这项工作是由美国国家科学基金会奖DBI的0852891。号PHY0120999根据合作协议，由加利福尼亚大学戴维斯分校生物光子科学与技术，一个指定的美国国家科学基金会科学技术中心管理中心还资助了这项工作的一部分。

1。在准备，并把这个系统内的拉曼样品，必须采取一定的照顾。 <ol><li>由于该系统通常使用非常高数​​值孔径物镜，工作距离很短，样品置于盖玻片上。生物样品通常是放置在1号安装盖玻片厚度Attofluor细胞室（Invitrogen公司，卡尔斯巴德，加利福尼亚州）。 </li><li>液体样品，特别是那些对人体无毒，被放置在一个小玻璃瓶，巩固有机硅环氧开幕盖玻片。瓶子，然后倒测量。 </li><li>因为我们的系统上取决于泵和脉冲信号的精确定时，我们不使用我们的显微镜的传统重点的调整，转换的目标，向上和向下的约束。这将增加和减去从我们的系统的光路。相反，我们我们的样本放在显微镜阶段，有其自身的独立的集中控制的顶部安装一个中学阶段。 </li></ol> 2。为了利用时间选通的拉曼光谱，激发光必须得到妥善的准备。 <ol><li>我们开始与光从2.4 W可调脉冲钛新兴：Sapph激光（变色龙，相干系统，美国加州圣克拉拉市）。在80 MHz的脉冲序列的每个脉冲有30个新泽西州的能源，有140 FS的时间宽度，并在808纳米〜6 nm的光谱带宽与中心的频谱。 </li><li>为了防止重新进入激光腔反射，光线通过一个法拉第隔离。法拉第隔离器前放置一个半波板允许进入系统发送的总功率连续调谐。 </li><li>由于6纳米是一个过于宽泛的带宽，以解决最拉曼模式，光束通过一个很窄（0.8 nm的FWHM，安多弗公司，新罕布什尔州Salem）带通滤波器为808 nm为中心的发送。 </li><li>消色差的双峰上一个5毫米的β-钡硼酸（BBO）晶体（CASIX，福州，福建，公关中国）从808纳米到404纳米的波长减半（F = 100毫米）的光，然后集中。 BBO晶体放置在尖端和倾斜控制山，也翻译舞台上安装。为了最大限度地提高波长转换的效率，必须放置水晶正是左右对称的双重焦点，其晶轴对齐传入的光束的偏振。 </li><li>由于偏振相关波长转换效率，控制发送到样品的光量可放置法拉第隔离器后的第二个半波片。通过旋转此波片，发送到样品的光的强度可以调整独立泵束（下面要讨论的）发送的强度。 </li><li>第二个消色差的双重选择退出束足够大，以补回的显微镜物镜光圈，和成一个倒置显微镜（IX - 71，奥林巴斯（F = 500 mm的），波长转换光recollimated中心谷，PA）通过两个转向镜子。 </li><li>显微镜的物镜，被安置在系统的最单片元件的光学元件，定义光轴。为了激发光对准轴，一面镜子放置在显微镜的样本平面。两个转向镜子，然后反复调整，同时观察上的CCD摄像头时代（μEye，IDS，Obersulm，德国），连接到显微镜的成像端口的背反射的激光束。假设，在相机上的形象是在显微镜的领域 - 的的观点为中心，梁是上的轴时，焦斑是在显微镜芯片和客观的翻译沿z轴为中心不会更改中心点散焦光束。 </li><li>当样品放置在样品平面照射激光光发生拉曼散射。放在显微镜下面的目标分色过滤器分离移波长激发光从拉曼散射光（荧光），指导拉曼散射光显微镜的侧面端口。显微镜进行了修改，以消除任何在这条道路的镜片，这样的信号光从平行显微镜出现。 </li><li>由于信号光从显微镜中出现的是较明确的格兰 - 汤普森偏振片光圈较大，一个0.47x望远镜建造两个消色差双峰（F1 = 75毫米，F2 = 35毫米），是用来缩小光束。 </li><li>光信号，然后由一个格兰 - 汤普森偏振片在0 °为导向，尊重在实验室内的垂直极化，并指示，在那里与泵束重组到分色镜。 </li></ol> 3。为了光闸以最高效率运作，必须小心编制以及泵的梁（克尔梁）。 <ol&gt; <li>泵脉冲是第一次由0.35x放大两个双峰（F1 = 35毫米，F2 = 100毫米）消色差望远镜的构造相匹配的信号光束的最后大小。 </li><li>泵束，然后送入放在一个线性的翻译阶段，可以调整，以确保泵和脉冲信号（下面要讨论的调整）的时间重叠成直角棱镜组成的延迟线。 </li><li>延迟线后，光束通过半波片和偏振片面向发送到垂直45 °在实验室内。这将确保泵束的适当的偏振态，当它到达非线性介质。 </li><li>然后光线反射的两个转向镜子，压电控制，用来微调泵束的位置，重叠信号光束空间。重叠是通过观察泵和信号光在两个地点，一个接近和一个远梁相结合，从分色镜。通过第一个转向镜在近点，和压电镜梁远点重叠重叠的两束，泵束可以完全共线与信号光束。 </li><li>随着两束相结合，科尔门和收集系统设置为最大限度地收集的时间选通信号。 </li></ol> 4。随着两束相结合，科尔门和收集系统设置为最大限度地收集的时间选通信号。 <ol><li>泵和信号光束先通过分色过滤器，在404纳米，外径6，防止任何令人兴奋的拉曼散射的残余激发光在非线性介质。 </li><li>泵和信号光束，然后都集中到1厘米光程包含非线性材料的石英比色皿（F = 35毫米）消色差双峰。任何非线性材料，有一个适当的高非线性指数（高于CS 2），并适当短的时间响应（&lt;2个PS），可以利用这里。对于这些实验中，我们使用二硫化碳，CS 2中 ，有一个非线性折射率n 2 = 3.1 × 10 -18米2 / W光recollimated焦距相同，第一个与第二双峰。 </li><li>梁，然后通过和格兰 - 汤普森在一个旋转的贴装分析仪，然后通过吸收和干扰过滤器相结合，有一个808纳米的OD 10。 </li><li>最后，信号光的重点是消色差的双峰（F = 35毫米到50μm多模光纤，其中光纤是安装在一个阶段，可在X，Y和Z的翻译）纤维，然后加上一个附加的CCD相机（SP2300i和PIXIS 100B，分别由普林斯顿仪器，特伦顿制造，新泽西州）的商业成像摄谱仪。 </li><li>要对齐收集系统，以最大限度地采集到的信号，分析仪设置为0 °和测试样本的甲苯在样品平面上。通过调节X，Y，Ž控制的光纤安装，收集的拉曼信号进行了优化。 </li><li>为了确保适当的空间和时间上的泵和信号光束的重叠，镜子被放置在显微镜的样本平面。 404纳米过滤器从系统中删除。随着分析仪旋转到90 ° retroreflected 404纳米束送入摄谱仪的调整等，它不饱和相机的强度。泵束的关闭，该分析仪是旋转，以尽量减少传输的404 nm的信号。泵束的重新开启，延迟阶段是慢慢调整，直至404纳米的光传输开始增加。然后，通过反复调整的延迟阶段，压电电镜，X，Y，和纤维的Z控制，最大限度地提高信号。 </li><li>由于复古反映404纳米的光束和拉曼散射光，可采取略有不同的路径，通过该系统，最终的调整是由放置在样品阶段和轻微的调整对齐，通过改变很强的拉曼散射（如甲苯）延迟阶段，piezeo电镜，X，Y和Z和光纤控制，以优化的拉曼信号。 </li></ol> 5。一个单一的时间选通拉曼光谱收集需要几个光谱采集到正确的系统工件。 <ol><li>随着设置为0 °，激发光束和关闭泵束的，“非门”频谱分析仪，代表没有时间浇注系统的好处，将收购的频谱。 </li><li>随着分析仪设置为90 °，激发光泵束的仍然关闭，一回地面频谱，杂散光通过偏振片和系​​统中的其他要素的泄漏量。 </li><li>分析仪，其余90 °和所有梁，采取“门控”频谱，允许通过交叉偏光片的拉曼散射光泵束的存在。 </li><li>最后，仍然在90 °的分析仪，激发光泵束，第二个背景谱是，代表“门”进入光谱仪的光为808 nm的残留量的频谱作出贡献。 </li><li>此外，频谱是采取关闭所有的激光器，特征基线“黑暗”的相机和电子信号水平。 </li><li>由于光谱往往需要漫长的整合时间，这是必须分解成数块（帧）的积分时间，允许适当的矫正宇宙射线 - 超过几分钟的时间跨度获取数据时常见的问题。 </li></ol> 6。收购完成后，几个处理步骤，有利于提高数据的质量和外观。 <ol><li>首先，所有的光谱都黑了纠正，减去“黑暗”的频谱。 </li><li>为了正确的宇宙射线，构成一个采集的几帧是一个像素由像素的基础上相互比较。对于任何任何以外，所有帧的像素中值的2位数偏差的帧的像素，该像素的值是在所有帧中值取代。 </li><li>宇宙射线纠正帧，然后平均通过Savitzky - Golay滤波平滑。 </li><li>要提取的“真实”的选通频谱，我们减去“激发只”和“泵仅”从选通频谱测量的光谱。 </li><li>最后，门和非门的光谱，再减去5阶多项式，确定使用Lieber和Mahadevan 扬森 2方法纠正的背景。 </li></ol> 7。代表性的成果： <img src="/files/ftp_upload/2592/2592fig1.jpg" alt="图1"/> 图1。克尔门控系统的示意图。泵束的路径显示为红色，而倍频路径以蓝色显示。拉曼光谱和荧光重叠的路径显示为绿色，而显示为黄色荧光时间已被过滤掉的路径。缩写如下：BPF，带通滤波器，CCD电荷耦合器件; DCM的分色镜; FI，法拉第隔离;λ/ 2，半波片;低通滤波器，长通滤波器; NLM时，非线性介质，P，偏振片二次谐波，二次谐波产生晶体。 <img src="/files/ftp_upload/2592/2592fig2.jpg" alt="图2"/> 图2顶：浸油溶解香豆素的原始光谱。红色曲线显示举行开放（最大传输分析仪）的门采取的频谱。黑色曲线显示对齐最小传输和泵束的应用（选通频谱）分析仪频谱。蓝色曲线显示与最小传输和无泵束的应用对齐分析仪采取的spectum（门举行闭门）。绿色曲线显示，只有泵束的应用而采取的频谱。虚线洋红色线显示面板图所示的光谱区域。所有的光谱已经与11点，三阶Savitzky - Golay滤波平滑。底部：香豆素的光谱，荧光背景减法油浸泡后溶解。红色曲线的频谱与门打开，和蓝色的曲线是选通频谱。选通频谱清楚地显示了令人费解的油的高波数峰值的特点。

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拉曼光谱生物医学领域已经看到在过去的几年中，作为其表现出的潜力解决一些困难的挑战，在生物诊断的越来越大的兴趣。例如，拉曼光谱已经证明，在癌症的检测3，4，5，6诊断价值。拉曼光谱技术也已用于细菌定量7，8和细菌对药物的反应9。它也发现在其他医学领域的应用范围从骨骼健康10 biofluid分析，11，12的广泛应用。 然而，...

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>产品编号</th><th>评论</th></tr><tr><td>镜头</td><td> ThorLabs </td><td>各种</td><td>所有的镜头涂以1％的最大传输损失</td></tr><tr><td>可调钛Sapph激光</td><td>连贯</td><td>变色龙</td><td>新泽西州，30日为200 fs，80 MHz的</td></tr><tr><td> 40X油浸泡目标</td><td>奥林巴斯</td><td> UApo/340 </td><td> NA = 1.35 </td></tr><tr><td>倒置显微镜</td><td>奥林巴斯</td><td> IX - 71 </td><td>修改，删除所有的镜头在侧面端口</td></tr><tr><td>半波片</td><td> Thorlabs </td><td> AHWP05M - 600 </td><td></td></tr><tr><td>格兰 - 汤普森偏振片</td><td> Thorlabs </td><td> GTH10M </td><td> 〜10％的传输损耗</td></tr><tr><td>光谱仪</td><td>有价证券投资阿克顿</td><td> SP2300i </td><td></td></tr><tr><td>防治荒漠化公约</td><td>有价证券投资阿克顿</td><td> PIXIS 100B </td><td></td></tr><tr><td> Mathmatical软件</td><td> The MathWorks公司</td><td> MATLAB </td><td>版本2008A </td></tr><tr><td>法拉第隔离器</td><td> EOT </td><td> BB8 - 5I </td><td></td></tr><tr><td>压电电镜</td><td>纽波特</td><td> AG - M100 </td><td></td></tr><tr><td> BBO晶体</td><td> CASIX </td><td>自定义</td><td> 1毫米的厚度</td></tr><tr><td>带通滤波器</td><td>安多弗</td><td> 008FC14 </td><td> 808 ± 0.4 nm的</td></tr><tr><td>分色镜</td><td> Semrock </td><td> FF662 - FDI01 </td><td>在662纳米带边</td></tr><tr><td>长通滤波器</td><td> Semrock </td><td> BLP01 - 405R </td><td>在417纳米带边</td></tr><tr><td>带通滤波器2 </td><td> Semrock </td><td> FF02-447/60 </td><td> 417-447纳米</td></tr><tr><td> CS 2 </td><td> Sigma - Aldrich公司</td><td> 335266 </td><td>纯度达99％ </td></tr><tr><td>香豆素30 </td><td> Sigma - Aldrich公司</td><td> 546127 </td><td>纯度达99％ </td></tr><tr><td>浸油</td><td> Cargille </td><td> 16242 </td><td> DF型</td></tr></tbody></table>

rejection of fluorescence background in resonance and spontaneous raman microspectroscopy

拉曼光谱仪是常常困扰着一个强烈的荧光背景，特别是对生物样品。如果样品是兴奋的超短脉冲，一个系统，可以暂时分离频谱重叠信号皮秒时间尺度可以隔离的火车，及时到达迟到荧光光的拉曼散射光。在这里，我们讨论一个复杂的非线性光学系统，采用全光开关在一个低功率光学克尔门的形式，隔离拉曼光谱和荧光信号的建设和运营。一个单一的808 nm的激光2.4 W的平均功率和80 MHz的重复率是分裂的，与约200毫瓦808纳米的光被转换为&lt;5兆瓦404 nm的光发送到的样本，以激发拉曼散射。其余未转换的808纳米的光，然后发送到非线性介质泵全光快门行为。快门打开和关闭了约5％的峰值效率在800 FS。使用这个系统，我们能够成功地分离拉曼光谱和荧光信号，在80 MHz的重复使用的脉冲能量和平均的权力，保持生物安全。由于该系统具有光功率没有余力，我们几个细节设计和调整的考虑，有助于最大限度地提高系统的吞吐量。我们还讨论了克尔介质内的信号和泵光束的空间和时间上的重叠，以及详细的协议光谱采集获得的协议。最后，我们报告了几个有代表性的拉曼光谱中存在强荧光，用我们的时间浇注系统取得的成果。

Watch this Scientific Journal Video about Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy at JoVE.com

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

我们讨论一个复杂的非线性光学系统，采用超快全光开关，隔离荧光信号的拉曼的建设和运营。使用这个系统，我们能够成功地分离的拉曼光谱和荧光信号，利用脉冲能量和平均的权力，保持生物安全。

波谱与自发拉曼显微荧光背景的抑制

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

rejection-fluorescence-background-resonance-spontaneous-raman

Research

JoVE Journal

Bioengineering

13.0K 次观看.  University of California, Davis. 我们讨论一个复杂的非线性光学系统，采用超快全光开关，隔离荧光信号的拉曼的建设和运营。使用这个系统，我们能够成功地分离的拉曼光谱和荧光信号，利用脉冲能量和平均的权力，保持生物安全。

视频: Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

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 ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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 ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

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 ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

体内 使用双光子荧光和受激拉曼散射显微镜对生物组织进行成像

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...