
			<td style="width:99px;">Python</td>
			<td style="width:119px;">N/A</td>
			<td>Version 2 necessary ; 2.7 recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Microsoft Excel</td>
			<td style="width:99px;">Microsoft</td>
			<td style="width:119px;">N/A</td>
			<td>Most recent version recommended</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">P3813</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated measurement of cryptococcal species polysaccharide capsule and cell body

这项技术的目的是为大量的多糖胶囊测量提供一个一致的, 准确的, 可管理的过程。
首先, 根据每个图像的唯一计算的强度值生成阈值图像。然后, 利用完备的圆霍夫变换 (红隧) 算法, 根据对象和背景的对比度来检测圆。最后, 根据中心坐标和半径大小对检测到的细胞胶囊和体进行匹配, 并在可管理的电子表格中将数据导出到用户。
这种技术的优点很简单但意义重大。首先, 由于这些计算是由一个算法而不是一个人来执行的, 所以精确度和可靠性都提高了。 无论分析多少样品, 准确度或可靠性都没有下降。其次, 此方法为 "隐球菌" 字段建立了一个潜在的标准操作过程, 而不是实验室中的胶囊测量变化的当前情况。第三, 由于手动胶囊测量是缓慢和单调的, 自动化允许快速测量大量的酵母细胞, 反过来又有利于高吞吐量数据分析和日益强大的统计。
该技术的主要局限性来自于该算法的功能。首先, 该算法将只生成圆。当隐球菌细胞及其胶囊采用圆形形态时, 很难将这种技术应用于非圆形物体的检测。其次, 由于如何检测圆, 红隧算法可以根据多个簇圆的外边缘检测出巨大的 pseudo-circles。但是, 在 pseudo-circle 中捕获的任何错误的单元格都可以很容易地检测到并从结果数据集中删除。
本技术是以印度油墨的光场显微镜为基础, 测定新生种的圆形多糖胶囊;虽然它可以应用于其他基于对比度的圆形物体测量。

图像首先由印度墨水幻灯片的显微镜, 使用明亮的野外显微镜和相机 (图 1a) 来获得。重要的是要使细胞分离, 并在足够低的密度不压倒的视野, 以及使用足够的污渍, 以创造对比细胞和背景。如协议中所述, 最佳图像的单元格的确切数目将因所使用的样本、显微镜和目标而异。重要的方面是确保细胞不聚集或重叠, 细胞的焦平面不明显变化,...

Watch this Scientific Journal Video about Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body at JoVE.com

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

自动测量隐球菌种多糖胶囊和细胞体

该技术描述了一种用于测量多糖胶囊和体半径的自动批处理图像处理器。最初设计为新生隐球菌胶囊测量时, 自动图像处理器也可应用于其他基于对比度的圆形物体检测。

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Research

JoVE Journal

Immunology and Infection

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

科研

免疫与感染

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

尺寸事项:新生隐球菌中胶囊直径的测定

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

红外测温仪在被动系统过敏反应和食品过敏评价中的应用

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

使用来自同一物种的抗体进行双标记免疫荧光研究宿主 - 病原体相互作用

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...