我们感谢中国科技部（2016YDF0600803）的财政支持。

样品制备<ol><li>从植物样品（ 例如，杨树茎）切下一个小的组织块（约3 mm x 3 mm x 5 mm）。 </li><li>将组织浸入沸腾的去离子水中30分钟。立即将其在室温（RT）下转移到去离子水中30分钟。重复此步骤，直到组织沉入容器的底部，表明组织中的空气已经被去除并且组织已经软化。 注意:对于在此步骤之前沉入底部的样品，通常重复此循环3-5次。 </li><li>在去离子水中制备20，50，70，90％（v / v）等分的聚乙二醇（PEG），以及纯PEG，并将溶液保持在65℃。 </li><li> 在一系列分级的PEG浴中孵育组织以置换水并允许PEG渗透。 注意:通常，分子量为2,000（PEG2000）的PEG用于嵌入。 <ol><li>用g处理组织如下:（a）20％PEG 1小时，（b）50％PEG 1.5小时，（c）70％PEG 2小时，（d）90％PEG 2小时，和（e）100％PEG 10小时。 </li></ol></li><li>在烤箱中预热65℃的盒子。将含有块的PEG倒入盒中，然后使用预热的镊子或针将组织块放置在所需位置。 </li><li>慢慢冷却盒子并将组织储存在室温下直至使用。 </li><li>使用锋利的剃须刀将含有目标组织的PEG块解剖成小块（约1cm×1cm×2cm），并将其安装在切片机上。 </li><li>从PEG块切割薄片（通常为3-10μm）。 </li><li>用手表玻璃中的去离子水冲洗该部分10次以从组织中去除PEG。 </li><li>将各部分用甲苯/乙醇（2:1，v / v）浸泡6小时以除去提取物。通过混合65mL去离子水，0.5mL乙酸和0.6g氯化钠来制备反应液在烧杯中举行仪式将一个部分和3 mL反应液加入到5 mL小瓶中。拧上小瓶的顶部。 </li><li>将小瓶在75℃的水浴中加热2小时以除去组织的木质素。用去离子水冲洗表面10次。 </li><li>将未处理/脱木质部分转移到玻璃显微镜载玻片上。用刷子或针头小心地打开部分。用薄纸去除额外的去离子水。 </li><li>将部分浸入D 2 O.用玻璃盖板盖住样品。用指甲油密封盖子，防止D 2 O蒸发。 </li></ol>光谱采集<ol><li>打开共焦拉曼显微镜的仪器操作软件。打开激光（波长= 532 nm），用100X显微镜物镜聚焦在晶体硅的表面，然后单击校准按钮校准仪器。 </li><li>切换仪器到光学显微镜模式并打开显微镜灯。将显微镜载玻片放在舞台上，盖子面对物镜。 </li><li>使用20X显微镜物镜查看样品，并找到感兴趣的区域。将浸油涂抹于盖玻片上并切换到浸没式显微镜物镜（60X，数值孔径NA = 1.35）。专注于样品表面。 </li><li>将仪器切换到拉曼测试模式，然后关闭显微镜灯。 </li><li>使用矩形工具选择一个映射区域。改变步长以确定获得的光谱数。 注意:注意步长（通常大于由物镜的数值孔径计算的光点直径;理论上大于1.22λ/ NA）。以下尺寸将导致过采样。 </li><li>设置最佳光谱参数，以在适当的采集时间（通常&lt;8 h）获得最佳信噪比（SNR）和光谱质量，了解样品适用性。通常，在仪器软件中输入激光（532 nm），滤光片（100％），孔（300），狭缝（100），光谱仪（1,840 cm -1 ），凹槽（1200t），物镜60X油），采集时间（2 s）。 </li><li>在数据处理之前保存光谱数据，并将其转换为通用格式（ 例如， TXT文件）。 </li></ol> 3.数据分析<ol><li>将光谱数据（TXT文件）加载到数据分析软件（ 例如 Matlab）中。在数据集上应用降噪技术来提高SNR（ 例如， Savitzsky-Golay算法或小波算法） </li><li>使用未处理的样品产生木质素和多糖的图像。对于木质素成像，由于芳环对称伸缩振动，考虑1,600cm -1附近的光谱峰。对于多糖成像（包括纤维素和半纤维素），使用光谱峰a圆2,889厘米-1 ，因为CH和CH 2伸展。 </li><li>使用脱木素样品产生纤维素和半纤维素的图像。对获取的光谱进行自我建模曲线分辨率（SMCR），以区分纤维素和半纤维素的光谱并对其分布进行成像。 </li><li>对获取的数据进行主成分分析（PCA）和聚类分析，以区分不同细胞壁层的拉曼光谱。 </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2.">Chapter 2.</a> Ionic Liquids in the Biorefinery Concept: Challenges and Perspectives. , 38-64 (2016).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+tissue+specific+compositions+of+bioenergy+feedstocks+Through+hyperspectral+Raman+imaging.">Understanding tissue specific compositions of bioenergy feedstocks Through hyperspectral Raman imaging.</a> Bio. 108 (2), 286-295 (2009).</li><li>Tolstik, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+prediction+of+HCC+tissues+by+Raman+imaging+with+identification+of+fatty+acids+as+potential+lipid+biomarkers.">Classification and prediction of HCC tissues by Raman imaging with identification of fatty acids as potential lipid biomarkers.</a> J. 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作者没有什么可以披露的。

植物细胞壁是组织成几层的复合材料，包括细胞角（CC），次生壁（SW，具有S1，S2和S3层）和复合中间片（CML，中间片加上相邻的原始细胞壁），这使得在样品制备期间难以获得平坦的表面。因此，具有比木材更复杂结构的植物样品，特别是草，通常需要被固化以允许精细切片。 PEG是一种用于切割和拉曼研究的理想硬质基质，因为它可溶于水。用去离子水漂洗可以很容易地去除。用于嵌入的PEG具?...

Plant biomass is the most abundant renewable resource on Earth; is mainly composed of lignin, cellulose, and hemicellulose; and is considered an attractive source of bioenergy and bio-based chemicals1. Unfortunately, it can resist degradation and confer hydrolytic stability or structural robustness to the plant cell wall. Such resistance is attributable to the accessible surface area, biomass particle size, degree of polymerization, cellulose crystallinity, and protective lignin2. A comprehensive understanding of the structural and chemical nature of the plant cell wall is thus significant from the viewpoint of plant biology and chemistry, as well as from that of commercial utilization. Commonly used wet chemistry analyses, such as chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, only provide average compositional data of the measured sample. Furthermore, these methods are invasive and destroy the original structure of the plant tissue3. 
The Raman imaging technique is a powerful tool for the nondestructive visualization of spatially resolved chemical information4. It uses a laser light to cause inelastic scattering with a photon and relies on changes in polarizability arising from the molecular vibrations. In this case, water causes weak Raman scattering, which makes this approach suitable for in situ investigations of biological samples5. The application of the Raman imaging technique to the plant cell wall can elucidate the structure and composition of plant cell walls in their native state, with the resolution on the scale of the single cell and even of the cell wall layers6. A typical Raman imaging analysis of a plant cell wall generally consists of three steps: 1) sample preparation, 2) spectral acquisition, and 3) data processing. 
Although one of the major advantages of Raman imaging is the ability to achieve label-free and non-destructive spectra with minimal sample preparation, physical sample sectioning is still necessary to expose the surface of interest. This process should be performed carefully to obtain a flat surface, since the technique depends on maintaining optical focus7. Spectral acquisition requires a balance between image quality and extensive acquisition times8. Data processing aims to effectively extract the chemical information from the image data, especially for the components with similar chemical structures, such as cellulose and hemicellulose. Due to the strong spectral overlap, the exact spectra are difficult to discern. In this case, multivariate analysis is a straightforward approach to effectively uncover the hiding structural and chemical information9. This work presents a general protocol describing the use of Raman imaging to visualize the main components in plant cell walls, including lignin, cellulose, and hemicellulose.

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:199px;">Microtome</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:199px;">Microm HM430</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Raman microscope</td>
			<td>Horiba Jobin Yvon</td>
			<td>Xplora</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oven</td>
			<td>Shanghai ZHICHENG</td>
			<td>ZXFD-A5040</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall

拉曼成像对植物生物量的应用正在增加，因为它可以提供水溶液的空间和组成信息。分析通常不需要大量样品制备;结构和化学信息可以在没有标记的情况下获得。然而，每个拉曼图像包含数千个光谱;这在提取隐藏信息时尤其是具有类似化学结构的组件提出了困难。这项工作引入了一个多变量分析来解决这个问题。该方案建立了可视化植物细胞壁内主要成分（包括木质素，纤维素和半纤维素）的一般方法。在该协议中，描述了样品制备，光谱采集和数据处理的过程。它在样品制备和数据分析方面高度依赖操作员的技能。通过使用这种方法，可以由非专业用户执行拉曼调查以获得high质量数据和植物细胞壁分析的有意义的结果。

图1给出了用于植物细胞壁的拉曼成像的典型的微拉曼系统的概述。例如，杨树（ Populus nigra L.）的原始拉曼光谱具有显着的基线漂移和峰值（ 图2a ）。在执行拉曼成像数据集（APRI）的自动预处理方法之后，这两个光谱污染物被成功地去除（ 图2b ）。 图3中显示了杨树的典型?...

Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

该方案旨在提出使用拉曼成像和多变量分析在植物细胞壁中可视化木质素，纤维素和半纤维素的一般方法。

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

combining-raman-imaging-multivariate-analysis-to-visualize-lignin

Research

JoVE Journal

Biochemistry

11.8K 次观看.  Beijing Forestry University. 该方案旨在提出使用拉曼成像和多变量分析在植物细胞壁中可视化木质素，纤维素和半纤维素的一般方法。 

视频: Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

科研

生物化学

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

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 ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...