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 border="0" cellpadding="0" cellspacing="0" width="827">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of the Material/Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:99px;">Catlog number</td>
			<td style="width:189px;">Comments/Description</td>
			<td style="width:183px;">Website</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paladin 1064nm laser</td>
			<td>Coherent&#160;</td>
			<td>N/A</td>
			<td>Prototype model not for sale</td>
			<td><a href="http://www.coherent.com/">http://www.coherent.com/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Levante Emerald Optical parametric oscillator</td>
			<td>APE Berlin</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.ape-berlin.de/en/products/levante/levante-emerald-opo#block-views-products-block-1">http://www.ape-berlin.de/en/products/levante/levante-emerald-opo#block-views-products-block-1</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IX 71 Microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.olympusamerica.com/seg_section/product.asp?product=1023">http://www.olympusamerica.com/seg_section/product.asp?product=1023</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoview 300 scanning unit</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
			<td>http://www.olympusamerica.com/seg_section/seg_product_print.asp?product=133</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photon multiplier tube R3896</td>
			<td>Hamamatsu</td>
			<td>N/A</td>
			<td></td>
			<td><a href="https://www.hamamatsu.com/jp/en/R3896.html">https://www.hamamatsu.com/jp/en/R3896.html</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Free standing optics / filters</td>
			<td>Thorlabs and Chroma</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.chroma.com/">http://www.chroma.com/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td><a href="http://www.thorlabs.de/index.cfm?">http://www.thorlabs.de/index.cfm?</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reglo peristaltic pump</td>
			<td>ISMATEC</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.ismatec.com/int_e/pumps/t_reglo/reglo.htm">http://www.ismatec.com/int_e/pumps/t_reglo/reglo.htm</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">USB2000+ spectrometer</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/products/usb2000+.asp">http://www.oceanoptics.com/products/usb2000+.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DT-MINI-2-GS light source</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/Products/dtmini.asp">http://www.oceanoptics.com/Products/dtmini.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FIA-Z-SMA-TEF Z shaped flow cell</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/Products/fiazsmaflowcells.asp">http://www.oceanoptics.com/Products/fiazsmaflowcells.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QP400-2-SR-BX optical fiber</td>
			<td>Ocean Optics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.oceanoptics.com/Products/premgradesol.asp">http://www.oceanoptics.com/Products/premgradesol.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic piping</td>
			<td>ISMATEC</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.ismatec.com/int_e/tubing/misc/tubing_home.htm">http://www.ismatec.com/int_e/tubing/misc/tubing_home.htm&#160;</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CARS dissolution tablet flow cell</td>
			<td>N/A</td>
			<td>N/A</td>
			<td colspan="2">Homebuilt at university - designed to hold 12mm diameter, 3mm thick tablets. The flowcell has a channel depth of around 0.5mm.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass beakers</td>
			<td>VWR</td>
			<td>D108980</td>
			<td></td>
			<td><a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4537423">https://us.vwr.com/store/catalog/product.jsp?product_id=4537423</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Theophylline anhydrate</td>
			<td>BASF</td>
			<td>30058079</td>
			<td></td>
			<td><a href="http://www.basf.com/group/corporate/en/brand/THEOPHYLLINE">http://www.basf.com/group/corporate/en/brand/THEOPHYLLINE</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ethylcellulose</td>
			<td>Colorcon</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.colorcon.com/products-formulation/all-products/film-coatings/sustained-release/ethocel">http://www.colorcon.com/products-formulation/all-products/film-coatings/sustained-release/ethocel&#160;</a></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

18.0K 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

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

从生物材料空间定向变化的光散射测量

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

科研

工程学

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

表面增强拉曼光谱检测生物分子利用EBL装配纳米衬底

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

从单电浆纳米粒子散射非线性测量

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the presence of carbon nanotubes. These nanowires are extreme in the sense that they are the limit of miniaturization of nanowires and their behavior is not always a simple extrapolation of the behavior of larger nanowires as their diameter decreases. The paper then describes the methods required to obtain Raman spectra from extreme nanowires and the fact that due to the van Hove singularities that 1D systems exhibit in their optical density of states, that determining the correct choice of photon excitation energy is critical. It describes the techniques required to determine the photon energy dependence of the resonances observed in Raman spectroscopy of 1D systems and in particular how to obtain measurements of Raman cross-sections with better than 8% noise and measure the variation in the resonance as a function of sample temperature. The paper describes the importance of ensuring that the Raman scattering is linearly proportional to the intensity of the laser excitation intensity. It also describes how to use the polarization dependence of the Raman scattering to separate Raman scattering of the encapsulated 1D systems from those of other extraneous components in any sample.

The paper describes a method for producing extreme nanowires by melt infiltration into carbon nanotubes and how 1D systems may be characterized and investigated using Resonance Raman Spectroscopy to determine vibrational and optical excitation energies.

至尊纳米线和其他维系统的共振拉曼光谱

This paper briefly describes how nanowires with diameters corresponding to 1 to 5 atoms can be produced by melting a range of inorganic solids in the ...

Stimulated Stokes and Antistokes Raman Scattering in Microspherical Whispering Gallery Mode Resonators

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres can be thought as a store of energy with a huge variety of applications: compact laser sources, highly sensitive biochemical sensors and nonlinear phenomena. A protocol for the fabrication of both the microspheres and coupling system is given. The couplers described here are tapered fibers. Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS). A proof of the cavity-enhanced phenomenon is given by the lack of correlation among the pump, signal and idler: a resonant mode has to exist in order to obtain the pair of signal and idler. In the case of hyperparametric oscillations (four wave mixing and stimulated anti-stokes Raman scattering), the modes must fulfill the energy and momentum conservation and, last but not least, have a good spatial overlap.

Efficient generation of nonlinear phenomena related to third order optical non-linear susceptibility &#935;(3) interactions in triply resonant silica microspheres is presented in this paper. The interactions here reported are: Stimulated Raman Scattering (SRS), and four wave mixing processes comprising Stimulated Anti-stokes Raman Scattering (SARS).

刺激斯托克斯和反斯托克斯拉曼散射在微球形回音壁模式谐振器

Dielectric microspheres can confine light and sound for a length of time through high quality factor whispering gallery modes (WGM). Glass microspheres ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

基于扫描光散射分析器（SLPS）的方法来量化评估来自眼内镜的前向和后向光散射

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

用于材料分析的高速连续波受激布里渊散射光谱仪

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Generation and Coherent Control of Pulsed Quantum Frequency Combs

We present a method for the generation and coherent manipulation of pulsed quantum frequency combs. Until now, methods of preparing high-dimensional states on-chip in a practical way have remained elusive due to the increasing complexity of the quantum circuitry needed to prepare and process such states. Here, we outline how high-dimensional, frequency-bin entangled, two-photon states can be generated at a stable, high generation rate by using a nested-cavity, actively mode-locked excitation of a nonlinear micro-cavity. This technique is used to produce pulsed quantum frequency combs. Moreover, we present how the quantum states can be coherently manipulated using standard telecommunications components such as programmable filters and electro-optic modulators. In particular, we show in detail how to accomplish state characterization measurements such as density matrix reconstruction, coincidence detection, and single photon spectrum determination. The presented methods form an accessible, reconfigurable, and scalable foundation for complex high-dimensional state preparation and manipulation protocols in the frequency domain.

A protocol is presented for the practical generation and coherent manipulation of high-dimensional frequency-bin entangled photon states using integrated micro-cavities and standard telecommunications components, respectively.

脉冲量子频率梳的产生与相干控制

We present a method for the generation and coherent manipulation of pulsed quantum frequency combs. Until now, methods of preparing high-dimensional ...

Scattering And Absorption of Light in Planetary Regoliths

Theoretical, numerical, and experimental methods are presented for multiple scattering of light in macroscopic discrete random media of densely-packed microscopic particles. The theoretical and numerical methods constitute a framework of Radiative Transfer with Reciprocal Transactions (R2T2). The R2T2 framework entails Monte Carlo order-of-scattering tracing of interactions in the frequency space, assuming that the fundamental scatterers and absorbers are wavelength-scale volume elements composed of large numbers of randomly distributed particles. The discrete random media are fully packed with the volume elements. For spherical and nonspherical particles, the interactions within the volume elements are computed exactly using the Superposition T-Matrix Method (STMM) and the Volume Integral Equation Method (VIEM), respectively. For both particle types, the interactions between different volume elements are computed exactly using the STMM. As the tracing takes place within the discrete random media, incoherent electromagnetic fields are utilized, that is, the coherent field of the volume elements is removed from the interactions. The experimental methods are based on acoustic levitation of the samples for non-contact, non-destructive scattering measurements. The levitation entails full ultrasonic control of the sample position and orientation, that is, six degrees of freedom. The light source is a laser-driven white-light source with a monochromator and polarizer. The detector is a mini-photomultiplier tube on a rotating wheel, equipped with polarizers. The R2T2 is validated using measurements for a mm-scale spherical sample of densely-packed spherical silica particles. After validation, the methods are applied to interpret astronomical observations for asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko (Figure 1) recently visited by the NASA Dawn mission and the ESA Rosetta mission, respectively.

Numerical and experimental methods are presented for multiple scattering of light in discrete random media of densely-packed particles. The methods are utilized to interpret the observations of asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko.

行星光的散射和吸收

Theoretical, numerical, and experimental methods are presented for multiple scattering of light in macroscopic discrete random media of densely-packed ...

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. ...