作者承认戈登和贝蒂摩尔基金会授予加州理工学院4037和4038资助这项工作。

1. 细菌的生长和计数 注意: 这适用于在适当培养基中生长的几乎任何细菌菌株 36 。在我们的例子中, 我们使用三菌株: 沙雷菌 作为一个常见的、容易识别的实验室菌株;和一个较小的, 高度能动的环境应变, 希瓦 oneidensis MR-1。为了比较检测的运动与 non-motile 细胞, non-motile 希瓦 突变体, 和 #916; FlgM, 也用于比较 37 。所有菌株都生长在性肉汤 (LB). 
<ol> <li> 准备无菌 LB 培养基 (每公升蒸馏水:10 克 bacto 胰, 5 克 bacto 酵母, 10 克氯化钠; 过滤器或高压釜)。准备标准的100毫米直径 LB 琼脂板 (相同的配方, 中等加 15 g 琼脂每升; 蒸压釜 (121, #176; C, 20 分钟), 以杀菌和倒入时, 冷却足够处理)。在冰箱中存放介质和盘子. </li>
	<li> 试验前的前一天 5-6 毫升的 #34; 硕士和 #34; 培养基从新鲜细菌菌落, 使用正确的无菌和生物安全技术。在30和 #176 的振动器上孵育 (120 rpm); C 为 ~ 12 h. 潜伏期将取决于应变和生长速率。在我们的实验中, 希瓦 菌株孵育12小时;但是, 沙雷 在8小时后收获, 以确保文化将显示 mid-logarithmic 的增长. </li>
	<li> 实验的当天, 采取分光光度读数 (OD 600 ) 的细菌的主文化, 这是预计的范围0.6 至0.7。它应该在对数增长阶段 (如先前所确定)。如果不是, 亚文化100和 #181; L 到5毫升培养基, 并允许细胞生长到 mid-log 阶段. </li>
	<li> 采用主区域性样本, 并使用 Petroff-豪塞尔计数室直接计数单元格。这将枚举活的和死的细胞. 
	<ol> <li> 提取10和 #181; 未稀释的文化的样品与微和转移入房间. </li>
		<li> 图像下的 high-dry 物镜显微镜 (40X 和 #160; 或 63X, NA 0.7-0.8) 使用相衬. </li>
		<li> 统计至少20平方和平均值的细菌数. 
		注: 只计算那些完全在一个正方形内的细菌, 只有那些越过顶部和左边的边界 (如果你愿意的话)。如果正方形由几行分隔, 请选择一个作为边界. </li>
		<li> 计算浓度为20平方 x 稀释因子的平均值。注意, 直接计数对浓度和 #60 不起作用; 10 7 /mL. </li>
	</ol> </li> <li> 用于对菌落形成单元 (CFU) 计数的区域性进行串行稀释。这将仅枚举活动单元格. 
	<ol> <li> 将每个选定的细菌样本与无菌0.9% 盐水溶液进行连续稀释。转移20和 #181; l 的细菌溶液和稀释它与180和 #181; l 盐水。重复直到最低浓度为 ~ 10 3 单元格/mL. </li>
		<li> 采取100和 #181; L 从至少两个稀释建议是 10 3 和 10 4 /mL 和 #160; #8212; 在适当的固体介质板上板。与无菌摊铺机。执行每次稀释的至少3复制. </li>
		<li> 在适当的温度下孵育细菌过夜或直到菌落生长. </li>
		<li> 计数殖民地和计算殖民地形成单位 (CFU) 根据 (# 的殖民地 x 稀释因子)/体积镀 = CFU/毫升。对复制的平均 CFU. </li>
	</ol> </li> </ol> 2。DHM 的高稀释样品的制备 <ol> <li> 在 LB 介质板上为 DHM 和后 DHM 计数的菌落形成单元稀释的主区域性的序列 (见 1.4.1-1.4.4)。执行这双盲, 使录音的人不知道每个测量样本的浓度. 
	<ol> <li> 将细菌稀释到 10-15 毫升的最小培养基中, 这将鼓励运动 (视情况而定), 但抑制细胞分裂, 以便在实验过程中细胞的浓度不会有明显变化。这可能是0.9% 盐水或更具体的运动介质。例如, 如果使用 大肠杆菌 , 则运动介质必须包含 EDTA 38 。对于这些例子, 我们使用0.9% 盐水. </li>
	</ol> </li> </ol> 3。录制 DHM 视频 <ol> <li> 使用无菌注射器, 抽取约10毫升的稀释利息. </li>
	<li> 使用无菌管件和油管将注射器连接到 DHM 样品室. 
	注: custom-made 微流控室是为了使样品的一致流动通过仪器的光学通路而建造的。有关详细信息, 请参见 结果 部分。在实验之前, 样品室的所有组分应该由灭菌消毒. </li>
	<li> 从注射器通过样品室源源不断地使用注射器泵. 
	注: 适当的流速取决于射流通道的尺寸、所需的吞吐量以及数据获取的限制. </li>
	<li> 当样品流经样品室时, 以适当的帧速率连续获取全息图。将创建一个时间文件, 记录每个图像捕获的时间, 以便以后在数据分析期间使用 (协议4节)。获取所有全息图在环境温度 (23 和 #176; C)。通过执行由照相机制造商提供的预先 c++ 可执行文件来记录全息图. 
	注: 适当的帧速率根据选择的流速而变化。对于这个实验, 建议使用一个帧速率, 这样细菌就会被成像和 #62; 2 倍, 因为它横跨仪器和 #39; s 的视野. </li>
	<li> 允许足够的时间让整个10毫升的样品流经 DHM. </li>
	<li> 为了确保在实验过程中细菌不会生长或死亡, 用100和 #181 对培养基中的富集进行接种; DHM 图像捕获。用无菌的吊具铺上, 并在适当温度下孵育24小时;计数殖民地存在. </li>
</ol> 4。计算细胞密度和检测限值 <ol> <li> 已获得的全息图视频, 分析数据中是否存在细菌. 
	<ol> <li> 计算全息图的时间序列的中值像素值, 然后从每个各自的像素中减去此中间数值, 以消除全息图像中的固定工件。这可以通过执行以下步骤在 ImageJ 中完成: <ol> <li> 转换为32位 (Image-Type-32 位) </li> <li> 计算中值 (图像堆栈-Zproject-中值) </li> <li> 从堆栈的其余部分减去中间值 (进程-ImageCalculator 减去) </li> </ol> </li> <li> 计算可见的通风环和焦点单元格的数目手动.在 ImageJ 中, 这是通过用点工具指向对象来完成的. </li>
		<li> 每系列全息图将附有一个时间文件 (在全息图像采集时记录)。使用时间文件计算在捕获图像时抽取的样本总量. </li>
	</ol> </li> <li> 通过将检测到的总体积的单元格总数除以图像的总数来计算单元格的密度。平均 5-10 帧用于精确统计. </li>
</ol> 5。图像重构到振幅和相位 <ol> <li> 选择每帧 10-200 单元格的代表性视频。装载原始 (不是中位减去) 全息图入重建软件 (例子: ImageJ 数值传播 39 ;洗发水 [GitHub]). 
	<ol> <li> 使用 ImageJ, 转到 OD-数值传播-数值衍射. </li>
		<li> 在提示时, 绘制一个傅里叶掩码以选择真实或虚拟图像, 并消除任何正弦噪声特征. </li>
		<li> 通过进入重建距离, 在适当的 z 步重建振幅和相位. </li>
	</ol> </li> <li> 中值减去振幅数据以减少噪音, 如4.1.1 中所示. </li>
	<li> 将数据绘制为3D 多维数据集或投影。转到插件和 #8212; 卷查看器. </li>
</ol>

<ol>
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作者没有什么可透露的。

全息图的数值重建: 用于全息图的数值重建, 采用了角谱法 (ASM)。这涉及到全息图的卷积与格林函数的 DHM。利用傅里叶卷积定理可以计算出在特定焦平面上的复杂波前, 如下所示:
<img alt="Equation 2" src="/files/ftp_upload/56343/56343eq2.jpg">(1)
其中, 是傅里叶变换运算符, 是全息图矩阵, 是 DHM 的绿色函数, 定义为: <img alt="Equation 3" src="/files/ftp_upload/56343/56343eq3.jpg"> <img alt="Equation 4" src="/files/ftp_upload/56343/56343eq4.jpg"> <img alt="Equation 5" src="/files/ftp_upload/56343/56343eq5.jpg">
<img alt="Equation 6" src="/files/ftp_upload/56343/56343eq6.jpg">(2)
其中, 是要重建的所需焦平面的距离 (沿光轴), 是光照波长、和分别是 x 和 y 方向的像素数, 以及和<img alt="Equation 7" src="/files/ftp_upload/56343/56343eq7.jpg"> <img alt="Equation 8" src="/files/ftp_upload/56343/56343eq8.jpg"> <img alt="Equation 9" src="/files/ftp_upload/56343/56343eq9.jpg"> <img alt="Equation 10" src="/files/ftp_upload/56343/56343eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56343/56343eq11.jpg"> <img alt="Equation 12" src="/files/ftp_upload/56343/56343eq12.jpg">分别是 x 和 y 方向的像素大小 (在探测器平面上)40。
从复波前, 强度可以通过的幅度平方来计算, 并且可以通过反正的虚和实部分之间的商的不确定来获得相位. <img alt="Equation 13" src="/files/ftp_upload/56343/56343eq13.jpg"> </img> <img alt="Equation 13" src="/files/ftp_upload/56343/56343eq13.jpg"> </img>
修改和故障排除 涉及稀疏细菌浓度计数的实验非常容易受到污染、误报和假阴性的影响。因此, 细菌的生长和计数, 以及 DHM 的高稀释样品的制备是本实验的关键步骤。在这个实验中, 必须谨慎地控制细菌生长和列举的环境, 以避免污染。适当的杀菌技术, 包括灭菌, 有助于防止污染, 以及假阳性。为了进一步防止误报, 细菌被选作这个实验, 表现出相对独特的特征 (例如, 菌在培养时具有非常鲜明的红色)。为了防止假阴性, 选择这个实验的细菌选择这样, 他们将能够生长在丰富的文化板块, 以及有特征的形状和/或运动模式, 通过 DHM 识别。通过电镀样品和通过平板计数对细菌进行定量, 定期检测污染和/或意外细胞生长是很重要的。
在进行此实验时, 预计每1毫升样本的数据大小大约为 2.4 GB。当然, 这个数字取决于所使用的相机和文件格式。报告的数据大小是使用 4 Mpx 检测器和标准 TIFF 文件格式。数据的后处理需要每1毫升样品大约6分钟的计算。在使用 3.5 GHz 和 32 GB RAM 的英特尔 i7 8 核处理器时, 观察到了处理时间。这些计算可以并行到 GPU, 从而大大缩短了处理时间, 但在这里没有完成。
技术的局限 虽然使用 DHM 检测极低浓度的细菌是可能的, 这种方法是非特异性的。单独的形态学不是鉴定细菌菌株的决定性手段。由于非目标细胞和聚合体的存在, 诊断应用程序将面临特定的挑战。然而, 双光束仪器的优势在于, 它能够在相对混浊的样品中成像细菌。在临床样品中检测细菌的能力, 以及所需样品的预处理程度仍有待确定。
协议中的关键步骤 
步骤 2:获取精确的稀释是定量枚举的关键。任何稀释做的都应该用板数来备份。步骤 3: 必须仔细选择流速和相应的帧速率, 以确保所有的电池都能被看到。在进行检测试验的极限之前, 应仔细调整泵的参数。步骤 4: 对于非常低的细菌浓度, 捕获足够的数据以确信检测是至关重要的。可能有必要重建一些数据, 以确定细胞, 而不是碎片, 正在看到。在极端情况下, 可能需要超过10毫升的解决方案。非能动菌株比运动菌株更容易发生歧义。步骤 5: 如果数据有任何歧义, 重建可以帮助证明全息图代表的是细胞而不是碎片。高分辨率的重建将显示经典的细胞形态, 和运动可以清楚地看到, 在轨道上的能动菌株。

在非常稀的样品中准确的细菌计数的测定在许多应用中是至关重要的: 几个例子是水和食品质量分析1,2,3;检测血液、脑脊液或痰液中的病原体4,5;生产医药产品, 包括无菌水6;和环境群落分析在营养环境中如开阔的海洋和沉积物7,8,9。在木星和土星的冰冷卫星上, 特别是木卫二10、11和土卫二12、13、 14, 已知有地下液态海洋。由于维京人在1978年试图在另一个星球上寻找现存的生命, 因此没有任何任务, 因此在空间飞行任务的过程中, 细菌识别和计数的技术和仪器的开发是有限的15。
传统的平板计数方法只发现培养细胞, 它能在环境菌株中代表少数物种, 有时和 #60; 1%16。盘子需要几天或几周的孵化来获得最大的成功, 这取决于应变。荧光显微镜在很大程度上取代了板数作为快速准确的微生物计数的金标准。核酸标记荧光染料, 如 4, 6-diamidino-2-吲盐酸盐 (DAPI), SYBR 绿色, 或吖啶橙绑定到核酸是典型的染料使用17,18,19, 虽然许多研究使用了克标志的荧光指标20,21,22,23,24。使用这些没有预步骤的方法, 每个 mL 会导致检测 (检测) 105单元格的限制。改进的 LoD 是可能的使用过滤。液体样品是真空过滤到膜上, 通常是聚碳酸酯和理想的黑色, 以减少背景。低背景染料, 如上面提到的 DNA 污渍, 可以直接应用于过滤器25。为了准确地按眼睛计数, 每个过滤器需要 105单元格, 这意味着对于每个 mL 的样本比 105单元更稀, 必须收集和筛选大量的样本卷。为了系统地探索过滤器的所有区域, 并因此减少了计数所需的单元数, 将检测范围推到每 mL26102单元格, 从而开发了激光扫描设备。然而, 这些在大多数实验室是不可用的, 并且需要先进的硬件和软件, 允许专家确认观察到的粒子是细菌而不是碎片。
作为参考, 成人脓毒症通常开始显示的症状和 #60; 100 细胞/毫升的血液, 和婴儿在和 #60; 10 细胞/毫升。从一个成年人的血液提取需要10毫升, 从一个婴儿, 1 毫升。pcr 方法被存在人和致病植物群脱氧核糖核酸和由 PCR 抑制的组分抑制在血液27,28。尽管有各种新兴技术, 文化仍然是诊断血流感染的金标准, 特别是在更多的农村地区或发展中国家。为了探测其他行星上的生命, 热力学计算可以估计生命的能量预算, 从而预测可能的生物量。1-100 细胞/毫升预计将热力合理的欧罗巴29。从这些数字可以很容易地看出, 大量的水溶液中的少量细胞的检测是一个重要的未决问题。
在本文中, 我们演示了检测沙雷菌和希瓦 oneidensis (野生类型和 non-motile 变种) 的浓度为 105, 104, 103, 和100细胞/毫升使用离轴数字全息显微镜 (DHM)。DHM 优于传统光学显微镜的关键优势是在高-的同时成像厚样本体积, 该实现, 样品室是0.8 毫米厚。这些样品室由烷 (soft-lithography) 由精密机械加工的铝模制成, 公差为±50µm。这代表大约100倍的改善在领域的深度在大功率光学显微镜。DHM 还提供了定量的相位信息, 允许测量光路长度 (折射率和厚度的乘积)。DHM 和类似的技术已用于监测细菌和酵母细胞周期和细菌干质量的计算30,31,32;散射差异甚至可以用来区分细菌菌株33。
我们使用的仪器是 custom-built 专门用于微生物, 如以前发布的34,35, 它的设计和构造是演示和讨论的。通过注射器泵连续向0.25 µL 容积样品室提供水溶液;流率是由相机帧率确定的, 以确保整个样本量的成像。统计计算预测的样本量必须被成像, 以便检测到一个相当数量的细胞在一定浓度。
对于细胞检测应用, 不需要将全息图重建为振幅和相位图像;对原始全息图进行了分析。这节省了大量的计算资源和磁盘空间: 重建时, 500 Mb 的全息图视频将是 1-2 Tb。然而, 我们确实讨论了通过样品的深度重建, 以确认全息图代表了所需的物种。DHM 的一个重要特点是它能够监测图像的强度和相位。在强度上几乎是透明的生物体 (如大多数生物细胞) 在相时清晰地显现出来。由于它是一种无标签的技术, 没有使用染料。这是一个dvantage 为可能的太空飞行应用, 因为染料可能不生存使命的条件, 并且-更重要地-不能假设与外星有机体一起使用, 可能不用脱氧核糖核酸或 RNA 为编码。这也是在诸如北极和南极等极端环境中工作的一个优势, 在那里, 染料可能难以带到偏远的地方, 并可能在储存时降解。图像的相位和振幅重建是使用一个开源软件包, 我们已经提供了 GitHub (洗发水) 或使用 ImageJ。

<table><tbody><tr><td>Bacto Yeast</td><td>BD Biosciences</td><td>212750</td><td></td></tr><tr><td>Bacto Tryptone</td><td>BD Biosciences</td><td>211705</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>7710</td><td>Many options for purchase</td></tr><tr><td>Bacto Agar</td><td>BD Biosciences</td><td>214010</td><td></td></tr><tr><td>10 cm Petri dishes</td><td>VWR</td><td>10053-704</td><td></td></tr><tr><td>15 mL culture tubes</td><td>Falcon (Corning Life Sciences)</td><td>352002</td><td>Loose-capped</td></tr><tr><td>Petroff-Hauser chamber</td><td>Electron Microscopy Sciences</td><td>3920</td><td></td></tr><tr><td>10 mL syringes</td><td>BD Biosciences</td><td>309604</td><td>Luer-Lok tip not necessary</td></tr><tr><td>Male Luer to 1/16&amp;rdquo; barbed fitting</td><td>McMaster-Carr</td><td>51525K291</td><td></td></tr><tr><td>Autoclavable 1/16&amp;rdquo; ID PVC tubing for flow</td><td>Nalgene</td><td>8000-0004</td><td>Sold by length, purchase accordingly</td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>PHD 2000</td><td></td></tr><tr><td>Sample Chamber</td><td>Custom</td><td>n/a</td><td>See Materials Section</td></tr><tr><td>Holographic Microscope</td><td>Custom</td><td>n/a</td><td>See Wallace et al.</td></tr><tr><td>Open-source software</td><td>Custom</td><td>https://github.com/bmorris3/shampoo</td><td></td></tr></tbody></table>

quantifying microorganisms at low concentrations using digital holographic microscopy (dhm)

准确地检测和计数稀疏细菌样品在食品、饮料和制药加工业、医疗诊断和机器人任务到太阳系其他行星和卫星的生命检测中有许多应用。目前, 稀疏细菌样品通过培养电镀或荧光显微镜进行计数。培养皿需要很长的孵育时间 (天到周), 荧光显微镜需要广泛的染色和样品的浓度。在这里, 我们演示如何使用离轴数字全息显微镜 (DHM) 在非常稀的文化中列举细菌 (100-104细胞/毫升)。首先, 讨论了定制 DHM 的构建, 并详细说明了如何构建低成本的仪器。讨论了全息术的原理, 根据仪器的光学性能特点和细菌溶液浓度 (表 2), 利用统计模型估计视频检测细胞的时间长度。.视频检测的细胞在 105, 104, 103, 和100细胞/毫升是实时演示使用 un-reconstructed 全息图。利用开源软件包对振幅和相位图像进行重建。

结果应表明, DHM 能在极低的水平上检测活细菌和死菌。计数的细菌数量应与使用 Petroff-豪瑟计数室和钢板计数所得的结果一致。标准统计方法提供了各种细菌浓度不同检测方法的准确性信息。
图 1显示了 Petroff-豪塞尔计数室, 以及用于直接枚举单元格的计数室的显微结构。图 2A显示了一个典型的富集文化板块, 允许有足够的时间为S. 菌生长菌落 (室温16小时)。图 2B显示了所使用的所有菌株的生长曲线。当精确分光光度计的测量值不同时, OD600与菌落计数之间的关系应在分光光度计的可靠范围内线性化;我们测量〜 OD600 = 0.1 为 ~ 108/毫升。直接 Petroff-豪塞尔计数和菌落计数应同意在10% 内, 直到细胞开始死亡, 由营业额在增长曲线看到。
图 3显示了自定义采样室的设计。本实验采用了自定义微流控腔, 使样品在全息显微镜的视场中连续流动。在样品室的建造中采用了标准的微流控技术和材料。在微流体装置中插入带有雄性口的钝尖不锈钢针, 为样品室提供入口和出口端口。微流体通道几何是由烷软光刻法建立的, 采用了机械加工的铝制主模, 通过铝框架在两个光学质量玻璃窗之间进行压缩。该公司建立了流动通道的几何形状, 并确保皮下注射针头, 提供流体输送到和从流动通道。这些样品室仅用于建立流道几何, 从而在仪器的光学路径中不含任何的硅橡胶。请注意, 由于离轴全息显微镜的光学性质, 两个微通道在样品室中相互平行成型。这是为了匹配干涉仪的两臂之间的光路长度 (称为 "标本" 和 "参考" 武器)。参考通道应只包含无菌介质, 不应受流量的限制。
图 4显示了数字全息显微镜在实验室实施过程中的示意图和图像。它由照相机提供的软件操作。图 5显示了用于细菌计数的具有代表性的 DHM 数据及其与每个视场中的细胞预测数的对应关系。从原始全息图, 没有重建, 可以看到和计数的细胞中位减法和手动跟踪。这大大降低了计算负担与重建所有 z 平面, 因为原始全息图显示的细胞在整个会议厅的深度。指出了不同浓度的文化形态及其与板数和 Petroff 数的对应关系。总的细胞计数是通过平均每个样本体积中所看到的细胞总数来确定的。检测的限制是由映像的样本卷总数决定的。在这个实验中, 我们对这个值设定了一个最大的限制, 最大总计为10毫升的样品成像0.25 µL 增量 (总共4万图像每个样本)。如果在20帧的每个帧中清晰地观察到单元格, 则在达到最大总音量之前停止录制。在前一篇论文中, 我们报告了一个公式, 用于估计检测细菌的数量, 在50% 的浓度从 1-105细胞/毫升 (表 1)。根据这一估计, 对所有4万帧的检查应允许检测 1/毫升的细胞, 虽然这种计算是密集的。
图 6显示了重建软件 (斐济)39的使用情况。一个单一的全息图打开, 软件 "OD-数值传播-数值衍射" 运行。在对话框中给出了成像参数;振幅、强度和相位都可以重建。可以选择单个 z 平面, 也可以使用批重建重建整个深度。
图 7显示了选定示例中不同深度的振幅和相位图像的重建。用于生成重建的傅里叶掩模, 连同中值减去强度图像、相位图像、通过相栈的最大投影以及强度叠加的体积投影。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56343/56343fig1.jpg"> 
图 1: 细菌细胞的直接计数.在细胞/毫升的细菌浓度计算计数的细菌总数计数室和结垢的数字核算的事实, Petroff-豪瑟室只包含 20 nL 样本。(A) Petroff-豪塞尔计数室。(B) 在10X 相位对比度下计数室显微结构的图像, 显示不同大小的盒, 对枚举不同大小的单元格有用。(C) 在计数室最小的框内出现S. 菌;40X 相衬。(D) 用于计数的方法, 它排除掉在上、右边缘 (N) 上的单元格, 而不是下边缘和左边的边 (Y)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56343/56343fig2.jpg"> 
图 2: 按菌落生长和光学密度计算细胞数.(A) S. 菌在室温下孵育16小时。在正确的稀释, 文化板材应该有 20-200 殖民地准确计数和统计。显示的板块有点过于拥挤, 无法可靠计数。(B) 用于3菌株的生长曲线。光学密度0.1 对应于 ~ 108单元/mL, 但精确值因仪器而异。直接 Petroff-豪瑟计数同意板块计数在10% 以内的线性增长范围。误差线表示5独立样本的标准偏差。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56343/56343fig3.jpg"> 
图 3: 样品室设计.(A) 微流体原理图 (样本和参考微标记)。(B) 完全组装的样品箱。(C) 分解 CAD 视图, 显示样品室的组成元素。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56343/56343fig4.jpg"> 
图 4: 数字全息显微镜的示意图和图像.(A) 示意图显示四主要元素: 来源, 样品 (标本路径被标记为规范. 和参考路径被标记 Ref.), 显微镜, 和传感器。(B) 硬件的实体模型。纤维馈源组件位于底部, 而成像相机位于顶部。显微镜光学-由两个非球面透镜和继电器透镜组成-包含在300毫米长的透镜管之内。三轴阶段之间的来源显微镜光学提供简单的手工操作的标本正在研究中。(C) 实验室中仪器的照片。图像下半部分的刻度线表示1英寸。示意图是重新从华莱士et al.34.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56343/56343fig5.jpg"> 
图 5: 增长曲线和具有代表性的 DHM 数据.(A) 根据365µm x 365 µm x 1 mm 的样本量预测每个视场的单元格。DHM 枚举的最佳范围显示在矩形中, 实际的示例表示为 (B) 和 (C)。(B) DHM 8 x 10 5单元/ml 的全息图, 由 Petroff-豪瑟和 7.7 x 105单元/ml 测量, 按盘数测量;这个图像显示110细胞, 非常适合预测。(C) DHM 2.0 0.1 x 10 5单元/ml 的全息图, 由 Petroff-豪瑟和 2.0 0.3 x 105单元/ml 测量, 按盘数测量;此图像显示27单元格。缩放栏 = 35 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56343/56343fig6.jpg"> 
图 6: 执行重建.(a) 在斐济打开全息图。(B) 运行数值传播并输入波长和图像大小。选择接近0的 z 距离开始。(C) 在提示选择真实图像或虚拟映像时绘制一个傅立叶掩码。(D) 浴处理允许在整个卷中进行重建。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56343/56343fig7.jpg"> 
图 7: 重建的外观.沙雷区域性的图像在 ~ 106单元格/mL。(A) 原始全息图。(B) 傅立叶变换。圆圈内的区域是要使用的区域;其余的都是蒙面的(C) 在单 z 平面上进行振幅重构。(D) 在单 z 平面上进行相位重建;刻度线显示相移的度数。(E) 通过相位堆栈中的所有 z 平面进行最大强度投影。(F) 全振幅堆栈 (365 x 365 x 800 µm) 的音量视图。缩放栏 = 35 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56343/56343fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>浓度 [每毫升细胞]</td>
			<td>需要的帧</td>
		</tr>
		<tr>
			<td>1.00E+00</td>
			<td>6686</td>
		</tr>
		<tr>
			<td>1.00E+01</td>
			<td>669</td>
		</tr>
		<tr>
			<td>1.00E+02</td>
			<td>67</td>
		</tr>
		<tr>
			<td>1.00E+03</td>
			<td>7</td>
		</tr>
		<tr>
			<td>1.00E+04</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1.00E+05</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
表 1:DHM 检测细菌所需样本量的最小数量 (检测置信度为 50%).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>属性</td>
			<td>值</td>
			<td>单位</td>
		</tr>
		<tr>
			<td>工作波长</td>
			<td>405</td>
			<td>nm</td>
		</tr>
		<tr>
			<td>物镜数值孔径</td>
			<td>0。3</td>
			<td>-</td>
		</tr>
		<tr>
			<td>系统放大倍数</td>
			<td>20</td>
			<td>-</td>
		</tr>
		<tr>
			<td>横向分辨率</td>
			<td>0。7</td>
			<td>μ</td>
		</tr>
		<tr>
			<td>样品成像量</td>
			<td>360 x 360 x 800</td>
			<td>μ m x μ m</td>
		</tr>
		<tr>
			<td>仪器长度</td>
			<td>400</td>
			<td>毫米</td>
		</tr>
	</tbody>
</table>
表 2:数字全息显微镜的光学和性能规范

Watch this Scientific Journal Video about Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM at JoVE.com

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

数字全息显微镜 (DHM) 是一种体积技术, 允许成像样品50-100X 厚比明显微镜在可比的分辨率, 与重点进行后处理。DHM 用于识别、计数和跟踪非常低密度的微生物, 并与光学密度测量、平板计数和直接计数相比较。

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

quantifying-microorganisms-at-low-concentrations-using-digital

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

10.2K 次观看.  California Institute of Technology. 数字全息显微镜 (DHM) 是一种体积技术, 允许成像样品50-100X 厚比明显微镜在可比的分辨率, 与重点进行后处理。DHM 用于识别、计数和跟踪非常低密度的微生物, 并与光学密度测量、平板计数和直接计数相比较。

视频: Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

科研

环境科学

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

癌症研究

Compact Lens-less Digital Holographic Microscope for MEMS Inspection and Characterization

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and automotive. However, effective inspection and characterization metrology systems are needed to ensure the functional reliability of MEMS. This study presents a system based on digital holography as a tool for MEMS metrology. Digital holography has gained increasing attention in the past 20 years. With the fast development and decreasing cost of sensor arrays, resolution of such systems has increased broadening potential applications. Thus, it has attracted attention from both research and industry sides as a potential reliable tool for industrial metrology. Indeed, by recording the interference pattern between an object beam (which contains sample height information) and a reference beam on a CCD camera, one can retrieve the quantitative phase information of an object. However, most of digital holographic systems are bulky and thus not easy to implement on industry production lines. The novelty of the system presented is that it is lens-less and thus very compact. In this study, it is shown that the Compact Digital Holographic Microscope (CDHM) can be used to evaluate several characteristics typically consider as criteria in MEMS inspections. The surface profiles of MEMS in both static and dynamic conditions are presented. Comparison with AFM is investigated to validate the accuracy of the CDHM.

We present a compact reflection digital holographic system (CDHM) for inspection and characterization of MEMS devices. A lens-less design using a diverging input wave providing natural geometrical magnification is demonstrated. Both static and dynamic studies are presented.

紧凑的镜头少数字全息显微镜的MEMS检测与表征

A micro-electro-mechanical-system (MEMS) is a widely used component in many industries, including energy, biotechnology, medical, communications, and ...

工程学

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜：测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

生物工程

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

将流体设备与显微镜和流式细胞学相结合，研究跨空间尺度的多孔介质中的微生物传输

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

免疫与感染

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

生物学

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

多式联运定量相成像数字全息显微准确评估肠道炎症和上皮伤口愈合

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

医学

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

的弱散射主题内嵌数字全息显微镜（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

<meta charset="utf8"></head><body>与真菌伴侣共培养蔗糖分泌蓝细菌的方案

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

<meta charset="utf8"></head><body>异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...