的MKK感谢英国工程和物理科学研究理事会（EPSRC）生命科学的个人奖学金的接口方案。我们也想承认由英国生物技术和生物科学研究理事会（BBSRC）的资金。

没有什么不同强度的荧光显微镜聚焦或宽领域的FLIM的样品制备的协议。其次是数据采集，数据分析的主要任务，即从原始数据中提取的荧光寿命。一旦这些已经获得的数据的解释，有助于验证或伪造的假设。 1。染色细胞与分子转子<ol><li>准备股票液（10毫升）约1毫克/毫升的染料溶解在适当的溶剂（如甲醇肾上腺素-12）6,7用一个精确的平衡和吸管。 </li><li>为的显微镜coverslide底面与被染色的细胞模型癌细胞线，在我们的例子中的HeLa生长在多孔板，在孵化器5％的CO 2气氛直到在37°C〜80％汇合。 </li><li>添加10 - 20μl原液的活细胞生长在一个多-WEL升板（SmartSlide 50微孵化系统，Wafergen），4毫升的Opti-MEM培养基（GIBCO公司），每6孔板。这就产生了在良好的微摩尔染料浓度。 </li><li>返回孵化器在37°C多孔板与5％的CO 2染色为10-45分钟的气氛。 </li><li>从孵化器的多孔板和洗细胞3-4次，用4毫升透明细胞培养液中（例如的Opti-MEM中），以去除多余的染料。 </li><li>显微镜阶段转移多孔板和连接到所需温度控制器/ 5％CO 2的进气口，在成像准备。 </li></ol> 2。 FLIM的，在细胞中的荧光分子转子<ol><li>显微镜舞台上放置样品，并获得传输和荧光图像识别荧光细胞。一个实验装置示意图如图。 1.Verify，地点艰难的荧光发散反恐执行局（如细胞膜，细胞质）。获得荧光光谱，并确认这是染料或预期的蛋白质，在这种情况下，分子转子的频谱。作为阴性对照，图像非彩色样本和验证，它并没有发出荧光。虽然这一步是没有必要专门FLIM的，它是在总体上是好的做法，并帮助验证该样本是什么你认为它是。 </li><li>切换到FLIM的模式 - 移动镜的荧光检测光路（“外部探测器”按钮“光束路径设置”面板上的Leica TCS SP2采集控制软件），这是很容易完成。一个适当的荧光过滤器来阻止任何兴奋的光芒到达探测器必须在荧光检测beampath的。 </li></ol><img alt="图1" src="/files/ftp_upload/2925/2925fig1.jpg" /> 图1时域使用AC FLIM的实验安排。onfocal激光扫描显微镜。 <ol start="3"><li>扫描样本和检查，对计算机的控制FLIM的收购，探测器的计数率（黑条上标有收购贝克尔Hickl SPC的830板控制软件CFD）是不超过激光重复率约1％（绿色栏标记同步采集控制软件）。如果是的话，减少中性密度过滤器放置在激光束路径的激光激发强度，例如，以避免收集桩扭曲的荧光衰减曲线。 </li><li>收购FLIM的形象，通常为3-5分钟，停止扫描并保存原始数据（三维数据的“魔方”组成的空间坐标x和y，时间）。 </li><li>打开荧光衰变分析软件包的原始数据，例如三2月14日 ，商业软件，显示荧光强度图像。这简直是​​综合性的荧光衰减，即荧光衰减曲线下的面积，在每个像素。 </li><li>选择一个典型的像素上放置光标，并检查该像素中的荧光衰减。如果峰值计数低于100，使用空间的像素分级。相邻像素的数量（例如3x3或5x5）被添加到中央的像素，以便获得更高的峰值计数有。这提供了一个为下一步的统计精度较高。另外，测量可重复采集时间较长（步骤5）。为30 - 50分钟，得到了一个高约10倍的峰值计数（总计数），但是这是远远太长引入文物由于样品运动，显微镜漂移，光的危险，因为大多数生物样品采集时间漂白。 </li><li>选择一个全球性的像素阈值（上方装有一个像素的衰减）和适用于一个单一的指数衰减拟合的图像。结果产生一个以上的门槛，每个像素的荧光寿命，然后将其编码颜色。每个像素的颜色与合适的结果，并获得1 FLIM的地图。检查卡方值的减少，各种像素 - 约1（1.3），表示一个不错的选择。检查相应的残留物，而应随机分布在零附近。 </li><li>荧光寿命直方图地块往往是某些荧光寿命与荧光寿命本身发生。调整色彩范围，荧光寿命分布到适合的颜色范围。 </li><li>如果单指数拟合不产生约1卡方值（1.3），有一个从零残差系统偏差，需要一个更复杂的模型。例如，尝试双指数模型拟合荧光衰变为两个不同的环境探测可能的适当人选也将产生，这给染料的相对量的指示，在一个前指数的因素或振幅帐户环境MENT或其他。另外，拉伸指数函数可能是适当的，占了荧光寿命的分布。 </li><li>的荧光寿命，指数前因子，寿命比，并为每个像素的指数前因子比对的结果，然后可以在色彩编码。每个像素是彩色的，根据其价值，由于荧光寿命，指数前因子及其比率的对比得出。再次，检查降低的卡方值（也可在彩色编码和图像显示） - 约1（1.3），表示一个不错的选择。检查残差，应随机分布在零附近。 </li><li>荧光寿命直方图应陪同平均荧光寿命值，方便的可视化和荧光寿命分布的所有图像。 </li></ol> 3。代表结果荧光衰变测量甲醇/甘油混合物的粘度增加，荧光分子转子如图。 2。是单指数荧光衰减，荧光寿命作为粘度的功能显着变化。它增加了从甲醇约300 PS（粘度0.6 CP）95％甘油（粘度950 CP）在3.4纳秒。 <img alt="图2" src="/files/ftp_upload/2925/2925fig2.jpg" /> 图2。肾上腺素-C的荧光衰变型材甲醇/甘油的混合物，不同粘度12 6。 数校准情节的荧光寿命τ与粘度η为荧光分子转子图。 3。福斯特·霍夫曼方程9的要求，它是一条直线<img src="/files/ftp_upload/2925/2925eq1.jpg" alt="式（1）" /> 其中 k是radiativé速率常数，Z和 X是常数，0 &lt;X &lt;1。对数以双方产量<img src="/files/ftp_upload/2925/2925eq2.jpg" alt="公式（2）" /> 其中 x是直线的梯度。 <img alt="图3" src="/files/ftp_upload/2925/2925fig3.jpg" /> 图3。的日志荧光寿命比日志粘度积肾上腺素-12产量直线按照福斯特霍夫曼方程。 观察活细胞的培养与荧光分子转子状染料分布的荧光图像。 HeLa细胞培养与细观取代肾上腺素染料FLIM的图像显示在图。 4。可以充分使用一个单一的指数衰减模型拟合图像的每个像素中的荧光衰变。 图4。（一）荧光强度和（二）与肾上腺素-12染色HeLa细胞FLIM的图像。明亮，圈点的地区表现出了比其他地区的寿命较短。这个短liftime对应的puncta在较低的粘度，可能是脂滴，根据福斯特霍夫曼方程。 通过绘制每个像素中提取的寿命，我们获得了整个图像的荧光寿命直方图如图。 5。 <img alt="图5" src="/files/ftp_upload/2925/2925fig5.jpg" /> 图5。荧光寿命从细观取代肾上腺素分子转子FLIM的HeLa细胞染色图像的直方图。

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没有利益冲突的声明。

FLIM的超过强度为基础的荧光成像提供了一些关键的优势。它可以报告光物理，是很难或不可能通过荧光成像观察的事件，因为它可以从荧光浓度的影响，他们分开。这是映射成像荧光分子转子内的粘度特别有用。荧光寿命可以很容易地被转换成使用粘度校准图，如图所示。 3，荧光分子转子的浓度无关。 FLIM的有可能是复杂的工件数据的解释。10器乐文物包括散射光，这将显示为荧光衰变开始上的高峰，可以用很短的衰减时间混淆，或后的小峰IRF的，可能会导致显微镜内部的反射。这些散射光可以认定为文物等因为他们可以区分光谱歧视 - 他们总是在激发光波长相同。记住，在空气中，光在1纳秒30厘米，有助于查明反射的起源。 过滤器或玻璃荧光也可以导致一个工件，特别是在低样品的荧光，但可以很容易地确定没有样品测量：如果衰减在这种情况下取​​得的，它是由于仪器无关与样品！另一方面，注意样品自发荧光，也可能有助于荧光衰变。 在时间相关单光子计数（的TCSPC），时间幅度转换器（TAC）的非线性可能会导致贫穷的配合，但可以通过阻断激发和环境光，如从传输的光源照射到样品标识测量时间。应该是恒定的背景获得图像的每个像素。从一个固定的背景发生偏差的地区，绝不会产生一个不错的选择，应避免测量，如果他们不能被淘汰的TCSPC卡通过调整参数。 一个臭名昭著的TCSPC神器是光子的堆积，这是由光子的检出率过高造成的。11,12这导致只有第一个光子被超时，忽略任何后续的光子，因为电子是忙碌的时间和处理的第一个光子。堆积导致的荧光寿命缩短，最好的办法来避免这种光子计数率保持在约1％的激光重复率。 展望有FLIM的各种实现，并根据不同的应用，每个人都有其优点和缺点。13理想的荧光显微镜下将收购整个多维荧光emissio列印强度，位置，寿命，波长和偏振单光子探测灵敏度，最大的空间分辨率和最小采集时间在一个单一的测量，轮廓。目前有没有这个功能的独特组合技术，构建一个仪器开发仍然是一个挑战。重要问题的新的物理技术在细胞生物学中的应用往往是意外的发现的路径，还有很长的路要走之前，我们已接近饱和的能力，为细胞生物学荧光成像。事实上，如寿命，频谱和偏振，以及更迅速地在更高的空间分辨率的三维成像，荧光成像参数，一定要揭示细胞生物学的新的方面。

样品用荧光分子转子硬件： 倒徕卡TCS SP2的共焦扫描显微镜相干米拉900钛蓝宝石飞秒激光与1威尔第V6泵激光或滨松PLP-10 470皮秒脉冲激光二极管激发光源贝克尔与Hickl SPC 830板，在3GHz的奔腾IV，1GB RAM与Windows XP的计算机贝克尔与Hickl水冷头PMC100-01探测器的基础上H5773P-01滨松光电倍增管安装在显微镜的x1端口，或混合探测器 DCC 100探测器控制模块软件： 三2月14日或由贝克尔和Hickl SPCImage 2.8

fluorescence lifetime imaging of molecular rotors in living cells

扩散往往是化学反应或生物过程的速率决定的一个重要一步，并在广泛的细胞内事件的作用。粘度是影响分子和蛋白质扩散的关键参数之一，并已与粘度变化在细胞水平上对疾病和故障。1-3方法来衡量散装粘度发达，成像微粘度仍然是一个挑战。微观物体，如单细胞的粘度地图，直到最近一直难以取得。测绘与荧光技术的粘度是有利的，因为类似的其他光学技术，它是微创，非破坏性，可应用于活细胞和组织。 荧光分子转子表现出荧光寿命和量子产率，这是其微粘度的功能。4,5分子内扭转或旋转导致从激发态返回到基态非辐射衰变。粘性环境减缓这种旋转或扭曲，限制访问这个非辐射衰变途径。这导致的荧光量子产率和荧光寿命的增加。改性疏水的肾上腺素染料作为荧光分子转子的荧光寿命成像（FLIM的）表明，这些探针的荧光寿命是其环境的微粘度的功能。6-8 A与溶剂的粘度产量的荧光寿命的对数图一条直线，服从福斯特·霍夫曼方程。9此图也可作为校准曲线转换成粘度的荧光寿命。 经过修改后的肾上腺素荧光分子转子活细胞的培养，点状染料分布观察荧光图像。在日获得的粘度值e在活细胞puncta高于水的100倍左右，和细胞质。6,7时间分辨荧光各向异性测量产生旋转相关时间，在与这些大型的微粘度值一致。测绘荧光寿命是独立的荧光强度，从而使分离探针浓度和粘度的影响。 总之，我们已经开发出一种务实和灵活的方法来映射基于荧光分子转子FLIM的细胞中的微粘度。

Watch this Scientific Journal Video about Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells at JoVE.com

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

荧光寿命成像（FLIM的）已经成为一项关键技术，对图像的环境和特定蛋白质在活细胞染料的相互作用。 FLIM的荧光分子转子允许映射在活细胞中的粘度。

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

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JoVE Journal

Bioengineering

24.8K 次观看.  King's College London. 荧光寿命成像（FLIM的）已经成为一项关键技术，对图像的环境和特定蛋白质在活细胞染料的相互作用。 FLIM的荧光分子转子允许映射在活细胞中的粘度。

视频: Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

科研

生物工程

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 ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

澳大利亚同步加速器串行晶体测量的利皮迪科注射协议

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...