Name: coherent anti-stokes raman scattering (cars) microscopy visualizes pharmaceutical tablets during dissolution
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房颤是由荷兰技术基金会污水处理厂，这是苏国的应用科学分工，经济事务部的技术方案支持。 （STW OTP 11114）。

<img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51847/51847fig1highres.jpg" width="500" /> 图1示出了示意图，通过溶解设置CARS显微镜设置与内在流动，这个数字已经被修改福塞尔等人 13。 1，系统启动<ol><li>打开20皮秒脉冲1,064 nm的CARS激光，让激光热身（约1.5小时）。 </li><li>打开氘灯的UV光源，并允许它预热（约10分钟）。 </li><li>通过设置快门开关置于"开"打开的氘灯的UV光源快门。 </li><li>转动显微镜控制PC上，并打开显微镜控制软件。 </li><li>打开紫外分光光度计PC上，打开光谱仪控制软件。 </li></ol> 2米croscope设置<ol><li>选择所需的显微物镜。使用20X/0.5 NA物镜来实现这项工作呈现的结果。 </li><li>设置过滤器在过滤器中设置炮塔发射的激发激光器和反映CARS信号。选择775 nm的长通分色镜和一个650nm的带通40nm的过滤器，复制在这项工作中所示的结果。 </li><li>将适当的过滤器的光电倍增管（PMT）检测器，其传输CARS信号和滤除不需要的光的前面。过滤的光具有750nm的短通滤波器和一个650nm的带通40nm的过滤器，复制在这项工作中所进行的实验。 </li></ol> 3，系统测试<ol><li>打开蠕动泵和泵溶出介质为通过Z形UV流动池在几分钟以清除先前的液体从管道。 </li><li>确定所述泵通过称重溶出介质的泵在2分钟的量的流量。调节水泵转速未直到所需的流率就达到了。泵的溶出介质为5毫升/分钟的流速，以达到报道了这项工作的结果。 </li></ol> 4，紫外溶出度测定<ol><li>在紫外线光谱仪控制软件，单击"文件"菜单，然后单击"新建吸光度测量"，打开一个窗口，其中列出了所有可用的光谱仪。 </li><li>点击正确的紫外光谱仪，然后单击"下一步"，打开其中显示了数据采集参数的窗口。 </li><li>同时定义积分时间和频谱平均。选择150毫秒，200均线复制在这项工作中所示的结果积分时间。 </li><li>单击标记为"下一步"按钮，弹出用来记录参考光谱的画面。 </li><li>点击显示为一个黄色的灯泡来记录一个参考光谱的按钮。这个测量过程中连续地抽取溶出介质。 </li><li>由开关设置为"关闭"关闭快门上的氘灯紫外光源。 </li><li>单击标记为"下一步"按钮，弹出用来记录暗频谱画面。 </li><li>点击显示为灰色灯泡录制暗谱的按钮。这个测量过程中连续地抽取溶出介质。 </li><li>按一下按钮，上面写着"完成"，开始紫外线吸光度测量。 </li></ol> 5，CARS溶解视频<ol><li>在CARS显微镜控制软件上点击，选择一个"XYT"测量按钮。 </li><li>单击下拉框中，以像素为单位选择图像尺寸。选择512×512像素的图像尺寸再现汇报这项工作的影像。 </li><li>拖动成像速度滑块无论是"快"，"中等"或"慢"的位置。使用扫描速度快（每幅图像的1.12秒）来实现结果在这项工作中所示。 </li><li>点击标有"放大"的箭头来调整缩放级别。选择"2倍"变焦复制放大的视图（350×350微米）用于这些结果和场的水平。 </li><li>点击下拉框，并选择使用的目的。 </li><li>单击输入框并输入所需的CARS溶解视频（取决于实验的长度）帧的数量。通过记录900帧重现这一工作中所示的结果进行溶解约15分钟。 </li></ol> 6。CARS波长调谐<ol><li>使用光学参量振荡器（OPO）控制器，直至在所需的拉曼频率最大激光输出达到调节OPO的设置，如温度，压电位置，Lyot型滤光器的位置。调整OPO 2960 -1记录相同的结果这篇文章中提出。 </li></ol> 7，溶出度试验<ol><li>放置一个药片放入自定义的样品架内置CARS流动池，螺杆样品架紧闭以防止泄漏。 </li><li>连接管道的汽车流通池连接汽车流通池包含溶出介质和蠕动泵的烧杯中。 </li><li>将包含在显微镜舞台上的平板汽车流通池。 </li><li>检查CARS流动池连接到溶出介质的烧杯中，蠕动泵，Z状的UV流动池和废物收集烧杯中。 </li><li>点击"XY重复"按钮，启动显微镜系统扫描，连续扫描模式。 </li><li>通过移动物镜调节显微镜的焦距，直到片剂的表面是在视场显微镜控制计算机屏幕上。 </li><li>点击标有"PMT"显微镜控制软件的滑块。通过增大/减小光电倍增管的电压，直到调节探测器的灵敏度一个满意的图像（既不过于黑暗，也不饱和）是在屏幕上可见。注意:注意不要使用高电压过载光电倍增管。对于这项工作中，我们使用了光电倍增管的电压约为600伏特，但这样可以根据所使用的光电倍增管而异。 </li><li>点击"停止"，在显微镜控制软件停止连续扫描。 </li><li>同时（或接近越好）开始抽溶出介质，开始记录单XYT扫描，并开始收集紫外吸收光谱。 </li><li>在溶解实验中，监控录像和手动调整显微镜焦距，以保证平板电脑是持续关注的焦点。 </li></ol> 8，溶解后<ol><li>通过关闭停止蠕动泵。 </li><li>单击"文件"菜单，然后单击"保存视频"在显微镜控制软件的XYT扫描保存为一个视频。 </li><li>单击"文件"菜单，然后单击"保存"，然后单击"停止导出"上的光谱仪控制软件，以阻止紫外吸收光谱的集合。 </li><li>除去汽车由显微镜载物台流动池，并从汽车流通池中取出药片。 </li><li>清洗车采用水和乙醇流动池，然后用干纸巾。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

When performing CARS microscopic dissolution experiments there are a few critical aspects that need to be monitored during the experiment. Firstly, introducing the dissolution medium to the CARS flow cell causes the focus to move. This means that the image is immediately lost and it takes a few microns of objective adjustment to find the surface again. Secondly, there is risk of liquid leakage from the CARS flow cell if the glass cover breaks during the experiment. This can potentially cause liquid damage to the optics, so it is important to listen for any cracking sound that could mean the glass has broken. Finally, there is also a small chance that the piping can become blocked due to particulate matter in the system during the experiment, this can be seen as a sudden unusual change in the UV spectra and also through periodically checking the flow during the experiment.
Particulate blockage of the piping is mainly an issue with tablets that have been designed to disintegrate during dissolution. This is one of the limitations for this technique as this system requires the surface of the tablet to remain intact throughout the dissolution to allow imaging. In addition to disintegrating tablets, it is currently not possible to image tablets that are designed to swell during dissolution as this can lead to breakage of the CARS flow cell.
Imaging tablets during dissolution provides a greater understanding of what is occurring on the surface of a dissolving tablet. Conventional pharmaceutical dissolution methods focus only on the drug content dissolved in the dissolution medium which can identify whether the tablet passes or fails the required standard. However, in the case of a failed test it is difficult to determine what caused the failure. The case of a failed dissolution test is potentially where in situ dissolution analysis using CARS microscopy can provide answers.
Future applications for in situ dissolution analysis using CARS microscopy could include investigations using more complicated tablets containing more than one drug or excipient, in particular non-swelling sustained or controlled release dosage forms during formulation development. Additionally, it could be possible to investigate samples using biorelevant dissolution media creating conditions more closely related to in vivo. 
In conclusion, this work shows that CARS microscopy is capable of rapid chemically specific imaging based on Raman vibrational frequencies allowing selective imaging of the drug in a tablet containing both drug and excipient. Additionally, CARS microscopy combined with inline UV absorption spectroscopy is a powerful tool capable of monitoring the surface of tablets undergoing dissolution and correlating surface changes seen using CARS with changes in dissolution rate.

在口服药物剂型，如片剂和胶囊的发展有较强的重视溶出度测试。口服剂型都必须溶解它们可以被吸收为治疗功效之前。难溶性药物一般都达到足够的浓度，这使得溶出度测试尤为重要1的问题。药典溶出度方法是最常用的溶出分析。在大多数情况下，这需要制备药物片剂或胶囊中，然后放入流动的溶解介质的烧杯中。溶解的药物浓度，然后通过使用标准的光谱技术，如UV吸收光谱仪2分析该溶出介质的样品来确定。这些传统的药物溶出方法不提供样品的任何直接分析或可能被对剂型的溶解表面发生任何变化。样品的溶解过程中直接​​分析可以提供关于溶解剂型的详细信息，并可能识别导致溶出试验失败的问题。 溶解剂型的直接分析，需要在它们能够监测溶解过程的原位分析技术的使用。溶解过程中就地记录的分析技术不能被溶解介质的存在的影响，该技术需要一个高的时间分辨率，以可靠地测量在秒数量级的变化，以溶解剂型。衰减全反射紫外光谱法已被证明是适合的溶解过程中测量的变化，但缺少由成像技术3中提供的空间分辨率。如扫描电子显微镜（SEM）和自发拉曼映射传统药物成像技术都具有限制因素防止它们的使用在原地解散。 扫描电镜成像是能够成像的药物剂型的表面的高分辨率快速成像技术。然而，通常在真空条件下进行的扫描电镜成像，需要样品涂层使得它在原位溶解成像不适宜。光纤耦合自发拉曼光谱结合了流通池和UV流通过吸收光谱，目前已进行溶解过程中监测各种药物系统在原位 ，包括茶碱4，卡马西平，和吲哚美辛5。拉曼光谱是能够识别溶解过程中发生的表面变化，但它提供了关于在地表变化没有发生空间信息。自发拉曼映射使用拉曼光谱，并提供有关样品的表面空间信息，但成像只需要几分钟的顺序来小时，这取决于图像区域，使得它在原地解散成像不适合。 相干反斯托克斯拉曼散射（CARS）显微术是一种快速成像技术，结合内嵌紫外吸收光谱，它使我们能够开发就地解散分析能力的技术。 CARS显微镜提供了快速而不受溶出介质中的存在使得其在原位溶解分析一个合适的技术影响化学选择性成像。 CARS技术大致分为两个组的基础上，激光器的脉冲持续时间;其中之一是窄带车（皮秒脉冲激光器），而另一个是宽带CARS（飞秒脉冲激光器）。一个典型的CARS显微镜系统由两个脉冲激光源，在倒置显微镜。以产生CARS信号时，脉冲激光器中的一个需要是可调谐的，以便有两个激光器相匹配的拉曼振动之间的频率差。此外，两个激光器必须重叠在空间（空间）和时间（时间），以从两个激光器到达样品在同一时间同一区域的脉冲。作为拉曼振动是特​​定化学和CARS信号仅在显微镜的聚焦体积内产生，CARS显微镜能够化学选择性成像，分辨率降低到衍射极限的。 使用单一的拉曼振动模式窄带CARS显微镜允许大约快100倍成像相比，自发拉曼成像技术，6。宽带CARS显微镜图像在更宽的光谱范围（600-3,200 -1对比〜4厘米-1），但具有较低的频谱分辨率（大约10 -1对比〜4厘米-1）和较慢的成像速度（50毫秒/像素对比〜5微秒/像素）则为窄带CARS显微镜7。 窄带CARS显微镜已被用于图像DRUG释放一些药物系统。在药物制剂领域，Kang 等 8-10成像药物加载的聚合物膜。最初它们成像的装药，其随后从静态溶出介质中的药物释放的成像分布。 Jurna 等 11和Windbergs 等 12更进一步和成像首先在随后使用动态溶出介质成像的药物溶解脂质剂型的茶碱分布。 我们已经开发了一种新的分析方法同时监测在数位板上进行溶解与窄带CARS显微镜的同时，录制溶解的药物浓度，用紫外吸收光谱表面的变化。我们说明了如何使用包含模型药物茶碱结合乙基纤维素进行溶解，以水为溶出介质，这种方法的成像片。

<table><tbody><tr><td>Paladin 1,064 nm laser</td><td>Coherent</td><td></td><td>Prototype model not for sale</td></tr><tr><td>Levante Emerald Optical parametric oscillator</td><td>APE Berlin</td><td></td><td></td></tr><tr><td>IX71 Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Fluoview 300 scanning unit</td><td>Olympus</td><td></td><td></td></tr><tr><td>Photomultiplier tube R3896</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>Free standing optics / filters</td><td>Thorlabs and Chroma</td><td></td><td></td></tr><tr><td>Reglo peristaltic pump</td><td>ISMATEC</td><td></td><td></td></tr><tr><td>USB2000+ spectrometer</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>DT-MINI-2-GS light source</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>FIA-Z-SMA-TEF Z shaped flow cell</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>QP400-2-SR-BX optical fiber</td><td>Ocean Optics</td><td></td><td></td></tr><tr><td>Plastic piping</td><td>ISMATEC</td><td></td><td></td></tr><tr><td>CARS dissolution tablet flow cell</td><td></td><td></td><td>Homebuilt at university - designed to hold 12 mm diameter, 3 mm thick tablets. The flowcell has a channel depth of around 0.5 mm.</td></tr><tr><td>Glass beakers</td><td>VWR</td><td>D108980</td><td></td></tr><tr><td>Theophylline anhydrate</td><td>BASF</td><td>30058079</td><td></td></tr><tr><td>Ethyl cellulose</td><td>Colorcon</td><td></td><td></td></tr></tbody></table>

coherent anti-stokes raman scattering (cars) microscopy visualizes pharmaceutical tablets during dissolution

传统的药物溶出试验测定药物通过测定溶出介质的药物含量溶解在一段时间的量。这种方法提供了有关正在发生的事情溶解药片表面上几乎没有直接的信息。作为片剂表面的组成和结构可以在溶解过程中改变，这是必不可少的溶出度试验中对其进行监视。在这项工作中相干反斯托克斯拉曼散射显微镜被溶解时用于图像片的表面，同时用紫外吸收光谱是同时提供溶解药物浓度的在线分析含有茶碱无水物和乙基纤维素的50％的混合物的片剂。该测量结果表明， 在原 CARS显微镜能够成像的选择性茶碱在乙基纤维素的存在。此外，无水茶碱转化为茶碱一水合物溶解过程中，用针形哭stals溶解过程中不断增长的数位板表面。茶碱无水物到一水合物的转化，结合的药物减少暴露于流动介质中的溶解导致降低溶出率。我们的研究结果表明， 在原位 CARS显微镜结合内嵌紫外吸收光谱是能够监测药物片剂溶出度和关联面的变化与改变溶解速度。

在使用CARS 原位溶解分析显微镜上包含模型药物茶碱无水物和乙基纤维素，用蒸馏水泵以5毫升/分钟的50:50的混合物作为溶出介质片剂（扁平面直径12毫米，）进行。 CARS图像（512×512像素），收集每1.12秒处的拉曼振动频率2960厘米-1是选择性的片剂的溶出度实验的持续时间的茶碱含量， 图2示出了选择的帧从溶解视频。在溶解开始时（ 图2，时间为0秒）有绿色示出了片的茶碱含量区和也有黑暗的地方有只乙基纤维素存在于片剂的表面上。在片剂的表面上的暗区也能够隐约看到乙基纤维素含量。这是因为，据报道，乙基CELLULO本身具有拉曼振动频率与最大值大约2,930和2975厘米-1 14。大约60秒后，似乎有茶碱一水合物晶体生长表面上的开始，可以被看作是窄的针状晶体生长向外从至少一种晶核为中心的框架（ 图2，时间60秒） 。一水合物晶体的生长可以更清楚地看出130秒后（ 图2，时间130秒）。此外，在时间点130秒可以看到，一水合物晶体。并没有完全分散在片剂的表面上。看来好像乙基纤维素区域的存在已经身体挡水合物针的延长。经过250秒时，可以看到，该表面的一水合物覆盖不是这表明一水合物晶体本身开始溶解作为突出。 图2。帧从汽车溶解视频。从溶解视频的无水茶碱与乙基纤维素平板电脑选择汽车等图像（2,960 -1）。在0秒的图像被记录在样品的一个区域，而60，130，和250秒的图像被记录在试样的另一区域。汽车视频可作为补充资料。比例尺为50微米。 紫外（UV）光谱是吸收光谱使用UV光作为激发源的形式。紫外光谱测量电子跃迁从基态到激发态15。茶碱具有广阔的峰值约270 nm处为中心，而乙基纤维素为溶出介质因此预计不会造成记录的UV光谱中几乎不溶。的解离分析使用内联Z形UV流动池禄介质使我们能够定量地确定药物的溶解过程中溶解的量， 图3示出了UV-溶出曲线的茶碱无水物与乙基纤维素的片剂的溶出度。紫外溶出曲线（ 图3）示出了茶碱无水物的溶解开始迅速达到大约90微克/内120秒毫升的最大浓度;该时间点之后的溶解速率开始下降。中的溶解速率的降低可能是由于茶碱一水合物的存在下（溶解度6毫克/毫升，在25℃16）这是比茶碱无水较少溶解（溶解度12毫克/毫升，在25℃16的表面上的晶体），并清楚地在该时间点出现在CARS溶解视频（ 图2）。在逐步减少溶出速率也部分为B解释雅减少茶碱暴露在流动介质。发生这种降低是因为乙基纤维素是几乎不溶于水，所以作为茶碱溶解剩余的乙基纤维素阻碍茶碱暴露于溶解介质。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51847/51847fig3highres.jpg" width="500" /> 图3。紫外溶出曲线。浓度对时间曲线的茶碱无水物结合的乙基纤维素片剂表示茶碱的浓度在溶出介质中的溶出实验中。

Watch this Scientific Journal Video about Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution at JoVE.com

Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

相干反斯托克斯拉曼散射（CARS）显微术是结合本征流通过溶解的设置，以允许在原位和药物片剂进行溶出度的表面的实时可视化。使用这种定制的设置，因此能够使用内联紫外吸收光谱记录药物溶出曲线相关CARS视频。

相干反斯托克斯拉曼散射（CARS）显微可视化药片剂溶出过程中

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This ...

coherent-anti-stokes-raman-scattering-cars-microscopy-visualizes

Research

JoVE Journal

Engineering

Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

18.1K 次观看.  University of Twente. 相干反斯托克斯拉曼散射（CARS）显微术是结合本征流通过溶解的设置，以允许在原位和药物片剂进行溶出度的表面的实时可视化。使用这种定制的设置，因此能够使用内联紫外吸收光谱记录药物溶出曲线相关CARS视频。 

视频: Coherent anti-Stokes Raman Scattering CARS Microscopy Visualizes Pharmaceutical Tablets During Dissolution

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

科研

生物化学

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

基于刺激拉曼散射的非线性显微镜的实现

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

工程学

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

基于宽带受激拉曼散射显微镜的多重化学成像

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

高光谱受激拉曼散射与相干反斯托克斯拉曼散射显微镜在化学成像中的应用的直接比较

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

化学

A Package of Established Analytical Tools to Investigate the Solid-State Alteration of Lipid-Based Excipients

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical manufacturing. However, the major challenge is their unstable solid-state, affecting the stability of the pharmaceutical product. Critical physical properties of lipids for their processing-such as melt temperature and viscosity, rheology, etc.-are related to their molecular structure and their crystallinity. Additives, as well as thermal and mechanical stress involved in the manufacturing process, affect the solid-state of lipids and thus the performance of pharmaceutical products thereof. Therefore, understanding the alteration in the solid-state is crucial. In this work, the combination of powder x-ray diffraction and differential scanning calorimetry (DSC) is introduced as the gold standard for the characterization of lipids' solid state. X-ray diffraction is the most efficient method to screen polymorphism and crystal growth. The polymorphic arrangement and the lamella length are characterized in the wide- and small-angle regions of x-ray diffraction, respectively. The small-angle x-ray scattering (SAXS) region can be further used to investigate crystal growth. Phase transition and separation can be indicated. DSC is used to screen the thermal behavior of lipids, estimate the miscibility of additives and/or active pharmaceutical ingredients (API) in the lipid matrix, and provide phase diagrams. Four case studies are presented in which LBEs are either used as a coating material or as an encapsulation matrix to provide lipid-coated multiparticulate systems and lipid nanosuspensions, respectively. The lipid solid-state and its potential alteration during storage are investigated and correlated to the alteration in the API release. Qualitative microscopical methods such as polarized light microscopy and scanning electron microscopy are complementary tools to investigate micro-level crystallization. Further analytical methods should be added based on the selected manufacturing process. The structure-function-processability relationship should be understood carefully to design robust and stable lipid-based pharmaceutical products.

This publication shows the application of x-ray diffraction and differential scanning calorimetry as gold standards for investigating the solid-state of lipid-based excipients (LBEs). Understanding the solid-state alteration in LBEs and its effect on the performance of pharmaceutical products thereof is the key factor for manufacturing robust lipid-based dosage forms.

一套成熟的分析工具，用于研究脂质赋形剂的固态变化

Lipid-based excipients (LBEs) are low-toxic, biocompatible, and natural-based, and their application supports the sustainability of pharmaceutical ...

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

生物学

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

生物工程

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

使用相干拉曼散射成像对皮肤内的药物化合物进行可视化和定量

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

医学

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

蓝宝石OPO基于激光的标准激光扫描显微镜:在TI相干反斯托克斯拉曼散射（CARS）系统的实现

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

相干反斯托克斯拉曼散射（CARS）显微可视化药片剂溶出过程中

摘要

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此视频中的章节

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

基于刺激拉曼散射的非线性显微镜的实现

基于宽带受激拉曼散射显微镜的多重化学成像

高光谱受激拉曼散射与相干反斯托克斯拉曼散射显微镜在化学成像中的应用的直接比较

一套成熟的分析工具，用于研究脂质赋形剂的固态变化

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

使用相干拉曼散射成像对皮肤内的药物化合物进行可视化和定量

蓝宝石OPO基于激光的标准激光扫描显微镜:在TI相干反斯托克斯拉曼散射（CARS）系统的实现

使用刺激拉曼散射显微镜对卡诺哈布迪炎电子人中的脂质存储动力学进行无标签成像

相干反斯托克斯拉曼散射（CARS）显微可视化药片剂溶出过程中

摘要

探索更多视频

此视频中的章节

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

基于刺激拉曼散射的非线性显微镜的实现

基于宽带受激拉曼散射显微镜的多重化学成像

高光谱受激拉曼散射与相干反斯托克斯拉曼散射显微镜在化学成像中的应用的直接比较

一套成熟的分析工具，用于研究脂质赋形剂的固态变化

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

使用相干拉曼散射成像对皮肤内的药物化合物进行可视化和定量

蓝宝石OPO基于激光的标准激光扫描显微镜:在TI相干反斯托克斯拉曼散射（CARS）系统的实现

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

使用刺激拉曼散射显微镜对卡诺哈布迪炎电子人中的脂质存储动力学进行无标签成像