فإن الكتاب أود أن أنوه الجينوم كندا وكولومبيا البريطانية الجينوم للتمويل منصة، والدعم. نشكر أيضا الدكتور كارول E. باركر لمراجعة نقدية للمساعدة مخطوطة والتحرير. شيلي أيضا بفضل كولومبيا البريطانية البروتيوميات الشبكة للحصول على الدعم.

ليس لدينا شيء في الكشف عنها.

أهم الاعتبارات لنجاح MALDI MSI هي: 1) إعداد الأنسجة؛ 2) اختيار مصفوفة، 3) تطبيق مصفوفة؛ و 4) تفسير البيانات وتحليلها. عندما يتم إعداد العينة والمصفوفة بشكل مناسب، هو الآلي والحصول على البيانات MS. تحليل البيانات من هذا النوع من التجربة هو تماما كثيفة العمالة. <p class="jove_content" sty...

الطيف الكتلي التصوير (MSI) هو تقنية تحليلية لتحديد توطين وتوزيع الأنماط المكانية من المركبات على سطح قسم الأنسجة 1،2. وقد استخدمت مصفوفة بمساعدة الليزر الامتزاز / التأين (MALDI) MSI لتحليل الببتيدات والبروتينات لأكثر من عقد من الزمان، وكانت هناك تحسينات كبيرة في طرق تحضير العينة، حساسية الكشف، القرار المكانية، استنساخ ومعالجة البيانات 3،4. من خلال الجمع بين المعلومات من أقسام ملطخة تشريحيا والتجارب MSI، الأطباء قادرون على ربط توزيع مركبات معينة مع ميزات مثيرة للاهتمام pathophysiologically 5. كما تم استجواب أنماط توزيع جزيئات صغيرة، بما في ذلك الأدوية الخارجية ونواتج تفاعلاتها 6،7 8-10 بواسطة MALDI MS-الأنسجة التصوير 11. الدهون هي ربما الأكثر دراسة القانون المدني على نطاق واسعSS من المركبات مع التصوير MALDI، سواء في MS 12-17 و MS / MS 18 وسائط. وقد تم استخدام MALDI MSI للتصوير جزيء صغير محدودة بسبب عدة عوامل: 1) المصفوفات MALDI هم أنفسهم جزيئات صغيرة (عادة م / ض &lt;500)، والتي تولد إشارات أيون فيرة. هذه الإشارات وفيرة يمكن قمع التأين من analytes الصغيرة جزيء وتتداخل مع 19،20 الكشف عنها. الخالية من المذيبات مصفوفة طلاء 21، مصفوفة التسامي 22، ومصفوفة precoated MALDI MS 23، من بين أمور أخرى، وضعت لتحسين MSI جزيئات صغيرة. المصفوفات الجديدة التي يمكن أن تحسن تحليل مركبات منخفضة ميغاواط ذات أهمية كبيرة في جزيء صغير MSI. وينبغي توفير هذه المصفوفات زيادة إشارات الحليلة مع إشارات مصفوفة انخفضت. في وضع إيجابي أيون، 2،5-dihydroxybenzoic حمض (DHB) وα-سيانو-4-hydroxycinnamic حمض (CHCA) هي التي تستخدم عادة اثنين من المصفوفات MALDI MS لMSI 24 </سوب&gt;. إن مصفوفة مثالية تشكل بلورات صغيرة، وذلك للحفاظ على توطين المكاني للالتحاليل. DHB يميل إلى تشكل بلورات أكبر، لذلك تم تطوير تطبيق المصفوفة باستخدام التسامي للتغلب على هذه المشكلة جزئيا، وسمحت استخدام هذه المصفوفة للتصوير الحساسة من الدهون الفوسفاتية 22،25. وقد استخدمت 9-Aminoacridine لMSI من analytes بروتيتش في وضع إيجابي أيون 26 والنيوكليوتيدات والدهون الفوسفاتية في سلبية ايون وضع 26-29. تم العثور على 2-Mercaptobenzothiazole لإعطاء الكشف MALDI كفاءة من الدهون 30، واستخدمت لتصوير الدماغ الماوس gangliosides 31. قرار عالية جدا من تحويل فورييه ايون سيكلوترون صدى (FTICR) الطيف الشامل يمكن أن يخفف إلى حد ما حل هذه المشكلة عن طريق إشارات من إشارات الحليلة مصفوفة 32. ميزة أخرى لاستخدام FTICR-MS هو أن كثافة من كتل مصفوفة متبدل الاستقرار هي احمرارالطبعة 33، مما يقلل أيضا هذه التدخلات 27. استخدام أنثرالين (DT؛ 1،8 ثنائي هيدروكسي-9 ،10-dihydroanthracen-9-واحد) في شكل مصفوفة MALDI للتصوير الأنسجة قد سبق وذكرت 34. في هذا العمل الحالية، يتم توفير بروتوكول مفصلة لاستخدام DT لMSI الدهون الذاتية على أسطح أقسام الأنسجة عدسة الأبقار، في وضع إيجابي أيون.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Rat Liver</td>
			<td>Pel-Freez Biologicals</td>
			<td>56023-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Calf Lens</td>
			<td>Pel-Freez Biologicals</td>
			<td>57114-2</td>
			<td>Sample should be decapsulated29 before use</td>
		</tr>
		<tr>
			<td>Dithranol (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>10608</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>&#945;-Cyano-4-hydroxy-cinnamic Acid (CHCA)</td>
			<td>Sigma-Aldrich</td>
			<td>70990</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>2,5-Dihydroxybenzoic Acid (DHB)</td>
			<td>Sigma-Aldrich</td>
			<td>85707</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>Reserpine</td>
			<td>Sigma-Aldrich</td>
			<td>83580</td>
			<td></td>
		</tr>
		<tr>
			<td>Terfenadine</td>
			<td>Sigma-Aldrich</td>
			<td>T9652</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>14265</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Formate</td>
			<td>Sigma-Aldrich</td>
			<td>14266</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma-Aldrich</td>
			<td>39253</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>34972</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Commercial Alcohols</td>
			<td></td>
			<td>95%</td>
		</tr>
		<tr>
			<td>ES Tuning Mix</td>
			<td>Agilent Technologies</td>
			<td>G2431A</td>
			<td></td>
		</tr>
		<tr>
			<td>ITO Coated Glass Slides</td>
			<td>Hudson Surface Technology</td>
			<td>PSI1207000</td>
			<td>Ensure that samples are placed on the electrically conductive side</td>
		</tr>
		<tr>
			<td>Wite-Out Shake-N-Squeeze Correction Pen</td>
			<td>Bic</td>
			<td>WOSQP11</td>
			<td></td>
		</tr>
		<tr>
			<td>Airbrush Sprayer</td>
			<td>Iwata</td>
			<td>Eclipse HP-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>ImagePrep</td>
			<td>Bruker</td>
			<td>249500-LS</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI adapter</td>
			<td>Bruker</td>
			<td>235380</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dithranol as a matrix for matrix assisted laser desorption/ionization imaging on a fourier transform ion cyclotron resonance mass spectrometer

الطيف الكتلي التصوير (MSI) يحدد توطين وتوزيع الأنماط المكانية من المركبات على سطح قسم الأنسجة، وذلك أساسا باستخدام MALDI (مصفوفة بمساعدة الليزر الامتزاز / التأين) القائم على التقنيات التحليلية. وهناك حاجة إلى مصفوفات جديدة للمشاريع الصغيرة جزيء MSI، والتي يمكن تحسين تحليل منخفضة الوزن الجزيئي (MW) مركبات،. وينبغي توفير هذه المصفوفات زيادة إشارات تحليلها، بينما تتناقص إشارات خلفية MALDI. بالإضافة إلى ذلك، استخدام الأدوات عالية جدا دقة، مثل تحويل فورييه ايون سيكلوترون صدى (FTICR) الطيف الشامل، لديه القدرة على حل الحليلة إشارات من إشارات المصفوفة، وهذا يمكن التغلب على العديد من المشاكل المرتبطة جزئيا مع الخلفية مصدرها MALDI المصفوفة. الانخفاض في شدة مجموعات مصفوفة متبدل الاستقرار من قبل FTICR MS يمكن أيضا أن يساعد على التغلب على بعض التدخلات المرتبطة قمم مصفوفة على غيرها من الصكوك. عالية الدقةصكوك مثل الطيف الشامل FTICR هي مفيدة لأنها يمكن أن تنتج أنماط توزيع العديد من المركبات في وقت واحد في حين لا يزال توفير الثقة في هويات الكيميائية. أنثرالين (DT؛ 1،8 ثنائي هيدروكسي-9 ،10-dihydroanthracen-9-واحد) قد سبق وذكرت في شكل مصفوفة MALDI للتصوير الأنسجة. في هذا العمل، على هجين رباعي عالية جدا دقة وقدمت FTICR أداة بروتوكول لاستخدام DT لMALDI التصوير الدهون الذاتية من أسطح أقسام الأنسجة الثديية، من خلال إيجابية أيون MALDI إلى MS.

<html> يجب أن عينات الأنسجة التي يتم مقطوع وذوبان الجليد التي شنت على الشرائح الزجاجية ايتو المغلفة تكون سليمة، ودون تمزق مرئية. بالنسبة للعديد من الأنسجة، والأنسجة ذوبان الجليد المباشر على تركيب شريحة الزجاج المطلي ايتو هو مقبول. بالنسبة لبعض أنسجة معينة مثل عدسة الأب...

Watch this Scientific Journal Video about Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer at JoVE.com

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

أنثرالين (DT؛ 1،8 ثنائي هيدروكسي-9 ،10-dihydroanthracen-9-واحد) قد سبق وذكرت في شكل مصفوفة MALDI للتصوير الأنسجة من الجزيئات الصغيرة؛ البروتوكولات لاستخدام DT للتصوير MALDI من الدهون الذاتية على يتم توفير سطح أقسام الأنسجة التي كتبها إيجابية أيون MALDI-MS على دقة عالية جدا رباعي-FTICR الصك هنا.

أنثرالين كما مصفوفة لمصفوفة بمساعدة الليزر الامتزاز / التأين التصوير على تحويل فورييه ايون السيكلوترون الرنين مطياف الكتلة

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

dithranol-as-matrix-for-matrix-assisted-laser-desorptionionization

Research

JoVE Journal

Biology

14.1K Views.  University of Victoria.  أنثرالين (DT؛ 1،8 ثنائي هيدروكسي-9 ،10-dihydroanthracen-9-واحد) قد سبق وذكرت في شكل مصفوفة MALDI للتصوير الأنسجة من الجزيئات الصغيرة؛ البروتوكولات لاستخدام DT للتصوير MALDI من الدهون الذاتية على يتم توفير سطح أقسام الأنسجة التي كتبها إيجابية أيون MALDI-MS على د?...

Video: Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI تحضير العينة : الترا أسلوب طبقة رقيقة

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

خلط الكمي باستخدام التصوير بالرنين المغناطيسي

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

قياس كمية Invadopodia بوساطة التحلل البروتيني المصفوفة خارج الخلية في سياقات متعددة الخلايا واحدة و

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

تحسين نسبة نجاح تبلور البروتين بواسطة عشوائية Microseed ماتريكس فحص

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

إثراء خارج الخلية مصفوفة البروتينات من الأنسجة والهضم في الببتيدات لتحليل الطيف الكتلي

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

بمساعدة الليزر تسليخ مجهري (لام) كأداة للالترانسكربتي التنميط من أنواع الخلايا الفردية

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

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

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

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

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

تنفيذ نظام متماسك مكافحة ستوكس رامان نثر (CARS) على تي: الياقوت وOPO بالليزر القائم ستاندرد الليزر الضوئي مجهر

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

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM Microcoil معايرة الأداء والاستخدام أظهرت على جذور طاعون Medicago في 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

تحميل عينة المبردة في زاوية سحرية الغزل مطياف الرنين المغناطيسي النووي الذي يحافظ على الجدوى الخلوية

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.