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 ...

fluorescence-lifetime-imaging-of-molecular-rotors-in-living-cells

Nanyang Technological University

Research

JoVE Journal

Bioengineering

24.9K 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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

<meta charset="utf8"></head><body>使用荧光寿命成像显微镜探测癌细胞的代谢和粘度

Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting endogenous fluorescence. Viscosity is measured using a fluorescent molecular rotor.

使用荧光寿命成像显微镜探测癌细胞的代谢和粘度

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and ...

癌症研究

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

生物化学

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

免疫与感染

Autofluorescence Imaging to Evaluate Cellular Metabolism

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.

This protocol describes fluorescence imaging and analysis of the endogenous metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H), and oxidized flavin adenine dinucleotide (FAD). Autofluorescence imaging of NAD(P)H and FAD provides a label-free, nondestructive method to assess cellular metabolism.

自发荧光成像评估细胞代谢

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

神经科学

用于基于 FLIM 的代谢和缺氧分析的多细胞球状体形成

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

作者聚焦:使用荧光寿命成像显微镜标准化用于代谢和氧合分析的球状体形成方法

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

在活细胞中的荧光寿命成像分子转子

摘要

探索更多视频

此视频中的章节

荧光检测方法，为微流体液滴平台

使用荧光寿命成像显微镜探测癌细胞的代谢和粘度

使用荧光寿命成像显微镜探测癌细胞的代谢和粘度

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

荧光寿命成像显微镜观察细胞内圈套的贩运

自发荧光成像评估细胞代谢

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

在活细胞中的荧光寿命成像分子转子

摘要

探索更多视频

此视频中的章节

荧光检测方法，为微流体液滴平台

使用荧光寿命成像显微镜探测癌细胞的代谢和粘度

使用荧光寿命成像显微镜探测癌细胞的代谢和粘度

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

荧光寿命成像显微镜观察细胞内圈套的贩运

自发荧光成像评估细胞代谢

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜 （FLIM）

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）

多细胞球体的生产和多参数活细胞荧光寿命成像显微镜（FLIM）