Name: imaging of biological tissues by desorption electrospray ionization mass spectrometry
Uploaded: 2013-07-12T12:00:00
Description: Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

Dette arbejde understøttes af ARRA NSF MRI Instrument Development tilskud # 0923179 til FMF. Vi takker Aqua Asberry, Lab koordinator for Parker H. Petit Institut for bioteknologi og Biosciences Histology Core, for at få hjælp med vævssektionering.

1.. Vævssektionering <ol><li> Store flash-frosne, hele væv i -80 ° C fryser indtil den er klar til skæring. </li><li> Tillad vævsprøve at nå den temperatur i cryomicrotome før sektionering (30 min). Indstil klingen og prøvens temperatur til -30 ° C. </li><li> Når væv har nået den temperatur, håndtering af prøven med en pincet, skære forsiden eller bagsiden af hjernen afhængigt af hvilken del af hjernen er af interesse for tilstrækkelig monteringsflade (dvs. hvis den forreste del af hjernen e...

<ol>
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S., 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=Classifying+Human+Brain+Tumors+by+Lipid+Imaging+with+Mass+Spectrometry.">Classifying Human Brain Tumors by Lipid Imaging with Mass Spectrometry.</a> Cancer Res. 72, 645-654 (2012).</li><li>Wiseman, J. M., Ifa, D. R., Venter, A., Cooks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+molecular+imaging+by+desorption+electrospray+ionization+mass+spectrometry.">Ambient molecular imaging by desorption electrospray ionization mass spectrometry.</a> Nat. Protocols. 3, 517-524 (2008).</li><li>Van Berkel, G. J., Ford, M. J., Deibel, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-Layer+Chromatography+and+Mass+Spectrometry+Coupled+Using+Desorption+Electrospray+Ionization.">Thin-Layer Chromatography and Mass Spectrometry Coupled Using Desorption Electrospray Ionization.</a> Anal. Chem. 77, 1207-1215 (2005).</li><li>Lane, A. 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=Desorption+electrospray+ionization+mass+spectrometry+reveals+surface-mediated+antifungal+chemical+defense+of+a+tropical+seaweed.">Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed.</a> Proc. Natl. Acad. Sci. 106, 7314-7319 (2009).</li><li>Kertesz, V., Van Berkel, G. 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Optimering af DESI kilde geometri er afgørende for en vellykket MSI eksperimenter. De mange variabler der bidrager til tilpasningen af ​​systemet direkte påvirker følsomheden og billedopløsning. Hvis der under optimering, forsøgslederen har vanskeligheder ved at få signal, anbefaler vi at bruge rød Sharpie spot trukket på diaset som et benchmark, farvestoffet, rhodamin 6G, m / z 443, producerer et stærkt signal i den positive ion-mode og kan bruges til indledende optimering. Derudover valg af opløsningsmid...

A correction was made to <a href="http://www.jove.com/video/50575" target="_parent">Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry</a>. There was an error with an author's name. The author's surname was appended with a missing character:
Rachel V. Bennett
instead of:
Rachel V. Bennet

Erratum: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Målrettede imaging ved massespektrometri letter erhvervelsen af ​​kemiske oplysninger for opdagelse og hypotese-genererende applikationer. Målrettet billeddannelse af en kendt kemisk interesse, på den anden side, kan fremme øget følsomhed og selektivitet gennem specifik metode udvikling. Massespektrometri imaging (MSI) er mest almindeligt udføres på væv ved hjælp af MALDI, 1 sekundær ion massespektrometri (SIMS), 2 og omgivende ionisering teknikker, herunder desorption elektrosprayionisering (DESI), 3 laserablation-elektrosprayionisering (LAESI), 4, 5 og flydende mikro-junction-overfladen prøvetagningssonde (LMJ-SSP). 6. MALDI og SIMS, prøverne skal fjernes fysisk fra prøven, og skal være flad og tynd, da de analyseres under højvakuum. MALDI kræver coating af prøven med en stråling-absorberende matrix, tilføje en ekstra og besværligt skridt til prøven forberedelse. SIMShar den højeste lateral opløsning, men bombardement med meget energirige partikler forårsager omfattende molekylære fragmentering. Derfor MSI ved omgivende metoder udfylde en niche, hvor blødt analyse med minimal prøveforberedelse er ønskelig. Men til dato er alle metoder stadig begrænset af kravet om flade prøve overflader. DESI anvender en pneumatisk assisteret opkrævet opløsningsmiddel spray rettet mod prøveoverfladen at desorbere og ionisere analytter. 7 arbejdsmodel til desorption og efterfølgende ionisering ved DESI er kendt som &quot;dråben pick-up-model&quot;. 8-10 De ladede primære dråber produceret af DESI sonde kolliderer med overfladen, fugtning det og danner en tynd film, i hvilken analysanden opløses ved en faststof-væske mikroekstraktion mekanismen 8 Efterfølgende dråbe kollisioner resulterer i momentum overførsel og start af sekundære dråber indeholdende det ekstraherede materiale fra overfladen . 9,10 sidste ende, gasfase ioner menes at blive produceret gennem ESI-lignende processer efter ion fordampning, charge restkoncentrationer modeller eller andre modeller, 11 dog den præcise iondannelse proces i DESI er endnu ikke eksperimentelt bevist. 12. DESI følsomhed er stærkt afhængig af opløseligheden af analytten i spray opløsningsmiddel som desorption afhængig af lokaliserede mikroekstraktion. 13. Når det bruges med en software-kontrolleret prøve fase prøven scannes ensrettet med lane stepping under DESI ionisering sonde, og gennem tidsdomænet er m / z information korreleret med kemiske arter af geografisk fordeling (figur 1). Siden den første proof of principle DESI-MSI eksperiment rapporteret af Van Berkel og Kertesz i 2006, er 14 den teknik modnet betragteligt, 15 med rapporterede programmer i lipidanalyser, 3,16 narkotika metabolitter, 17,18 diseaSE biomarkører, 19 hjernevæv, 3,18,20 lungevæv, 18 nyrevæv, 18 testis væv, 18 binyrerne, 17 tyndtlagschromatografi plader, 21 og alger overflader. 22. Den rutinemæssige opløsning af billeder, opnået ved DESI-MSI er 100-200 um, hvilket i sidste ende bestemmes af det effektive overfladeareal ekstraheret med spray, men beslutninger så lavt som 40 um er blevet rapporteret. 23-25 ​​sådan opløsning og nem analyse gør DESI-MSI passende til hurtig og simpel analyse af biologiske vævsprøver med arealer i 0,5-5 cm 2 rækkevidde, der muliggør erhvervelse af værdifulde geografisk information til bedre at forstå biologiske processer 26.. Her, som et eksempel på en typisk DESI-MSI program gennemgår vi de proceduremæssige detaljer at gennemføre et vellykket eksperiment involverer billeddannelse af lipider i rottehjernevæv. De to mest kritiske trin i protokollen ervævspræparatet 27 og DESI ionkilde optimering, som beskrevet nedenfor.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura-Finetek</td>
			<td>4583</td>
			<td><a href="http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;subcat=48" target="_blank">http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;subcat=48</a></td>
		 </tr>
		 <tr>
			<td>Acetonitrile</td>
			<td>EMD</td>
			<td>AX0156-6</td>
			<td>OmniSolv, LC-MS Grade</td>
		 </tr>
		 <tr>
			<td>Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>695092-500 ml</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cryostat microtome</td>
			<td>Thermo Scientific</td>
			<td>CryoStar* NX70</td>
			<td>Any available microtome can be used for tissue sectioning <a href=" http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;groupType=PRODUCT&amp;searchType=0&amp;storeId=11152&amp;from=search&amp;ca=cryostar" target="_blank"> http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;groupType=PRODUCT&amp;searchType=0&amp;storeId=11152&amp;from=search&amp;ca=cryostar</a></td>
		 </tr>
		 <tr>
			<td>Omni Spray;DESI Spray Head</td>
			<td>Prosolia Inc.</td>
			<td></td>
			<td>Can also use the 2-D Omni Spray; Source kit instead of assembling components of imaging experiment <a href=" http://www.prosolia.com/sources.php" target="_blank"> http://www.prosolia.com/sources.php</a></td>
		 </tr>
		 <tr>
			<td>High Voltage Power Supply</td>
			<td>Stanford Research Systems, Inc.</td>
			<td>PS350/5000V-25W</td>
			<td><a href="http://www.thinksrs.com/products/PS300.htm" target="_blank">http://www.thinksrs.com/products/PS300.htm</a></td>
		 </tr>
		 <tr>
			<td>Rope heater, RTD, controller</td>
			<td>Omega</td>
			<td></td>
			<td><a href="http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;book=Heaters" target="_blank">http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;book=Heaters</a></td>
		 </tr>
		 <tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td>Version 7.1 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Translational stage</td>
			<td>Prior Scientific</td>
			<td>Optiscan II</td>
			<td><a href="http://www.prior.com/productinfo_auto_motorized_optiscan.html" target="_blank">http://www.prior.com/productinfo_auto_motorized_optiscan.html</a></td>
		 </tr>
		 <tr>
			<td>AccuTOF Mass Spectrometer</td>
			<td>JEOL</td>
			<td>JMS-T100LC</td>
			<td>Can use any mass spectrometer equipped with an extended capillary atmospheric pressure interface</td>
		 </tr>
		</tbody>
 </table>

imaging of biological tissues by desorption electrospray ionization mass spectrometry

Massespektrometri imaging (MSI) indeholder ikke-målrettet molekylær information med den højeste specificitet og rumlig opløsning til at undersøge biologisk væv ved hundredvis til snesevis af mikron skalaen. Når de udføres under omgivelsesbetingelser, prøve forbehandling bliver unødvendige, hvilket forenkler protokollen, samtidig med at den høje kvalitet af de indhentede oplysninger. Desorption elektrosprayionisering (DESI) er en spray-baseret omgivende MSI teknik, der giver mulighed for direkte prøvetagning af overflader i den åbne luft, selv in vivo. Når det bruges med en software-kontrolleret prøve fase prøven bitmapobjekt under DESI ionisering sonde, og gennem tidsdomænet er m / z information korreleret med kemiske arter &#39;rumlige fordeling. Nøjagtigheden af ​​DESI-MSI udgang afhænger af kilden orientering og positionering i forhold til prøvens overflade og massespektrometer indløb. Heri gennemgår vi, hvordan man forbereder vævssnit for DESI imaging og yderligere eksperimentelle betingelser, der direkte påvirker billedkvaliteten. Konkret beskriver vi protokollen for billedbehandling af rotte hjernevæv sektioner ved DESI-MSI.

Figur 3 viser et repræsentativt spektrum opnået fra en ubehandlet rottehjerne sektion. I positiv modus massespektret domineret af phosphatidylcholiner grundet deres høje ionisering effektivitet (tilskrevet det positivt ladede kvaternære ammonium-gruppe). Den samlede ion billede af vævssnittet er også vist i figur 3, der viser rigelige signal over hele hjernen sektion. Vigtige detekterede lipider er identificeret i tabel 1, ved litteratur sammenligninger. <p cl...

Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Desorption elektrospray massespektrometri (DESI-MS) er en omgivende metode, som prøver, herunder biologiske væv, kan afbildes med minimal prøveforberedelse. Ved rastering prøven under ionisering sonden, giver denne spray-baseret teknik tilstrækkelig rumlig opløsning til at skelne molekylære egenskaber af interesse inden vævssnit.

Imaging af biologisk væv ved Desorption elektrosprayionisering massespektrometri

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Research

JoVE Journal

Bioengineering

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

Cellular Lipid Ekstraktion for målrettet stabile isotoper Fortynding Liquid Chromatography-massespektrometri Analyse

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Prøve Forberedelse Strategier for massespektrometri Imaging af 3D cellekulturmodeller

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Kvalitativ Karakterisering af den vandige fraktion fra Hydrotermisk fortætning af alger ved hjælp af 2D gaskromatografi med Time-of-flight massespektrometri

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

Hele kroppen massespektrometri Imaging af Infrared Matrix-assisteret laser Desorption elektrosprayionisering (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Engineering Tredimensionale epitelvæv indlejret i ekstracellulær matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence