这项工作得到了国家卫生研究院的国家综合医学研究所的支持, R35GM124868 和杜克大学颁发的奖项。

1. 设置布局和对齐
<ol>
	<li>跟踪励磁激光器的安装与准直<ol>
		<li>使用国产安装将激光器粘贴到光学工作台上。该安装是一个简单的铝板与安装孔的激光头和光学表。激光应牢固地附着在金属支架上, 以稳定散热。对于这项工作, 使用 488 nm 固体激光器照明 (图 1), 虽然波长可以选择适合特定的荧光或实验。一个关键的因素是, 激光波长适合 2 d-爆炸物的工作范围, 这是由两个器 (图 1: W2) 之间的波板主要确定。488 nm 激光可以有效激发绿色或黄色荧光蛋白 (GFP/YFP), 这是常见的荧光标签在活细胞实验。</li>
		<li>使用一对介质镜像 (图 1: M) 调整激光束的高度和方向. 确保激光光束平行于光学工作台, 并将其调整到合适的高度。 注: 应根据显微镜平台选择高度。对于本协议中使用的所有后续镜像, 都应保留此高度, 但不会明确声明。</li>
		<li>准用一对透镜 (图 1: L1 和 L2) 对光束进行了分析。焦距的镜头, 应谨慎选择, 以避免激光斑点被修剪的光圈 2 d-爆炸物。通过 2 d-爆炸物排爆后, 激光束的形状变化明显。在这里的准直的质量是非常重要的, 因为像差将被随后的透镜放大, 任何分歧将恶化的性能 2 d-爆炸物的偏转。理想情况下, 通过将光束聚焦到至少20米以外的点来准光束。</li>
	</ol>
	</li>
	<li>将一个针孔 (图 1: PH) 放置在第一个准直透镜 (图 1: L1) 的焦点上。<ol>
		<li>选择合适尺寸的针孔。可根据以下计算选择针孔尺寸: 
		<img alt="Equation 1" src="/files/ftp_upload/56711/56711eq1.jpg"> 其中, λ是激光的波长, f是第一个透镜的焦距, D是输入光束的直径。</li>
		<li>在3D 平移阶段安装针孔, 使其能够精确放置在第一准直透镜的焦点上。</li>
		<li>调整针孔的位置。将激光功率计放在针孔后, 调整 XYZ 的针孔位置, 使电能表读数最大化。如果观察到一个衍射环, 用虹膜放置在 2 d-排爆前。</li>
	</ol>
	</li>
	<li>安装格兰-汤普森偏光片<ol>
		<li>将格兰-汤普森偏光片 (图 1: GP) 放在第二个准直透镜 (图 1: L2) 之后, 以清除激光束的偏振。旋转偏光片, 以找到最大的传输功率计。</li>
	</ol>
	</li>
	<li>安装 EODs<ol>
		<li>使用 2 EODs (图 1: EOD1 和 #38; EOD2) 将激光偏转到 X 和 Y 方向。通过调整每个爆炸物的偏航、俯仰和高度来最大化激光传输。</li>
		<li>使用制造商在每个偏转器一侧提供的校准标记对齐两个 EODs。</li>
		<li>将测试模式应用于2维爆炸物的控制器, 以检查爆炸物的输出。这可以是骑士的巡回 (图 2) 通过下面所描述的 FPGA 或由函数发生器提供的一个简单的正弦波。为了得到最佳性能, 每个偏转器的偏转应与压电 nanopositioner 的 X 或 Y 方向平行。这个方向是由器关于传输轴的角方向决定的。在不移动2维爆炸物的情况下旋转偏转方向, 在2维爆炸物排爆后放置一只鸽子棱镜, 使偏转轴与压电 nanopositioner 轴对准。</li>
	</ol>
	</li>
	<li>波板安装<ol>
		<li>将波板 (图 1: W1) 放在格兰-汤普森偏光片 (图 1: GP) 和 2 d-爆炸物排爆器之间, 使入射激光束极化与 2 d-爆炸处理的偏转轴线对准。放置一个长焦距镜头 (300 毫米) 后, 2 d 的爆炸物, 并放置一个 sCMOS 相机在激光焦点。接下来, 打开 2 d-爆炸物, 以生成下面描述的骑士之旅 (图 2)。旋转波板, 直到在相机上观察到均匀的方形激光分布。</li>
	</ol>
	</li>
	<li>光束扩展透镜对和继电器透镜副的安装<ol>
		<li>放置一对镜片 (图 1: L3 和 L4) 在 2 d-排爆后扩展光束以填充物镜的后孔 (图 1: OL) 和另一对镜片 (图 1: L5 和 L6) 以将焦平面中继到显微镜物镜。确保在这两对镜片之间留有足够的空间, 因为在后面的步骤中, 它们之间会安装标签镜头。</li>
	</ol>
	</li>
	<li>安装分色滤光片, 10/90 分束器, 聚焦透镜, APD, 物镜, 荧光发射过滤器, 完成检测通路。<ol>
		<li>安装分色滤镜 (图 1: DC) 以反射激光朝向物镜。将激光对准物镜的中心, 使用长透镜管的顶部和底部有两个虹膜。</li>
		<li>安装10/90 分束器 (图 1: BS), 其中10% 的光被反射到 sCMOS 相机 (如下所述) 和90% 的通过到 APD 进行跟踪。</li>
		<li>对准 APD将激光聚焦到物镜上的片上。使用片的反射来对齐所有下游元素, 通过在目标焦点上放置一个片并检查 APD 读数的强度。在分束器后, 在3D 平移阶段安装 apd 聚焦透镜 (图 1: L7), 然后是 apd。定位透镜, 使激光反射从物镜进入透镜中心。</li></ol></li></ol>在片上, 通过调整3D 位置, 使激光反射的强度最大化, 可以优化 APD 的位置。当探测器位置在任意方向移动时, APD 位置处于最佳位置, 从而降低了强度。
		<li>安装一个 longpass 荧光发射过滤器 (图 1: F), 然后将光束分配器移除反射和散射光。</li>
	
	
	<li>标记透镜的安装<ol>
		<li>将标记镜头放在两对镜片之间 (图 1: L4 和 L5之间), 如前所述, 并显示在图 1中。调整偏航, 俯仰, 高度和横向位置, 使光束通过垂直通过标记透镜的中心。</li>
	</ol>
	</li>

2. 样品制备。
<ol>
	<li>固定粒子制备<ol>
		<li>在 PBS 中稀释 190 nm 荧光珠到 ~ 5 x 108珠/毫升。添加400µL 珠解决方案上的片和安装在压电舞台的样品持有人 (珠的体积将取决于样品持有人; 这里使用的样品盒的直径是18毫米)。在这里使用的珠子, PBS 导致微粒存放在片。</li>
	</ol>
	</li>
	<li>自由运动微粒准备<ol>
		<li>稀释 110 nm 荧光珠到〜 5 x 108珠/毫升与 DI 水。添加400µL 珠解决方案到一个片和安装到样品持有人的压电阶段。</li>
	</ol>
	</li>
</ol>
3. 优化跟踪参数
<ol>
	<li>光栅扫描固定粒子<ol>
		<li>在显微镜上放一个固定的微粒样品。</li>
		<li>打开激光, 微控制器, 压电 nanopositioner 控制器, 标签镜头控制器, 和爆炸物控制器。请注意, 该顺序并不重要。在闭合回路中运行压电 nanopositioner。</li>
		<li>光栅扫描 XYZ 中的示例使用自定义扫描软件程序 (图 S3, 根据作者的请求提供的软件), 它在骑士之旅 (图 2) 中驱动 2 d-排爆, 从 APD 收集计数, 驱动压电nanopositioner, 并执行位置计算 (图 S2)。步长为 40 nm, 扫描范围为2µm。<ol>
			<li>将片放在目标的焦点上, 去掉荧光发射滤波器, 利用激光从片的反射来最大化信号强度作为微 Z 位置的函数。在将样品放置在焦平面上后, 将荧光发射过滤器放回。</li>
			<li>打开扫描程序。通过在 "开始"、"完成" 和 "步骤" 中键入数字来设置扫描范围和步骤大小。首先设置一个大的扫描范围和步长, 以找到一个粒子 (例如, 10 x 10 µm 范围与 200 nm 步大小)。找到微粒后, 缩小扫描范围并减小步长 (例如, 2 x 2 µm 范围, 100 nm 步长)。单击 "扫描" 按钮执行3D 光栅扫描以查找荧光焦点。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>标记镜头设置 (另请参见图 3)<ol>
		<li>点击标签镜头控制软件。依次单击 "连接"、"通电"。</li>
		<li>为了改变触发信号的输出相位, 必须改变输出触发模式。首先将模式从 "RGB" 更改为 "多", 然后将其更改回。现在可以更改该阶段 (图 3b)。</li>
		<li>将输出阶段设置为0°、90°和270°。虽然可以使用覆盖相空间的任何三阶段, 但发现这三的工作经验很好 (图 3c)。</li>
		<li>选择68500赫兹频率设置。</li>
		<li>为了找到最佳频率, 经常需要改变频率搜索范围。单击 "高级"、"设置"。更改 "最大值"。频率 (hz) 的0列从7万赫兹到 71500 hz。这可以调整到任何频率范围是适当的。单击 "保存校准"、"退出校准" (图 3d)。</li>
		<li>为了使新的校准生效, 请将共振切换到另一个频率 (例如, 189150 hz), 然后切换回68500赫兹频率设置 (图 3e)。</li>
		<li>将振幅逐渐改变为35%。单击 "锁定共振"。锁定频率后, 单击 "解锁共振" (图 3e)。标记透镜现在可以使用了。</li>
		<li>要校准, 更改估计中使用的参数, 使估计位置等于实际位置, 如图 4e所示, f. 输入 x、y 和 z 的扫描范围, 然后单击 "扫描" 按钮以在 XYZ 中扫描示例, 以移动粒子通过移动激光光斑。粒子的位置, 因为它是通过激光聚焦扫描应该同意的估计粒子位置确定的位置估计回路 (图 S1)。估计位置和实际位置 (图 4d) 之间的关系可以从估计位置图像 (图 4f) 中提取。如果位置不一致, 请调整位置估计循环中使用的激光位置 (ck) 的值 (图 S1)。</li>
		<li>对齐标签镜头<ol>
			<li>当标签镜头控制器关闭, 光栅扫描粒子图像 (如下所述) 在安装标记镜头和安装标记透镜后, 应该看起来相同。接下来, 通过移动目标与 z nanopositioner 进行荧光粒子 z 堆栈扫描, 以调整标记透镜的位置和角度 (图 4)。调整标记透镜的位置, 直到沿 z 方向的 xy 平面中的粒子图像没有漂移, 这是当粒子的 xy 位置没有变化 (图 4d) 的函数时实现的。跟踪系统可以在标签镜头对准后使用。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>微粒监测用 sCMOS 相机的安装<ol>
		<li>安装一个 sCMOS 相机, 以可视化粒子, 而他们被跟踪。仅在安装并优化了跟踪系统的所有组件后才安装 sCMOS。</li></ol></li></ol>在显微镜上加载一个固定的荧光粒子样本, 并运行跟踪程序, 锁定一个粒子在目标的焦点体积。然后, 安装 100 mm 焦距透镜 (图 1: L8) 和 sCMOS。调整 sCMOS 位置, 使粒子的图像聚焦到最小和最亮的点。
	
	

4. 自由扩散纳米粒子的实时3D 跟踪
<ol>
	<li>将 110 nm 的自由运动微粒样品放在显微镜上。</li>
	<li>打开激光, 微定位控制器, 压电 nanopositioner 控制器, 标签镜头控制器, 和爆炸物控制器。在开环中运行压电 nanopositioner。根据步骤3.2 设置标签镜头软件。</li>
	<li>打开并运行跟踪软件 (可根据作者的要求提供)。将位置估计参数设置为其优化值, 在3节中找到。</li>
	<li>将片放在目标的焦点上, 去掉荧光发射滤波器, 利用激光从片的反射来最大化信号强度作为微 Z 位置的函数。在找到片后, 增加焦点位置15µm 将激光从片聚焦到解决方案中。放回荧光发射过滤器。</li>
	<li>设置整型控制常数。积分控制常数可以设置为低值开始, 并慢慢增加, 直到振荡可以看到在粒子的位置读数。一旦观测到振荡, 将积分控制常数设置为引起振荡的值的80%。典型值将是约0.012 的 XY ' 积分增益 ' 和0.004 的 Z ' 积分增益 '。</li>
	<li>在 "跟踪阈值" 和 "最小跟踪" 中设置跟踪阈值, 并通过单击 "搜索" 和 "自动跟踪" 启动跟踪实验。终止跟踪的阈值设置略高于后台级别, 触发跟踪的阈值大约比背景高两倍。在这里, 触发跟踪的阈值是3赫, 终止轨迹的阈值是1.5 赫。</li>
</ol>

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作者声明没有竞争的金融利益。

尽管近年来出现了许多3D 单粒子跟踪方法, 但在低光子计数率下, 以简单的设置进行的高速3D 扩散的鲁棒 real-time 跟踪仍然是一个挑战, 这限制了它在重要的生物问题.本协议中描述的3维 DyPLoT 方法以多种方式解决了这些难题。首先, 将激励和检测路径与其他实现简单而健壮的方法相比, 大大简化了。其次, 移动激光光斑和位置估计算法为反馈回路提供精确的位置估计, 使反馈更加稳定。第三, 动态移动激光光斑的有效大探测范围 (1 x 1 x 4 µm) 可以跟踪快速移动的粒子。为了了解这一大探测区域的重要性, 考虑压电 nanopositioner 的固有响应时间非常重要。高速压电级有共振1赫的顺序, 将响应时间限制为1毫秒在 1 ms 中, 100 纳米纳米粒子的扩散系数为4µm2/秒, 将从衍射中心的平均 90 nm 扩散公司检测量。这只是平均位移, 真正的随机运动将显示比这更大的步骤, 导致粒子离开焦点体积和结束当前的轨道。对于较小的粒子来说, 情况更糟, 因为越来越大的热扩散步骤导致了跟踪损失。有效的大探测区使跟踪系统克服了固有的压电级滞后, 从粒子位置的大扩散跳跃中恢复, 提高了跟踪机构的整体鲁棒性。最后, 大扫描区域允许系统容易地拾起新的微粒, 允许连续的轨道快速地获取和大数据集汇编。
用户应该记住, 有一些关键的步骤。首先, 2 维爆炸物排爆和标签镜头的对准是至关重要的。两者必须保持良好的对齐才能获得最佳的精度。其次, 在固定粒子扫描步骤中用于位置估计的参数必须得到很好的校准 (请参见图 4)。所估计的粒子位置应与激光扫描范围的中心对应的粒子位置匹配。最后, 反馈积分常数 (KI) 应仔细调整, 从一个小值开始, 然后向上向上, 直到观察到振荡, 然后备份到该值的大约80%。
根据所需的应用程序, 有几个关于3维 DyPLoT 的警告要记住。而优化的位置估计是设计用于布朗运动, 它也很适合于方向运动。该算法可以直接应用于任何类型的运动, 因为它只是假设的位置不确定的高斯, 而不是真正的粒子位置。对于需要持续线性运动的情况, 可以在位置估计算法中添加一个附加术语 (请参见辅助信息中的公式 3 )。
在设置3维 DyPLoT 时, 应仔细考虑 2 d-爆炸物和标签的参数选择。特别重要的是用于骑士的旅行扫描的 2 d-爆炸物的 bin 时间。在理想的情况下, 骑士的巡回赛将同步到标签镜头的时期, 以确保有效的取样。然而, 重要的是要考虑的反应时间 2 d-爆炸物的步骤变化的电压。对于这里使用的单位, 有一个2-3 µs 的响应时间后, 电压的应用, 达到预期的点。在20µs bin 时间, 这是约10-15% 的收集时间。减少时间, 使其匹配的标签镜头周期为14µs 增加了 bin 时间的分数, 这是 2 d-爆炸物排爆的滞后时间。这导致了不正确的激光位置值和不正确的位置估计。
另一个让实验者牢记的因素是时间分辨率。虽然此处显示的数据是以 100 kHz 的数据速率收集的, 但最终的时间分辨率是由数据用于位置测量的最终定义的。如果将 nanopositioner 的读数用作粒子位置 (图 6), 则时间分辨率将取决于积分控制常量的值。例如, 对于图 6a-d中显示的整型控件, 舞台响应的顺序是1毫秒, 而对于图 6e-h,它的顺序是10毫秒. 如果需要更快的时间分辨率, 则位置估计算法的光子-光子结果将与光子计数率相关联。例如, 一个10赫发射率产生100µs 的时间分辨率和100赫发射率产生一个10µs 的时间分辨率。此外, 由于精度遵循关系<img alt="Equation 2" src="/files/ftp_upload/56711/56711eq2.jpg"> 41, 因此空间和时间精度是耦合的。因此, 探测器的亮度决定了空间和时间精度。
在本协议中, 我们描述了高速 real-time 3D 单粒子跟踪方法的设置、布局和对准, 它利用2维爆炸物和标记透镜动态移动聚焦激光光斑以实现快速粒子位置估计。然后介绍了样品制备和参数优化方法。3 d-DyPLoT 提供了一个强大的和相对简单的方法来锁定快速移动和低排放扩散粒子。简单的光学布局使它可以很容易地添加到任何现有的显微镜支架的侧面端口, 以便它可以与成像模块结合起来。通过这个协议, 我们希望更多的研究者能够更广泛地实施 RT-3D-SPT, 以解决快速的, 三维的生物过程。

先进的成像技术的出现, 打开了一个窗口, 看到更详细的细胞现象的结构, 一路下降到分子水平。方法如随机光学重建显微镜 (风暴)1,2,3, 照片激活的本地化显微镜 (PALM)4,5,6,7, 结构化照明显微镜 (SIM)8,9,10,11, 和受激辐射损耗显微镜 (STED)12,13, 14已经远远超出了衍射极限, 从而将空前的细节提供给了活细胞的结构和功能。然而, 充分了解这些系统的行为需要动态信息和结构信息。上面列出的超分辨率方法涉及空间分辨率和时间分辨率之间的权衡, 从而限制了动态过程的探测时间精度。提供高空间精度和时间分辨率的方法是 RT-3D-SPT15、16、17、18、19、20 21,22,23,2425262728在这里, 我们将传统的3维的30和 RT-3D-SPT 区分开来. 传统的3维单标需要一个时间序列的三维图像数据 (可以用共焦显微镜或荧光显微镜获取)给定正确的配置)。在传统的3维标度中, 通过在每个图像堆栈中定位粒子并在连续的卷中连接位置来创建轨迹, 来确定粒子的坐标。对于这些方法, 最终的时间分辨率取决于容积成像率。对于共焦显微镜, 这是很容易在几秒到几十秒的规模。对于荧光的方法 , 其中的光学路径纵 , 使轴向位置信息可以提取 , 时间分辨率是有限的相机曝光或读取时间。这些 epifluorescent 的方法是有限的范围内轴向信息可以收集, 虽然最近的进展, 在傅里叶平面相掩模设计和自适应光学是将这些范围扩展到10µm 或更多31,32,33,34。
相比之下, RT-3D-SPT 不依赖于获取3D 图像堆栈并在事实之后找到粒子。相反, real-time 的位置信息是通过单点检测器提取的, 通过使用高速压电阶段, 反馈被应用到有效地 "锁定" 物镜焦距中的粒子。这就允许连续测量粒子的位置限制, 只有多少光子可以收集。此外, 这种方法能够在粒子在长距离移动时对其进行光谱审讯。RT-3D-SPT 的效果类似于一个无力的纳米物体的光阱, 其中粒子是连续探测和测量在 real-time 不需要大的激光功率或光学力量。鉴于 RT-3D-SPT 提供了一种连续审讯快速扩散对象的方法 (最多20µm2/秒)25,29在低光子计数率三,20, 35, 它应提供一个窗口, 进入快速或瞬态的生物过程, 如细胞内货物运输, 配体受体结合, 和细胞外动力学的单一病毒。然而, 在这一点上, RT-3D-SPT 的应用仅限于少数几个致力于推动这一技术的团体。
一个障碍是 RT-3D-SPT 方法所要求的光学布局的复杂性, 这是多种多样的。对于大多数方法, 光反馈是由压电工作台提供的。当粒子在 X、Y 或 Z 中产生小的运动时, 从单点探测器的读数转换为错误函数, 并以高速向压电 nanopositioner, 这反过来又移动样品来抵消粒子的运动, 有效地将其锁定在相对于物镜的位置。要测量 X、Y 和 Z 中的小位置移动, 可以使用多个检测器 (4 或5取决于实现)15、18、21或多个励磁点 (2-4), 如果锁定放大器用于使用旋转激光光斑提取 X 和 Y 位置)25,28被应用。这些多个检测和发射点的重叠使得系统难以对齐和维护。
在这里, 我们提出了一个高速目标锁定 3 d-标的方法与简化光学设计称为 3 d-DyPLoT29。3 d-DyPLoT 使用 2 d-爆炸物和标记透镜36,37,38 , 以高速率 (50 赫 XY, 70 赫 Z) 动态移动聚焦激光光斑。结合激光聚焦位置和光子到达时间, 使粒子的3D 位置可以迅速获得, 即使在低光子计数率。2 d-爆炸物排爆行动驱动激光焦点在骑士的游览样式39以正方形大小 1 x 1 µm 在 x Y 平面和标记透镜移动激光焦点在轴向范围 2-4 µm。3D 粒子位置得到优化的位置估计算法29,40在3D。在现场可编程门阵列 (FPGA) 上对3D 动态移动光斑、雪崩光电二极管 (APD) 的光子计数、real-time 粒子位置计算、压电级反馈和数据记录进行了控制。在本协议中, 我们描述了如何建立一个3维 DyPLoT 显微镜 step-by 步, 包括光学对准, 校准与固定粒子, 最后自由粒子跟踪。作为一个示范, 110 纳米荧光珠连续跟踪在水中的时间分钟。
本文所描述的方法是一个理想的选择, 在任何应用, 它是希望连续监测快速移动荧光探针在低光水平, 包括病毒, 纳米颗粒, 和小泡, 如体。与以往的方法相比, 只有一个单一的励磁和单一的检测路径, 使对准和维护一目了然。此外, 这个大的探测区域使这个显微镜能够很容易地快速地拾取粒子, 而在低信号水平 (下至10赫) 跟踪的能力使得这种方法适合微光应用程序29。

<table border="0" cellpadding="0" cellspacing="0" width="1075">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">2D Electro-optic Deflector&#160;</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">M310A</td>
			<td style="width:541px;">2 required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Power supply for EOD</td>
			<td style="width:145px;">ConOptics</td>
			<td style="width:160px;">412</td>
			<td>Converts FPGA ouput to high voltage for EOD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">TAG&#160; Lens</td>
			<td style="width:145px;">TAG Optics</td>
			<td style="width:160px;">TAG 2.0</td>
			<td>Used to deflect laser along axial direction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">XY piezoelectric nanopositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-PDQ275HS</td>
			<td>Used for moving the sample to lock the particle in the objective focal volume in&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Z piezoelectric nanoposiitoner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">Nano-OP65HS</td>
			<td>Used to move the objective lens to follow the diffusing particle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Micropositioner</td>
			<td style="width:145px;">MadCity Labs</td>
			<td style="width:160px;">MicroDrive</td>
			<td>Used to coarsely position sample and evaluate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Objective Lens</td>
			<td style="width:145px;">Zeiss</td>
			<td style="width:160px;">PlanApo</td>
			<td>High numerical aperture required for best sensitivity. 100X, 1.49 NA, M27, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">sCMOS camera</td>
			<td style="width:145px;">PCO</td>
			<td style="width:160px;">pco.edge 4.2</td>
			<td>Used to monitor the particle&#39;s position</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">APD</td>
			<td style="width:145px;">Excelitas</td>
			<td style="width:160px;">SPCM-ARQH-15</td>
			<td>Lower dark counts beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Field programmable gate array</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">NI-7852r</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Software</td>
			<td style="width:145px;">National Instruments</td>
			<td style="width:160px;">LabVIEW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tracking excitation laser</td>
			<td style="width:145px;">JDSU</td>
			<td style="width:160px;">FCD488-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-150-A-ML</td>
			<td>L1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Pinhole</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">P75S</td>
			<td>PH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Glan-Thompson Polarizer</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">GTH5-A</td>
			<td>GT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Half-wave plate</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">WPH05M-488</td>
			<td>WP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-75-A-ML</td>
			<td>L3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-250-A-ML</td>
			<td>L4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Lens</td>
			<td style="width:145px;">ThorLabs</td>
			<td style="width:160px;">AC254-200-A-ML</td>
			<td>L6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Dichroic Mirror</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">ZT405/488/561/640rpc</td>
			<td>DC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Fluorescence Emission Filter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">D535/40m</td>
			<td>F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">10/90 beamsplitter</td>
			<td style="width:145px;">Chroma</td>
			<td style="width:160px;">21012</td>
			<td>BS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">190 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/9942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">110 nm fluorescent nanoparticles</td>
			<td>Bangs laboratories</td>
			<td>FC02F/10617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-545A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Powermeter</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">PM100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">CMOS</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">DCC1545M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Iris</td>
			<td style="width:145px;">Thorlabs</td>
			<td style="width:160px;">SM1D12D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:145px;">Mad City Labs</td>
			<td style="width:160px;">RM21</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for real-time 3d single particle tracking

实时三维单粒子跟踪 (RT-3D-SPT) 有可能在蜂窝系统中对快速的3D 过程产生影响。尽管近年来提出了各种 RT-3D-SPT 方法, 但在低光子计数速率下跟踪高速3D 扩散粒子仍然是一个挑战。此外, RT-3D-SPT 设置通常是复杂和难以实施, 限制其广泛应用于生物问题。该协议提出了一个 RT-3D-SPT 系统, 名为3D 动态光子定位跟踪 (3 维 DyPLoT), 它可以跟踪高扩散速度 (高达20µm2/秒) 在低光子计数率 (下至10赫) 的粒子。3 d-DyPLoT 采用2D 电光偏转器 (2 d-爆炸物) 和可调谐的声学梯度 (标签) 镜头, 以驱动一个单一的聚焦激光点动态在3D。结合一种优化的位置估计算法, 3 维 DyPLoT 可以锁定在高跟踪速度和高定位精度的单粒子上。由于单一的励磁和单一的检测路径布局, 3 维 DyPLoT 是健壮的, 易于设置。本协议讨论如何逐步构建3维 DyPLoT。首先, 介绍了光学布局。然后, 通过光栅扫描190纳米荧光珠与压电 nanopositioner 对系统进行了标定和优化。最后, 为了证明 real-time 3D 跟踪能力, 110 nm 荧光珠在水中跟踪。

固定粒子扫描 (图 4) 和自由扩散 110 nm 荧光粒子跟踪 (图 5) 是按照上述协议执行的。粒子扫描是通过移动压电 nanopositioner 和 bin 光子, 同时计算在扫描的每个点的粒子的估计位置。扫描图像显示一个均匀强度的平方 (图 4a), 估计的位置显示一个与粒子的实际位置的线性关系, 在 x、y 和 z 方向上的 1 x 1 x 2 µm 范围 (图 4bf)。
为了演示 real-time 跟踪, 110 nm 的荧光粒子在水中被3维 DyPLoT (图 5a, b) 跟踪。均方位移分析显示了布朗运动的典型线性行为特征 (图 5c)。30弹道分析表明, 平均水动力直径 110 nm, 与制造商规范的大小的荧光纳米粒子被跟踪 (图 5d)。此外, 电影1显示了 real-time 压电 nanopositioner 读数和同步 sCMOS 图像为一个2分钟的自由扩散 110 nm 荧光粒子的弹道。
除了能够跟踪高速, 慢速运动粒子可以本地化高精度。图 6a-d显示使用与高速跟踪相同的反馈参数的3维 DyPLoT 系统对固定粒子的应用, 分别显示 X、Y 和 Z 中的17.6、26.4 和 53.4 nm 的精度, 其光子计数率为 105。图 6e-h显示在反馈控制下的精度降低了 10, 有效地交换了精度, 在 X、Y 和 Z 中分别展示了6.5、8.3 和 10.5 nm 的精度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56711/56711fig1.jpg"> 
图1。3D 跟踪系统示意图.2 d-爆炸物排爆 (EOD1和 #38; EOD2) 和标记透镜 (标记) 分别将激光沿 XY 和 Z 方向偏转。apd (apd) 用于收集荧光光子, 它们被发送到 fpga (fpga)。FPGA 用于位置计算算法, 光子计数, 控制 2 d-爆炸物, 以及控制和读取压电 nanopositioner (NSxy和NZz)。图中标记的其他组件: 镜像 (M);镜片 (L #);针孔 (PH);格兰-汤普森偏光片 (GP);波板 (W1);分色滤镜 (DC);物镜 (OL, 100X NA = 1.49);荧光发射过滤器 (F);分束器 (BS);XY 微阶段 (MSxy);Z 微阶段 (MSz)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/56711fig1large.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/56711/56711fig2.jpg"> 
图2。在3维 DyPLoT 中实施的骑士之旅的坐标.轴1和轴2应沿 X 或 Y 轴对齐 2 d-爆炸物排爆。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/56711fig2large.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/56711/56711fig3.jpg"> 
图3。标记镜头软件设置.有关详细信息, 请参阅4.2 节标签镜头设置。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/56711fig3large.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/56711/56711fig4.jpg"> 
图4。粒子扫描和位置估计.(a) 扫描图像 190 nm 荧光珠与 2 d 的爆炸物驱动激光在 1 x 1 µm 方骑士的旅游模式。荧光强度由颜色表示。单位: 赫。(b) 估计 xk, 该粒子的位置相对于 2 d-爆炸物排爆扫描的中心, 以微米计。b、 c和d中的颜色表示估计的位置。单位: µm (c) 在微米中对 yk的估计。(d) 估计 zk, 该粒子的位置相对于标签透镜扫描的轴向中心的微米。(e) 估计粒子位置 yk作为舞台位置的函数, 从 (c) 的整个网格进行平均。请注意, 从位置估计算法 (yk) 获得的估计粒子位置与实际位置一致。(f) 估计的粒子位置 zk作为舞台位置的函数, 从 (d) 的整个网格进行平均。估计的位置显示了一个线性关系与粒子的实际位置超过 1 x 1 x 2 µm 范围在 X, Y 和 Z 方向。请注意, 从位置估计算法 (zk) 获得的估计粒子位置与实际位置一致。估计的位置显示了一个线性关系与粒子的实际位置超过 1 x 1 x 2 µm 范围在 X, Y 和 Z 方向。(a-c) 中的白色刻度线表示 500 nm, 而在 (d) 中的黑色刻度线表示2µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/56711fig4large.jpg" target="_blank">请点击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56711/56711fig5.jpg"> 
图5。在水中跟踪110纳米荧光粒子, 3 维 DyPLOT。(a) 在水中自由扩散110纳米荧光纳米粒子的3D 弹道。(b) 荧光强度作为轨迹在 (a) 中的时间函数。(c) 在 (a) 中的轨迹。蓝线是测量的, 而虚线红线是最适合线性回归线。(d) 对30轨迹进行了分析, 显示了 110 nm 的平均水动力直径, 与所跟踪的荧光纳米粒子的大小吻合得很好。用斯托克斯-爱因斯坦关系计算了粒子的直径。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/56711fig5large.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/56711/56711fig6.jpg"> 
图6。固定粒子跟踪的精度.(a) 在不同的计数速率下, 用3维 DyPLoT 跟踪固定粒子。精度是 (b) 17.6 nm 在 X, (c) 26.4 nm 在 Y, 和 (d) 53.4 nm 在 Z 的发射率为100赫。(e) 在探测较慢的进程时, 可以将反馈控制常量 (KI) 降低10倍, 以提高精度。在这个减少的反馈控制下, 精确度是 (f) 6.5 毫微米在 X, (g) 8.3 毫微米在 Y 和 (h) 10.5 毫微米在 Z 以100赫的放射率。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/56711fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图 S1.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/fig_S1_position_estimation-01.pdf">请单击此处下载此文件.</a>
图 S2.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/fig_s2_TAG_operation.pdf">请单击此处下载此文件.</a>
图 S3.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/fig_s3_software.pdf">请单击此处下载此文件.</a>
补充信息<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56711/170802_software_description.pdf">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about A Protocol for Real-time 3D Single Particle Tracking at JoVE.com

A Protocol for Real-time 3D Single Particle Tracking

本协议详细介绍了一种 real-time 3D 单粒子跟踪显微镜的构造和操作, 能够跟踪高扩散速度和低光子计数率的纳米荧光探针。

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

a-protocol-for-real-time-3d-single-particle-tracking

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.8K 次观看.  Duke University. 本协议详细介绍了一种 real-time 3D 单粒子跟踪显微镜的构造和操作, 能够跟踪高扩散速度和低光子计数率的纳米荧光探针。

视频: A Protocol for Real-time 3D Single Particle Tracking

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

科研

生物工程

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

在原位生物过程单细胞形态实时测定的显微镜

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...