我们感谢分子生物科学 (手机) 博士计划和中田纳西州立大学 (MTSU) 的生物学系为这项研究提供资金。该项目还部分由 MTSU 基金会授予 D.E.N. 的特别项目赠款提供经费。

注意: C. 隐球菌是生物安全级别 2 (BSL-2) 病原体, 研究人员必须采取适当的预防措施。有关如何与 BSL-2 病原体安全工作的详细程序, 可在疾病控制中心网站上找到, 但重要的是要注意, 所有接触到C. 隐球菌的人都应该接受适当的训练来处理致病剂应始终佩戴适当的个人防护用品 (PPE), 一般是乳胶或丁腈橡胶手套。此外, 离心机上的转子应密封, 以防止 aerosolization 的样品和任何泄漏清除立即使用10% 漂白剂解决方案28。
1. 胶囊诱导
注: 除非另行注明, 所有步骤均应在室温下进行。
<ol><li>在酵母蛋白胨葡萄糖 (YPD) 汤 (pH 值 6.5 +/-0.2) 中培养C. 隐球菌, 并在37摄氏度孵育, 直到它们处于对数阶段 (约 18-36 h)。</li>
	<li>离心细胞在 870 x g为5分钟在21°c 和洗涤三次与磷酸盐缓冲盐水 (PBS)。</li>
	<li>使用 hemocytometer (或自动单元格计数器) 在 Dulbecco 最小的基本介质 (DMEM) 中, 在 2 x 105 cells/2 mL 上计数单元格和并用重悬。</li>
	<li>诱导胶囊, 孵育在37°c 和 5% CO2为 18 h。 注意: 在介质中缺少营养素和 CO2的存在相结合会刺激C. 隐球菌胶囊的生长。</li>
	<li>孵化后, 通过离心 (如上所述) 收割细胞, 除去除5-10 µL 上清的全部。同时, 收获相应的非诱导细胞样本, 为每个菌株进行测量。使用这些作为负控制。 注: 细胞小球将非常小, 可能很难看到。吸上清液时要小心。</li>
	<li>用印度墨水 (2.1 节) 或荧光染料染色 (2.2 节)。</li>
</ol>2. 染色
<ol><li>
只用印度墨水染色
	<ol>
<li>为了用印度墨水染色酵母, 并用重悬在剩余的清液中的颗粒, 并将 4μL (大约一半) 的细胞悬浮物和4μL 的印度墨水添加到玻璃显微镜滑梯上。轻轻搅拌, 避免起泡。</li>
		<li>应用13毫米玻璃盖玻片 (#1 5 厚度) 和密封边缘与无毒密封胶 (清除指甲油), 以防止样品干燥。</li>
	</ol>
</li>
	<li>
只用荧光染料染色
	<ol>
<li>用荧光染料染色酵母, 并用重悬50µL PBS 中的细胞颗粒, 1% 牛血清白蛋白 (BSA)。</li>
		<li>用等量的 Calcofluor 白色 (CFW) (1 µg/毫升) 和蒴果单克隆 18B7 (参见材料表), 在37摄氏度的30分钟内, 孵化出与 AlexaFluor488 (AF488) (10 µg/毫升) 相结合的培养物。</li>
		<li>离心细胞在 870 x g 5 分钟在21°c 后孵化和吸出上清。</li>
		<li>并用重悬细胞颗粒在10µL PBS 与1% 牛血清白蛋白 (BSA)。</li>
		<li>吸管8µL 细胞悬浮在玻璃显微镜幻灯片上。</li>
		<li>应用13毫米玻璃盖玻片 (#1 5 厚度) 和密封边缘与无毒密封胶 (清除指甲油), 以防止样品干燥。</li>
	</ol>
</li>
	<li>
用印度墨水和荧光染料染色
	<ol>
<li>为了更直接地比较印度墨水染色与荧光染料的功效, 用两种着色剂对同一细胞种群进行染色。要做到这一点, 首先孵化细胞与荧光胶囊和细胞壁染色, 每步 2.2.1-2.2.3。</li>
		<li>接下来, 将荧光染色的电池悬浮液和印度墨水的等量容积 (4 µL) 移到玻璃滑梯上。轻轻地混合。</li>
		<li>应用13毫米玻璃盖玻片 (#1 5 厚度) 和密封的边缘与无毒密封胶 (清除指甲油), 以防止样品干燥。</li>
	</ol>
</li>
</ol>3. 图像采集/显微术
<ol><li>
用印度墨水染成的成像细胞。
	<ol>
<li>使用标准光显微镜 (100X 放大倍数) 获取图像。</li>
		<li>每个条件下至少有50个单元格的图像。 注意: 每个字段的单元格数会有所不同, 这是可以接受的。然而, 重要的是, 重叠的单元格保持在最低限度, 因为它们很难测量 (手动和自动软件)。对于这些实验, 图像获得了在深度16位, 曝光时间优化, 以最大限度地提高图像对比度, 同时尽量减少的模糊, 因为小的移动的细胞。</li>
	</ol>
</li>
	<li>
荧光染料染色的成像细胞
	<ol>
<li>通过荧光显微镜对细胞进行成像, 打开显微镜成像软件, 选择合适的显影激发和发射波长 (CFW 和 AF488 分别使用405nm 和 488 nm 灯激发)。使用荧光显微镜获取图像。</li>
		<li>每个条件下至少有50个单元格的图像。 注意: 每个字段的单元格数会有所不同, 这是可以接受的。然而, 重要的是, 重叠的单元格保持在最低限度, 因为这些都难以测量 (手动和自动软件)。</li>
		<li>获取单元格的 z 堆栈图像序列, 以确保获得给定字段中每个单元格的最大胶囊直径。<ol>
<li>在显微镜控制软件中, 点击 "z 栈" 栏打开 "z 栈" 控制面板, 然后选择面板左上角的 "第一个/最后一个" 选项。</li>
			<li>使用显微镜上的精细对焦控制, 集中在一个单一酵母细胞的最宽点以下的水平, 并在当前的视野中的所有胶囊, 并点击 "设置第一"。</li>
			<li>使用精细焦点控件将焦点放在同一个单元格体的最宽点之上, 并在当前字段中的所有单元格中, 单击 "最后设置"。</li>
			<li>然后, 单击 "获取" 选项卡上的 "开始实验" 以获取当前位置的 z 堆栈。在多个位置重复该过程, 直到获得至少50个单元格的 z 堆栈图像为止。 注: 对于这些实验, 共焦针孔被设置为 1.0 AU 和一个重叠 z 系列10-20 片覆盖0.7 µM 每一个在选定的位置获得。非通风单元。</li>
		</ol>
</li>
		<li>接下来, 使用适当的图像分析软件将每个 z 栈系列转换为最大强度投影 (MIP)。 注意: MIPs 项目在堆叠图像29的每一个平面上的最大强度。创建 MIPs 使一个人能够产生2维的图像表示形式, 对应于捕获的3维堆栈中的最大单元格和胶囊直径。</li>
	</ol>
</li>
	<li>
用印度墨水和荧光染料染成的成像细胞
	<ol>
<li>使用 widefield (40X 放大) 或共聚焦显微镜 (63X 或100X 放大) 获取图像。对于印有印度墨水和荧光染料的细胞, 使用 widefield 显微镜捕捉图像, 并配备计算机控制阶段和油浸泡目标。 注: 使用的显微镜应使用成像软件 CFW 控制。通过 340-380 nm 激发过滤器激发出的荧光, 发射的光应该通过从 400 nm 的分色镜中反射出来的 435-485 nm 屏障过滤器检测出来。AF488 荧光可以通过465-495 纳米励磁过滤器激发, 通过一个 505 nm 的分色镜反射的 515-555 nm 屏障过滤器可以检测出发射光。</li>
		<li>每个染色方法的每种条件下至少有50个细胞。</li>
	</ol>
</li>
</ol>4. 胶囊的手动测量
<ol><li>对于染有印度墨水或荧光污渍的细胞, 使用细胞测量软件手动测量胶囊和细胞直径 (参见材料表)。要做到这一点, 请转到 "文件" > "打开图像" > "度量值", 并使用游标在整个单元格的最宽点 (总直径) 中绘制一条直线。</li>
	<li>接下来, 再次单击 "测量", 并通过单元格体 (细胞体直径) 绘制一条直线。 注意: 测量被自动保存到单元格中。</li>
	<li>对所有单元格重复此过程 (最小为 50/组)。</li>
	<li>要在测量后保存图像, 请单击 "文件" > "另存为", 并相应地命名图像。</li>
	<li>选择新测量的文件并将数据导出到电子表格软件。</li>
	<li>用方程计算平均胶囊直径: ([总直径]-[细胞体直径])。</li>
</ol>5. 胶囊的自动测量
注意: 对于成功的自动测量, 请使用具有较高对比度的16位图像, 并将其与适当的图像分析软件适当地集中在一起 (请参见材料表)。当成像C. 隐球菌用于胶囊测量时, 重点放在细胞壁和胶囊之间的边界上的暗环上是很重要的。这使得软件能够正确地描绘细胞和胶囊。
<ol><li>使用适当的图像编辑软件反转并创建所有印有墨迹的彩色细胞图像的副本 (请参阅材料表)。为此, 请单击 "文件" > "打开" 以打开印度墨迹着色图像, 然后 "控制" + "I" 反转图像。单击 "文件" > "另存为" 以保存反转图像。</li>
	<li>要将原始图像和倒图导入到软件中, 请单击 "文件" > "导入序列" > "加载图像" > "定义序列 (手动)" > "维度 (通道)" > "导入" > "应用"。 注意: C. 隐球菌细胞体和胶囊可以用特定的算法 (称为 "食谱") 来识别和屏蔽, 该软件包括在内。</li>
	<li>使用 "菌落分析器 (荧光)" 食谱对倒置图像进行检测和掩模, 然后单击 "应用"。在反转图像中, 定义C. 隐球菌细胞边界的暗环比周围的胶囊更亮。</li>
	<li>使用 "菌落分析器 (荧光)" 食谱对倒置图像, 以分割C. 隐球菌细胞体从他们的胶囊, 然后单击 "应用"。这将创建计数掩码。通过右键单击标记为 "计数掩码" 的标签, 重命名此计数掩码 "单元格体"。 注意: 该食谱由多个图像处理步骤组成: 背景删除、对象检测、对象分离和子集筛选。</li>
	<li>接下来, 使用原始图像上的 "细胞增殖" 食谱来检测和遮盖胶囊, 然后点击 "应用"。这将创建计数掩码。通过右键单击标记为 "计数掩码" 的标签, 重命名此计数掩码 "胶囊"。</li>
	<li>使用食谱, ' 胶囊分区 ', 创建一个新的面具上的原始图像, 以分割相邻的胶囊彼此。为此, 请从食谱下拉菜单中单击 "胶囊分区"。在 "胶囊分区" 框中, 为胶囊区域选择现在重命名的计数掩码 "胶囊" (5.5)。为加密单元格选择现在重命名的计数掩码 "单元格体" (5.4)。为输入通道选择 "原件", 并 "为已分区的胶囊创建掩码。单击 "应用 注: 配方产生细胞和胶囊直径, 定义为穿过细胞和胶囊中心的最长和最短轴的平均值, 以及从检测掩模处测量的面积。查找此软件中 "电子表格" 选项卡下的输出。对所有图像重复此过程 (最少50个单元格/组)。该配方的参数可以调整, 以优化单个图像的检测或优化的批量检测多图像。额外的测量可以计算的配方, 综合表征细胞和胶囊的形态。</li>
	<li>将数据导出到电子表格软件的选择中进行进一步分析。</li>
</ol>

<ol>
	<li>Park, B. 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=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>Coelho, C., Bocca, A. L., 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=The+intracellular+life+of+Cryptococcus+neoformans.">The intracellular life of Cryptococcus neoformans.</a> Annu Rev Pathol. 9, 219-238 (2014).</li><li>Rajasingham, R., 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=Global+burden+of+disease+of+HIV-associated+cryptococcal+meningitis:+an+updated+analysis.">Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis.</a> Lancet Infect Dis. 17 (8), 873-881 (2017).</li><li>Limper, A. H., Adenis, A., Le, T., Harrison, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+infections+in+HIV/AIDS.">Fungal infections in HIV/AIDS.</a> Lancet Infect Dis. 17 (11), e334-e343 (2017).</li><li>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=Crisis+in+Infectious+Diseases:+2+Decades+Later.">Crisis in Infectious Diseases: 2 Decades Later.</a> Clin Infect Dis. 64 (7), 823-828 (2017).</li><li>McClelland, E. E. C., Eisenmann, A., H, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+6.">Ch 6.</a> New Insights in Medical Mycology. , 131-157 (2007).</li><li>Leopold Wager, C. M., Wormley, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+versus+alternative+macrophage+activation:+the+Ying+and+the+Yang+in+host+defense+against+pulmonary+fungal+infections.">Classical versus alternative macrophage activation: the Ying and the Yang in host defense against pulmonary fungal infections.</a> Mucosal Immunol. 7 (5), 1023-1035 (2014).</li><li>Kwon-Chung, K. J., Rhodes, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+and+melanin+formation+as+indicators+of+virulence+in+Cryptococcus+neoformans.">Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans.</a> Infect Immun. 51 (1), 218-223 (1986).</li><li>Vecchiarelli, 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=Elucidating+the+immunological+function+of+the+Cryptococcus+neoformans+capsule.">Elucidating the immunological function of the Cryptococcus neoformans capsule.</a> Future Microbiol. 8 (9), 1107-1116 (2013).</li><li>Murphy, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cryptococcal+antigens+on+cell-mediated+immunity.">Influence of cryptococcal antigens on cell-mediated immunity.</a> Rev Infect Dis. 10 Suppl 2, S432-S435 (1988).</li><li>Cherniak, R., Morris, L. C., Belay, T., Spitzer, E. D., 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=Variation+in+the+structure+of+glucuronoxylomannan+in+isolates+from+patients+with+recurrent+cryptococcal+meningitis.">Variation in the structure of glucuronoxylomannan in isolates from patients with recurrent cryptococcal meningitis.</a> Infect Immun. 63 (5), 1899-1905 (1995).</li><li>Collins, H. L., Bancroft, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+Cryptococcus+neoformans+impairs+antigen-specific+T-cell+responses.">Encapsulation of Cryptococcus neoformans impairs antigen-specific T-cell responses.</a> Infect Immun. 59 (11), 3883-3888 (1991).</li><li>Yasuoka, A., Kohno, S., Yamada, H., Kaku, M., Koga, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+molecular+sizes+of+Cryptococcus+neoformans+capsular+polysaccharide+on+phagocytosis.">Influence of molecular sizes of Cryptococcus neoformans capsular polysaccharide on phagocytosis.</a> Microbiol Immunol. 38 (11), 851-856 (1994).</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>Cordero, R. J., Bergman, A., 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=Temporal+behavior+of+capsule+enlargement+by+Cryptococcus+neoformans.">Temporal behavior of capsule enlargement by Cryptococcus neoformans.</a> Eukaryot Cell. 12 (10), 1383-1388 (2013).</li><li>O'Meara, T. R., Alspaugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cryptococcus+neoformans+capsule:+a+sword+and+a+shield.">The Cryptococcus neoformans capsule: a sword and a shield.</a> Clin Microbiol Rev. 25 (3), 387-408 (2012).</li><li>McClelland, E. E., Smith, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+specific+differences+in+the+immune+response+to+infection.">Gender specific differences in the immune response to infection.</a> Archivum Immunologiae et Therapiae Experimentalis. 59 (3), (2011).</li><li>McClelland, E. E., Perrine, W. T., Potts, W. K., 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=Relationship+of+virulence+factor+expression+to+evolved+virulence+in+mouse-passaged+Cryptococcus+neoformans+lines.">Relationship of virulence factor expression to evolved virulence in mouse-passaged Cryptococcus neoformans lines.</a> Infect Immun. 73 (10), 7047-7050 (2005).</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+CO(2).">Induction of capsule growth in Cryptococcus neoformans by mammalian serum and CO(2).</a> Infect Immun. 71 (1), 6155-6164 (2003).</li><li>Vartivarian, S. E., 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=Regulation+of+cryptococcal+capsular+polysaccharide+by+iron.">Regulation of cryptococcal capsular polysaccharide by iron.</a> J Infect Dis. 167 (1), 186-190 (1993).</li><li>McFadden, D. C., Fries, B. C., Wang, F., 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=Capsule+structural+heterogeneity+and+antigenic+variation+in+Cryptococcus+neoformans.">Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans.</a> Eukaryot Cell. 6 (8), 1464-1473 (2007).</li><li>Garcia-Hermoso, D., Dromer, F., Janbon, G. <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+capsule+structure+evolution+in+vitro+and+during+murine+infection.">Cryptococcus neoformans capsule structure evolution in vitro and during murine infection.</a> Infect Immun. 72 (6), 3359-3365 (2004).</li><li>Gates, M. A., Thorkildson, P., Kozel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+the+Cryptococcus+neoformans+capsule.">Molecular architecture of the Cryptococcus neoformans capsule.</a> Mol Microbiol. 52 (1), 13-24 (2004).</li><li>Pontes, B., Frases, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cryptococcus+neoformans+capsule:+lessons+from+the+use+of+optical+tweezers+and+other+biophysical+tools.">The Cryptococcus neoformans capsule: lessons from the use of optical tweezers and other biophysical tools.</a> Front Microbiol. 6, 640 (2015).</li><li>Shen, H., 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=Automated+tracking+of+gene+expression+in+individual+cells+and+cell+compartments.">Automated tracking of gene expression in individual cells and cell compartments.</a> J R Soc Interface. 3 (11), 787-794 (2006).</li><li>Dorn, J. F., Danuser, G., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+processing+and+analysis+of+dynamic+fluorescence+image+data.">Computational processing and analysis of dynamic fluorescence image data.</a> Methods Cell Biol. 85, 497-538 (2008).</li><li>Nketia, T. A., Sailem, H., Rohde, G., Machiraju, R., Rittscher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+live+cell+images:+Methods,+tools+and+opportunities.">Analysis of live cell images: Methods, tools and opportunities.</a> Methods. , 65-79 (2017).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories. , 33-38 (2015).</li><li>Kwon, O., Kang, S. T., Kim, S. H., Kim, Y. H., Shin, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximum+intensity+projection+using+bidirectional+compositing+with+block+skipping.">Maximum intensity projection using bidirectional compositing with block skipping.</a> J Xray Sci Technol. 23 (1), 33-44 (2015).</li><li>Janbon, G., 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=Analysis+of+the+genome+and+transcriptome+of+Cryptococcus+neoformans+var.+grubii+reveals+complex+RNA+expression+and+microevolution+leading+to+virulence+attenuation.">Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.</a> PLoS Genet. 10 (4), e1004261 (2014).</li><li>Bisson, G. P., 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+use+of+HAART+is+associated+with+decreased+risk+of+death+during+initial+treatment+of+cryptococcal+meningitis+in+adults+in+Botswana.">The use of HAART is associated with decreased risk of death during initial treatment of cryptococcal meningitis in adults in Botswana.</a> J Acquir Immune Defic Syndr. 49 (2), 227-229 (2008).</li><li>van Teeffelen, S., Shaevitz, J. W., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+analysis+in+fluorescence+microscopy:+bacterial+dynamics+as+a+case+study.">Image analysis in fluorescence microscopy: bacterial dynamics as a case study.</a> Bioessays. 34 (5), 427-436 (2012).</li><li>Granger, D. L., Perfect, J. R., Durack, D. T. <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), 508-516 (1985).</li></ol>

作者 Hoyin, 是 DRVision 的产品经理, 出版了自动化图像分析软件 Aivia。

数十年来, 该胶囊一直是研究的主要焦点, 对 mycologists 和临床医生感兴趣的C. 隐球菌和球菌由于其作用的主要致病因素的病原体。用显微显微镜测量不同生长条件下的胶囊大小差异, 可以提供有关病原体及其对各种刺激的反应的重要信息 (即, 不同的环境条件,潜在的药物治疗,等) 在这里, 我们概述了一种方法, 以诱导生长的胶囊在C. 隐球菌, 并比较了两种不同的胶囊染色协?...

每年影响一季度百万人, 每年造成18万以上死亡,新生隐球菌是致病性, 胞内酵母和球菌1,2,的致病剂3. 受影响最严重的是贫穷国家的艾滋病毒阳性患者, 他们没有现成的抗逆转录病毒疗法, 使他们敏锐地易患疾病4,5,6。疾病控制中心的数据表明, 在撒哈拉以南非洲地区, 比起在记录中的 1上的任何埃博拉病毒爆发, 每年都有更多的人死于肺结核。最常见的接触途径是吸入在环境中常见的干燥孢子7。进入肺部后, 有几种致病因素有助于在受感染的个体中获得C. 隐球菌的成功。多糖胶囊被认为是微生物的主要毒力因子, 因为 acapsular 菌株不是毒性的8。
隐球菌胶囊由三个主要成分组成: glucuronoxylomannan (GXM)、galactoxylomannan (GalXM) 和 mannoproteins (MPs)9。虽然 MPs 是胶囊中相对较小的细胞壁相关组件, 但它们是免疫的, 可以促进一种主要支持炎症的响应9,10。相比之下, GXM 和 GalXM 构成了胶囊的大部分 (> 90% 的重量), 并有免疫抑制效应11。除了免疫调节作用外, 胶囊的快速扩增在体内形成了一个机械屏障, 可通过宿主吞噬细胞 (即、中性粒细胞和巨细胞)12摄取。C. 隐球菌胶囊及其合成是复杂的, 但总的来说, 增加的胶囊直径与增加的毒力6,13,14相关。考虑到这一点, 对于C. 隐球菌研究人员来说, 能够快速准确地量化胶囊测量是很重要的。
C. 隐球菌细胞及其多糖胶囊都是动态结构, 随着时间的推移15显示变化。该胶囊可以在密度、大小和组件上发生变化, 以响应主机环境中的更改16,17,18。低铁或营养水平, 接触血清, 人体生理 pH 值和增加 CO2是已知的启动胶囊生长16,18,19,20。此外, 研究人员还显示结构变化导致感染期间免疫反应有显著差异, 在其主机2122上为C. 隐球菌提供了优势。这是众所周知的, 因为C. 隐球菌胶囊的体系结构已经以多种方式进行了分析。例如, 电子显微镜显示, 该胶囊具有一个具有内部电子密层的异构矩阵, 在外层, 更透水层的下面是23。光散射和光镊的使用使研究人员得以进一步阐明其大分子特性24。通过对静态和动态光散射测量结果的分析, 我们知道多糖胶囊具有复杂的分支结构23。光学镊子用于测试结构的刚度, 并评估其抗体反应性24。然而, 到目前为止, 对C. 隐球菌胶囊最常用的分析是对其大小的测量。
为了量化胶囊的大小, 研究者使用了一个简单的测量方法: 胶囊的线性直径。数字显微镜用于捕获多个C. 隐球菌细胞 (一般上百个) 的图像, 它们都是印度墨水或荧光染料染色的。测量每个细胞体和周围胶囊的大小。通过对整个细胞直径 (细胞体 + 胶囊) 的减去细胞体直径, 计算出该胶囊的平均直径。直到这一点, 这些测量已经手动完成。虽然一般准确, 这种方法有缺点的研究人员。大型数据集可能需要几天甚至几周的时间来进行手工分析。由于这些测量是手工完成的, 所以主观和人为的错误可能会影响结果。
自动计算图像分析已成为许多分子细胞生物学领域的研究人员不可缺少的工具, 能够更快更可靠地分析生物图像25,26,27。精确的图像分析技术对于从通常复杂而庞大的数据集挖掘定量信息是必要的。然而, 一些测量, 特别是对C. 隐球菌胶囊的测量, 一直难以实现自动化。准确地识别细胞壁与胶囊之间的界面, 在相衬显微镜下成像时通常以暗环的形式出现, 用一个简单的阈值来解决会很麻烦。此外, 在文化中的C. 隐球菌细胞往往聚集在一起, 准确测量细胞的精确分割是必要的。
该项目的目的是 (i) 说明在C. 隐球菌中胶囊诱导的标准协议之一, (ii) 比较和对比印度墨水和荧光染色, 因为它们涉及胶囊直径测量, (iii) 发展简单,用印度墨水染色的图像测量胶囊直径的计算方法使用图像分析软件, (iv) 评估手动测量胶囊直径和使用软件自动化的好处和局限性。我们发现, 这两种染色方法, 荧光标记的细胞壁和胶囊, 而更费时, 提供了最一致的结果之间的实验。然而, 这两种方法都使我们能够成功地区分出不同胶囊大小的实验室和临床C. 隐球菌菌株。此外, 我们能够自动测量胶囊直径从印度墨水染色图像, 并发现这是一个可行的替代手工测量胶囊。

<table><tbody><tr><td>&lt;strong&gt;Capsule Induction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;C. neoformans cells&lt;/em&gt;</td><td></td><td></td><td>The clinical lab strain, H99S, was a kind gift from Dr. John Perfect (Duke University).&amp;nbsp; The clinical strains, B18 and B52, were kind gifts from Dr. Greg Bisson (University of Pennsylania).&amp;nbsp;</td></tr><tr><td>Yeast Peptone Dextrose Broth (YPD)</td><td>Fisher Scientific</td><td>DF0428-17-5</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td>This is made in the lab using standard recipe (137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4O, 2 mM Kh2PO4O)</td></tr><tr><td>DMEM/high-glucose with L-glutamine, without sodium pyruvate</td><td>GE Life Sciences</td><td>SH30022.01</td><td></td></tr><tr><td>6-well plates</td><td>Falcon</td><td>CL5335-5EA</td><td></td></tr><tr><td>Shaking incubator</td><td>Thermo Scientific&amp;nbsp;</td><td></td><td>MaxQ6000</td></tr><tr><td>CO2 incubator</td><td>Fisher Scientific</td><td></td><td>Isotemp</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td></td><td>Legend XTR</td></tr><tr><td>&lt;strong&gt;Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Scientific</td><td></td><td>Legend Micro 21R</td></tr><tr><td>India ink</td><td>Fisher Scientific</td><td>14-910-56</td><td></td></tr><tr><td>Calcofluor white</td><td>Sigma-Aldrich</td><td>18909-100ML-F</td><td></td></tr><tr><td>18B7 mouse anti-GXM antibody conjugated to Alexafluor 488</td><td></td><td></td><td>A kind gift from Dr. Arturo Casadevall (Johns Hopkins University)&amp;nbsp;</td></tr><tr><td>PBS with 1% Bovine Serum Albumin (BSA)</td><td></td><td></td><td>PBS is the same recipe listed above (line 4) with 1% BSA added and filter sterilized.</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Superfrost microscope slides</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Glass coverslips</td><td>Corning</td><td>2855-18</td><td>#1.5 thickness</td></tr><tr><td>Clear nail polish or other non-toxic sealant</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Image Acquisition&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Immersion oil</td><td>Cargille&amp;nbsp;</td><td>16484</td><td></td></tr><tr><td>Light microscope with immersion oil objective</td><td>Zeiss</td><td></td><td>Zeiss Axio A1 with a Plan - NEOFLUAR 100x oil immersion NA 1.30 objective</td></tr><tr><td>Light microscope camera</td><td>Zeiss</td><td></td><td>Zeiss Axiocam ErCD camera</td></tr><tr><td>Confocal microscope with oil immersion objective</td><td>Zeiss</td><td></td><td>LSM 700 laser scanning confocal equipped with a Plan-Apochromat 63X NA 1.4 oil immersion DIC M27 objective.&amp;nbsp;</td></tr><tr><td>Confocal microscope software</td><td>Zen 2009</td><td></td><td></td></tr><tr><td>Confocal microscope camera</td><td>Nikon</td><td></td><td>Nikon Ti-Eclipse with a Intensilight epifluorescence illuminator (Nikon), CoolSNAP MYO microscope camera (Photometrics), Plan Apo 60x NA 1.40 oil immersion objective (Nikon) and 1.5x magnification changer.&amp;nbsp;</td></tr><tr><td>Widefield imaging software</td><td>Nikon Elements (Nikon)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Capsule Measurement&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image editing software</td><td>Photoshop (Adobe)</td><td></td><td></td></tr><tr><td>Microscope software for manual measurement</td><td>Axiovision (Carl Zeiss)</td><td></td><td></td></tr><tr><td>Image analysis software for automated meesurement</td><td>Aivia (DRVision Technologies)</td><td></td><td></td></tr><tr><td>Spreadsheet software</td><td>Excel (Microsoft)</td><td></td><td></td></tr></tbody></table>

size matters: measurement of capsule diameter in cryptococcus neoformans

新生隐球菌多糖胶囊是致病性酵母的主要毒力因子和最常见的研究领域之一。囊粒大小在菌株间有很大差异, 在引入压力或低养分条件时, 具有快速生长的能力, 与菌株的毒力呈正相关。由于这些原因, 胶囊的大小对C. 隐球菌研究人员非常感兴趣。在表型测试中诱导了C. 隐球菌胶囊的生长, 以帮助了解不同处理方法对酵母的影响以及菌株之间的大小差异。这里我们描述了胶囊诱导的标准方法之一, 并比较了两种被接受的染色和测量胶囊直径的方法: (i) 印度墨水, 一个负的污点, 使用与常规光镜和 (ii) 共染色与荧光染料的细胞壁和胶囊后, 共聚焦显微镜。最后, 我们展示了如何通过计算图像分析来自动测量来自印度墨水染色样品的胶囊直径。

为了说明胶囊诱导、细胞染色、影像学和测量技术, 我们使用了三株C. 隐球菌:常见的, 特点很好的实验室菌株, H99S30, 和前两个临床分离菌株未知的胶囊直径, B18 和 B5231。
使用印度墨水的胶囊感应、染色和图像采集工作流显示在图 1A中。从所有三种菌株中取出的?...

Watch this Scientific Journal Video about Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans at JoVE.com

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

多糖胶囊是新生隐球菌的主要毒力因子, 其大小与菌株的毒力密切相关。胶囊直径测量用于表型检测和测量疗效。本文提出了一种标准的胶囊诱导方法, 并比较了两种染色法和测量直径法。

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

size-matters-measurement-capsule-diameter-cryptococcus

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

13.4K 次观看.  Middle Tennessee State University. 多糖胶囊是新生隐球菌的主要毒力因子, 其大小与菌株的毒力密切相关。胶囊直径测量用于表型检测和测量疗效。本文提出了一种标准的胶囊诱导方法, 并比较了两种染色法和测量直径法。

视频: Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

用不连续密度梯度用胶囊分离细菌

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

科研

遗传学

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

免疫与感染

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

可视化的非溶胞胞吐作用<em&gt;新型隐球菌</em&gt;从巨噬细胞用数码光学显微镜

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

自动测量隐球菌种多糖胶囊和细胞体

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

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

生物工程

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

肉汤稀释体外筛选: 一种简便快速检测新抗真菌化合物的方法

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

生物学

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

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

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

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

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 ...

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

评估软体动物粗提取物和澄清提取物的假定抗隐球菌特性

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

尺寸事项:新生隐球菌中胶囊直径的测定

摘要

探索更多视频

此视频中的章节

用不连续密度梯度用胶囊分离细菌

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

可视化的非溶胞胞吐作用

巨噬细胞胆固醇消耗及其对的吞噬作用

自动测量隐球菌种多糖胶囊和细胞体

数百个 C.白化病细胞生物原子力显微镜测量自动化

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

肉汤稀释体外筛选: 一种简便快速检测新抗真菌化合物的方法

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

评估软体动物粗提取物和澄清提取物的假定抗隐球菌特性

尺寸事项:新生隐球菌中胶囊直径的测定

摘要

探索更多视频

此视频中的章节

用不连续密度梯度用胶囊分离细菌

使用的细胞图像分析仪量化致病真菌与宿主细胞协会

可视化的非溶胞胞吐作用

巨噬细胞胆固醇消耗及其对的吞噬作用

自动测量隐球菌种多糖胶囊和细胞体

数百个 C.白化病 细胞生物原子力显微镜测量自动化

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

肉汤稀释体外筛选: 一种简便快速检测新抗真菌化合物的方法

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

评估软体动物粗提取物和澄清提取物的假定抗隐球菌特性

数百个 C.白化病细胞生物原子力显微镜测量自动化

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