Name: observation and analysis of blinking surface-enhanced raman scattering
Uploaded: 2018-01-11T14:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

作者感谢 Prof. y Ozaki (Kwansei 学院大学) 和 Dr. (国家高等工业科技研究院) 对这项工作进行了卓有成效的讨论。这项工作得到了教育、文化、体育、科学和技术部 (No. 16K05671) 的 KAKENHI (补助金) 的支持。

1. 样品制备
<ol><li>
制备银胶体纳米粒子20<ol>
<li>制备银胶体纳米粒子, 溶解0.030 克硝酸银和0.030 克柠檬酸三钠, 在150毫升水中的200毫升圆形底烧瓶。</li>
		<li>将烧瓶与回流 (Dimroth) 冷凝器结合。</li>
		<li>用磁力搅拌器和搅拌棒在烧瓶中搅拌溶液。然后, 在150° c 的油浴中, 在烧瓶中加热搅拌溶液60分钟。 注: 溶液将变成黄色, 然后是乳白色的灰色。</li>
		<li>在室温下冷却悬浮液, 并保持在冰箱内用铝箔覆盖的烧瓶中的悬浮。 注意: 在此点可以暂停协议。在一个月内, 用胶体纳米微粒储存在冰箱里。</li>
	</ol>
</li>
	<li>
多色闪烁发射样品的制备11<ol>
<li>要准备一个显微镜幻灯片, 用手用肥皂冲洗玻璃盘子, 并用清水冲洗。</li>
		<li>在玻璃板上加入0.1% 多聚 l-赖氨酸水溶液, 用鼓风机将溶液取出。</li>
		<li>将银胶体悬浮液添加到玻璃板上, 然后用鼓风机将悬浮液除去。</li>
		<li>用液体阻挡笔将玻璃板上的拖放区域括起来。</li>
		<li>将蒸馏水滴在玻璃板上, 用另一玻璃板盖上, 以制造显微镜滑动, 防止水蒸发。</li>
	</ol>
</li>
	<li>
为单调的彩色闪烁 SERS 进行样品制备7,8,9,10<ol>
<li>要准备一个显微镜幻灯片, 用手用肥皂冲洗玻璃盘子, 并用清水冲洗。</li>
		<li>将银胶体悬浮液与 thiacyanine 或 thiacarbocyanine 染料 (分别为25或4µM) 和氯化钠 (10 毫米) 水溶液混合, 体积比为 2:1: 1。</li>
		<li>将样品悬浮液放在玻璃板上, 然后用鼓风机将悬挂装置卸下。</li>
		<li>用液体阻挡笔将玻璃板上的拖放区域括起来。</li>
		<li>将氯化钠 (1 米) 的水溶液滴在玻璃板上, 使银纳米颗粒固定, 并用另一玻璃板盖住, 以制造显微镜滑动板, 防止溶液蒸发。</li>
	</ol>
</li>
</ol>2. 闪光银纳米粒子的观察
<ol><li>
样品照明
	<ol>
<li>将使用协议1.2 或1.3 的样品玻璃板放在倒置显微镜的舞台上。</li>
		<li>通过暗场冷凝器照亮样品玻璃板, 用物镜 (60X) 在玻璃板上聚焦各种彩色斑点 (蓝色、绿色、黄色和红色)。</li>
		<li>用衰减光束照射样品玻璃板, 其相对于样品表面的角度为30°, 从二极管泵浦固态 (DPSS) continuous-wave (cw) 激光器通过干涉滤光片。</li>
		<li>利用激光照射在同色的环境中观察银粒子为单调的彩色斑点, 将激光照射区移至视图中心, 并通过调整 z 方向上的舞台来聚焦在玻璃板上的斑点上。</li>
	</ol>
</li>
	<li>
闪烁的观察
	<ol>
<li>在物镜后插入一个 long-pass 过滤器, 并通过干涉滤光片, 在30°相对于样品表面的情况下, 用 DPSS 的连续激光光束照射样品玻璃板。</li>
		<li>通过在 x 和 y 方向移动舞台, 查找图 1中显示的闪烁点 (也请参见辅助材料中的图 S1 )。</li>
		<li>用倒置显微镜记录闪烁斑点的视频, 耦合到一个冷却的数字电荷耦合器件 (CCD) 相机, 它的时间分辨率为 61-120 毫秒, 为20分钟。</li>
	</ol>
</li>
</ol>3. 闪光 SERS 的分析
<ol><li>
从视频中推导时间剖面
	<ol>
<li>在控制 CCD 摄像机的软件中, 打开视频文件。</li>
		<li>若要选择闪烁的斑点和暗区, 请拖动单独覆盖视频图像中有斑点的区域的区域。</li>
		<li>若要从视频中的闪烁点和暗区域派生信号强度时间配置文件, 请在分析中选择时间分析, 然后在时间分析窗口中单击计算。</li>
		<li>将数据另存为文本文件。</li>
	</ol>
</li>
	<li>
时间剖面分析
	<ol>
<li>通过从暗区和/或与多项式函数的拟合中减去时间轮廓来拼合时间模板的基线, 如图 2A和2B所示。</li>
		<li>计算由大约2000点、 I基和基线强度标准偏差 (σ) 组成的平均基线强度, 如图 2C和2D中所示。</li>
		<li>使用比I基+ 3 的阈值更大的强度来区分明亮事件和暗事件, 并记录每个事件的持续时间。例如, 在图 3中, 将事件从0记录为黑色事件 (其持续时间为t = 3.5476 秒), 并将事件从3.5476 个到4.0981 秒记录为明亮事件 (持续时间为t = 0.5505 秒)。重复过程, 如表 1所示。</li>
		<li>按照表 2的第一和第二行中的表示, 计算每个持续时间内的亮和暗事件数。</li>
		<li>对每个工期的事件数求和, 但事件少于持续时间t。例如, 在表 2的第二和第三行中表示, 对每个持续时间的事件数进行求和 ( t = 0.0612 秒的事件除外) 为 41 + 18 + 9 +..。结果等于t = 0.1223 s 的求和,即103。</li>
		<li>按每个工期划分总结, 并对其进行规范化。例如, 如表 2所示, 将持续时间的总和t = 0.0612 s 除以持续时间0.0612 秒。结果是3351.5791。然后, 将结果除以表 2中第四行中的结果总数。概率分布被导出为0.64494。</li>
		<li>将明亮事件的概率分布绘制为Pon(t), 并将其持续时间t在对数对数图中, 并适合日志10P(t) 的日志10()<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">在上为特定的闪烁点推断幂定律指数α。<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">如果Pon(t) 是由安装的, 则拟合的行将在P(t)上的小数值上偏离图形, 如图 4A中的虚线所示。</li>
		<li>按日志10将暗事件的概率分布绘制为P关(t), 并将其持续时间t在对数对数图中, 并适合日志10P关闭(t)。从相同的闪烁点推断出幂律指数α <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> off和截断时间τ 。如果Poff(t) 是由安装的, 则拟合的曲线将从位于<img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> </img> P关闭(t) 的小值中偏离图形。</li>
		<li>对视频中的其他闪烁点重复3.2.1 到3.2.8。</li>
	</ol>
</li>
</ol>

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作者没有什么要透露的。

从银 nanoaggregate 结, SERS 被发射。因此, 我们需要准备粒子而不是胶体纳米粒子, 这是被柠檬酸阴离子覆盖。银聚合体是由添加多聚 l-赖氨酸所产生的盐析效应形成的, 它具有-NH3+ , 是来自氯化钠的 SERS、Na+阳离子的来源, 如补充材料的图 S2所示。此外, 为了照亮宽区的许多斑点, 不聚焦的激光光束是通过一个不附着在显微镜上的透镜, 相对于样品表面, 以30°的角度?...

表面增强拉曼散射 (SERS) 是高灵敏度的拉曼光谱从一个高贵的金属表面。由于拉曼光谱提供了基于尖峰位置的分子结构的详细信息, 通过分子中官能团的振动模式, 可以研究金属表面上的单个分子的信息使用 SERS1,2,3。从银 nanoaggregate 与吸附在分子水平, 一个闪烁的信号被 观察1,2,3,45,7,8,9,10,11,12,13,14,15, 16, 频谱波动1,2,3,4,5,67,8, 9,10,11,12,13,14。眨眼可以由一个单一的分子, 随机进出一个增强电磁 (EM) 领域在一个纳米银 nanoaggregate 交界处。因此, 闪烁被认为是分子检测的简单证据, 与使用 SERS 强度的泊松分布和双分析物2、3、17的技术相比。然而, 闪烁和波动频谱的详细机制, 可能强烈依赖于分子行为在 Ag 表面, 仍然是有争议的。
在先前的研究中, 用自相关函数分析了闪烁的 SERS, 它可以计算在增强的 EM 场中进出的分子的扩散系数和浓度12,13,14.此外, 一个标准化的标准偏差评分, 代表了不稳定性的总强度, 是从信号的时间剖面的15。然而, 这些分析方法可能是基于一些分子的行为。相比之下, 在一个超分辨率成像的闪烁 SERS, 分子行为在增强 EM 领域可以识别16。但是, 这些技术只能在增强的 EM 域中获得这些参数。单个分子在广泛范围内的随机行为 (例如, 在闪烁的 SERS 中) 可以表示为幂定律, 而不是平均4,5,6,7,8 ,9,10,11, 类似于单个半导体量子点 (数字)18,19中闪烁的荧光。通过使用幂律分析4、5、6、7、8、91011, 分子行为可以估计在明亮状态 (在增强的 EM 领域) 和黑暗状态10;那是, 分子的行为在整个银色表面可以估计。
对于此技术, 银胶体粒子使用4,5,6,,8,9,10,11。这些粒子显示各种局部的表面等离子共振 (LSPR) 波段, 强烈影响增强的电磁场时, 他们是在一定的波长兴奋。因此, SERS 活性银纳米粒子存在于胶体悬浮液中, 并能立即获得一些数据。在简单的纳米结构的情况下, 有特定的大小, 形状和安排, LSPR 依赖的 SERS 闪烁可以隐藏其他依赖性7;也就是说, 如果使用了 LSPR 的好的或坏的纳米结构, 则参数将是恒定的, 因此其他的依赖将被隐藏。幂律分析已被用于发现从银胶体粒子的各种依赖的闪光 SERS, 4,5,6,7,89,10,11。

<table><tbody><tr><td>Silver nitrate, 99.8%</td><td>Wako</td><td>194-00832</td><td></td></tr><tr><td>Trisodium citrate dihydrate, 99. %</td><td>Wako</td><td>191-01785</td><td></td></tr><tr><td>Poly-L-lysine aqueous solution, 0.1%</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>3,3&#039;-disulfopropylthiacyanine triethylamine</td><td>Hayashibara Biochemical Laboratories</td><td>NK-2703</td><td>a kind of thiacyanine dyes</td></tr><tr><td>3,3&#039;-diethyl-5,5&#039;-dichloro-9-methylthiacarbocyanine iodine salt</td><td>Hayashibara Biochemical Laboratories</td><td>SMP-9</td><td>a kind of thiacarobocyanine dyes</td></tr><tr><td>Sodium chloride, 99.5%</td><td>Wako</td><td>191-01665</td><td></td></tr><tr><td>Dimroth condenser</td><td>Iwaki</td><td>61-9722-22</td><td>perchased from AS ONE</td></tr><tr><td>Magnetic stirrer</td><td>Corning</td><td>DC-420D</td><td></td></tr><tr><td>Oil bath</td><td>Advantech</td><td>OS-220</td><td></td></tr><tr><td>Glass plate</td><td>Matsunami</td><td>S-1112</td><td>Microscope slide</td></tr><tr><td>Blower</td><td>Hozan</td><td>Z-288</td><td>Air duster</td></tr><tr><td>Liquid blocker pen</td><td>Daido Sangyo</td><td></td><td>LIQUID BLOCKER (Super Pap Pen). Ready-to-use hydrophobic barrier pen designed for immunohistochemistry applications</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-70</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>LCPlanFl 60&amp;times;</td><td>NA 0.7</td></tr><tr><td>Dark field condenser</td><td>Olympus</td><td>U-DCD</td><td>NA 0.8&amp;ndash;0.92</td></tr><tr><td>Cooled digital CCD camera</td><td>Hamamatsu</td><td>ORCA-AG</td><td>controlled by software Aqua Cosmos</td></tr><tr><td>Software for the cooled digital CCD camera</td><td>Hamamatsu</td><td>AquaCosmos</td><td>used for also derivation of the time-profiles from the blinking spots in the video&amp;nbsp;</td></tr><tr><td>Color CCD camera</td><td>ELMO</td><td>TNC-C920</td><td>not used for analysis</td></tr><tr><td>DPSS laser</td><td>RGB laser system</td><td>NovaPro532-75</td><td>&amp;lambda; = 532 nm;&lt;br/&gt; 60 mW (corresponds to a power density of 600 W/cm2)</td></tr><tr><td>Interference filter</td><td>Semrock</td><td>LL01-532-12.5</td><td></td></tr><tr><td>Long pass filter</td><td>Semrock</td><td>BLP01-532R-25</td><td></td></tr><tr><td>Software for the distinguishment and counting of the bright/dark events</td><td>home-maid</td><td></td><td>programmed by C++</td></tr><tr><td>Software for the fitting by a power law</td><td>LightStone</td><td>Origin6.1</td><td></td></tr></tbody></table>

observation and analysis of blinking surface-enhanced raman scattering

从一个单一的分子在银 nanoaggregate 结, 闪烁的表面增强拉曼散射 (SERS) 观察。本文介绍了如何制备 SERS 活性银 nanoaggregate, 在显微图像中记录某些闪烁点的视频, 并分析闪烁的统计数据。在这个分析中, 幂律再现了明亮事件相对于其持续时间的概率分布。黑暗事件的概率分布由幂律和指数函数拟合。幂定律的参数代表了光明和黑暗状态的分子行为。随机游走模型和分子在整个银色表面的速度可以估计。即使使用平均值、自相关函数和超分辨率 SERS 成像, 也难以估计。在未来, 幂律分析应与频谱成像相结合, 因为这种分析方法不能单独证实闪烁的起源。

从1.2 号协议制备的多聚 l-赖氨酸银粒子, 观察到了 SERS 和表面增强荧光的多色闪烁斑点, 如图 111所示。相比之下, 由 1.37、8、9、10所制备的染料分子, 在银粒子上观察到来自 SERS 的单调彩色闪烁点。有两种类型的 "阴性" 结果: 连续的结果...

Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

该协议描述了由于在银色表面上使用幂定律的单个分子的随机游走而引起的表面增强拉曼散射的分析。

闪烁表面增强拉曼散射的观测与分析

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

observation-and-analysis-of-blinking-surface-enhanced-raman-scattering

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SCIENTISTS IN ACTION

7.3K 次观看.  Kwansei Gakuin University. 该协议描述了由于在银色表面上使用幂定律的单个分子的随机游走而引起的表面增强拉曼散射的分析。

视频: Observation and Analysis of Blinking Surface-enhanced Raman Scattering

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

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 ...

科研

生物工程

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 ...

工程学

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, ...

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 ...

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. ...

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

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 ...

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 ...

闪烁表面增强拉曼散射的观测与分析

摘要

探索更多视频

此视频中的章节

波谱与自发拉曼显微荧光背景的抑制

表面增强拉曼光谱检测生物分子利用EBL装配纳米衬底

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

用于材料分析的高速连续波受激布里渊散射光谱仪

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

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

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

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

闪烁表面增强拉曼散射的观测与分析

摘要

探索更多视频

此视频中的章节

波谱与自发拉曼显微荧光背景的抑制

表面增强拉曼光谱检测生物分子利用EBL装配纳米衬底

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

用于材料分析的高速连续波受激布里渊散射光谱仪

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

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

用于组织诊断的小鼠脑实时双色受激拉曼散射成像

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