עבודה זו נתמכת על ידי מכשיר MRI ערה NSF מענק פיתוח # 0923179 לFMF. אנו מודים אקווה Asberry, רכזת מעבדה לח&#39; פטי מכון פארקר Bioengineering וBiosciences היסטולוגיה הליבה, לקבלת סיוע בחתך רקמה.

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

אופטימיזציה של הגיאומטריה מקור דסי היא קריטית עבור ניסויי MSI מוצלחים. המשתנים מרובים תורמים ליישור של מערכת משפיעים ישירות על רגישות ורזולוציה של תמונה. אם במהלך חיפוש, יש לו את הקשיים בקבלת אות הנסיין, מומלץ להשתמש בנקודת טוש אדומה מצוירת על השקופית כאמת מידה; הצבע, r...

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

הדמיה לא ממוקדת על ידי ספקטרומטריית מסה מאפשרת רכישת המידע כימי ליישומי גילוי והשערה מניבים. הדמיה ממוקדת של חומר כימי בשמו של עניין, ומצד שני, יכולה להקל על רגישות וסלקטיביות מוגברת באמצעות פיתוח שיטה ספציפי. ספקטרומטריית מסת הדמיה (MSI) מתבצעת לרוב על רקמות באמצעות טכניקות יינון סביבה, כוללים יינון desorption electrospray (דסי), 3 יינון אבלציה-electrospray לייזר (LAESI), 4, MALDI, יון משנית 1 ספקטרומטריית מסה (SIMS), 2 ו 5 ונוזל מייקר צומת משטח דגימת בדיקה (LMJ-SSP). 6 בMALDI וסימס, יש דוגמאות להסרה פיזית מהדגימה, וצריכה להיות שטוחה ודק, כפי שהם ניתחו תחת ואקום גבוה. MALDI דורש ציפוי של המדגם עם ספיגת קרינת מטריצה, והוסיף נדבך נוסף ומסורבל להכנת המדגם. SIMSיש לו את הרזולוציה לרוחב הגבוהה ביותר, אך הפגזה עם חלקיקים אנרגטיים ביותר גורמת לפיצול מולקולרי נרחב. לכן, על ידי שיטות MSI הסביבה למלא נישה שבה ניתוח רך עם הכנת מדגם מינימאלית רצוי. עם זאת, נכון להיום, כל השיטות עדיין מוגבלות על ידי הדרישה של משטחי מדגם שטוחים. דסי משתמש בספריי ממס פניאומטית בסיוע מחויב כלפי שטח המדגם לdesorb ויינן analytes. 7 מודל העבודה לdesorption ויינון לאחר מכן על ידי דסי שמכונה &quot;טיפת מודל איסוף&quot;. 8-10 הטיפין העיקריות המופקדות הופק על ידי חללית דסי מתנגש עם פני השטח, להרטיב אותו ויוצר סרט דק שלתוכו אנליטי הוא מומס על ידי מנגנון microextraction מוצק נוזל תוצאת 8 לאחר התנגשויות טיפה בהעברת מומנטום והמראה של טיפות משניים המכילות את החומר המופק מהמשטח . 9,10 סופו של דבר, גזיוני שלב הם האמינו להיות מיוצרים באמצעות תהליכים כמו &#39;חדשות ארכיאולוגיות בעקבות אידוי יון, מודלים שאריות חיוב או דגמים אחרים, 11 לעומת זאת את תהליך היווצרות יון המדויק בדסי טרם יהיה בניסוי מוכח. רגישות דסי 12 היא מאוד תלויה במסיסות של אנליטי בתרסיס הממס, כdesorption מסתמך על microextraction המקומי. 13 כאשר בשימוש עם תוכנת שלב דגימה מבוקרת, המדגם נסרק unidirectionally עם נתיב קפיצה מתחת לחללית יינון דסי, ודרך מישור הזמן, מידע מ &#39;/ z הוא מתואם עם הפריסה המרחבית המינים כימיים&#39; (איור 1). מאז את ההוכחה הראשונה של ניסוי דסי-MSI עיקרון שדווח על ידי ואן Berkel וקרטס בשנת 2006, 14 הטכניקה התבגרה במידה ניכרת, 15 עם יישומים שדווחו בניתוח של שומנים, 3,16 מטבוליטים סמים, 17,18 diseaסמנים ביולוגיים SE, רקמות, רקמות מוח 19 3,18,20 ריאות, רקמת הכליה 18, רקמת אשך 18, 18 בלוטת יותרת הכליה, 17 צלחות כרומטוגרפיה שכבה דקות, 21 ומשטחי אצות. 22 הרזולוציה השגרתית של תמונות שהושגו על ידי דסי-MSI הוא 100-200 מיקרומטר, אשר נקבע על ידי סופו של דבר את שטח הפנים היעיל שחולצו על ידי ספריי, אבל רזולוציות נמוכות כמו 40 מיקרומטר כבר דיווחה. החלטה כזו 23-25 ​​והקלות של ניתוח הופכת דסי-MSI המתאים לניתוח המהיר ופשוט של דגימות רקמה ביולוגיות עם שטח פנים בטווח 0.5-5 ס&quot;מ 2, המאפשר הרכישה של מידע מרחבי רב ערך כדי להבין טוב יותר את התהליכים ביולוגיים 26. כאן, כדוגמה ליישום דסי-MSI טיפוסי, אנו בודקים את הפרטים פרוצדורליים של ניהול ניסוי מוצלח הכולל הדמיה של שומנים ברקמות מוח חולדה. את שני שלבים הקריטיים ביותר בפרוטוקול הםהכנת 27 ודסי אופטימיזציה מקור יון רקמות, כפי שיתואר להלן.

<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

ספקטרומטריית מסת הדמיה (MSI) מספקת מידע מולקולרי לא ממוקד עם סגוליות הגבוהה ביותר והרזולוציה מרחבית לחקירת רקמות ביולוגיות במאות עד עשרות מיקרונים קנה מידה. כאשר היא מבוצעת בתנאי הסביבה, מדגם טרום טיפול הופך להיות מיותר, ובכך לפשט את הפרוטוקול תוך השמירה על האיכות הגבוהה של מידע שהושג. יינון electrospray desorption (דסי) הוא טכניקת MSI סביבה מבוססת ספריי המאפשרת דגימה הישירה של משטחים באוויר הפתוח, גם in vivo. כאשר בשימוש עם תוכנת שלב דגימה מבוקרת, מדגם rastered מתחת לחללית יינון דסי, ודרך מישור הזמן, מידע מ &#39;/ z הוא מתואם עם הפריסה המרחבית המינים כימיים &quot;. נאמנותו של פלט דסי-MSI תלויה בנטיית המקור והמיצוב ביחס למדגם השטח וכניסת ספקטרומטר מסה. בזאת, אנו בודקים כיצד להכין סעיפי רקמות לדסי אניmaging ותנאי ניסוי נוספים שמשפיעים ישירות על איכות תמונה. באופן ספציפי, אנו מתארים את הפרוטוקול להדמיה של חלקי רקמות מוח חולדה על ידי דסי-MSI.

איור 3 מציג ספקטרום נציג המתקבל מסעיף מוח חולדה שלא טופל. במצב החיובי, הספקטרום ההמוני הוא נשלט על ידי phosphatidylcholines בשל היעילות גבוהה שלהם היינון (מיוחסת לקבוצת אמוניום רבעוני הטעונה חיובי). תמונת יון הכוללת של קטע הרקמה היא גם מוצגת באיור 3, מראה אות ב?...

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 electrospray יינון ספקטרומטריית מסה (דסי-MS) היא שיטה שבאמצעותו דגימות סביבה, כולל רקמות ביולוגיות, יכולות להיות צילמו עם הכנת מדגם מינימאלית. על ידי rastering המדגם מתחת לחללית היינון, טכניקה מבוססת ספריי זה מספקת רזולוציה מרחבית מספיק כדי להבחין במאפיינים מולקולריים של עניין בתוך סעיפי רקמות.

הדמיה של רקמות ביולוגיות על ידי Desorption electrospray יינון המוני ספקטרומטריית

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

imaging-biological-tissues-desorption-electrospray-ionization-mass

Research

JoVE Journal

Bioengineering

18.4K Views.  Georgia Institute of Technology. Desorption electrospray יינון ספקטרומטריית מסה (דסי-MS) היא שיטה שבאמצעותו דגימות סביבה, כולל רקמות ביולוגיות, יכולות להיות צילמו עם הכנת מדגם מינימאלית. על ידי rastering המדגם מתחת לחללית היינון, טכניקה מבוססת ספריי זה מספקת רזולוציה מרחבית מספיק כדי להבחין במאפיינים מולקולרי?...

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

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 sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

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

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

נדיפים חיידקית משנית אפיון באמצעות Ionization electrospray ספקטרומטריית מסה (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

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

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 tomography ( CT).

ברזולוציה גבוהה 3D הדמיה של Ex-Vivo דגימות ביולוגיות באמצעות מיקרו-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 environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Immobilization חיידקית עבור הדמיה על ידי מיקרוסקופית כוח אטומי

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

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ליפידים מיצוי נייד עבור דילול ממוקד איזוטופ יציב ניתוח כרומטוגרפיה נוזלית ספקטרומטריית-Mass

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 antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

ניתוח הפנמה נייד של חלקיקים וחיידקים על ידי Cytometry Multi-Flow דימות ספקטרלי

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

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

לדוגמא הכנת אסטרטגיות להדמית ספקטרומטריית המסה של מודלי תרבית תאי 3D

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

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

אפיון איכותי של השבר מימית מ הידרותרמיות עיבוי של אצות באמצעות גז כרומטוגרפיה 2D עם זמן של הטיסה ספקטרומטריית מסה

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)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

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

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

הדמיה של כל הגוף ספקטרומטריית מסה על ידי אינפרא אדום מטריקס בסיוע לייזר Desorption electrospray יינון (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

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

הנדסה תלת מימדי רקמות אפיתל Embedded בתוך מטריקס

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

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).