The authors thank Prof. Peter Imming and Diana M&#252;ller for synthesis of the Trityl spin probe and Prof. Sebastian Polarz, Martin Wessig and Andreas Schachtschneider for the synthesis of the porous materials. Financial support by the DFG (DR 743/7-1) and within the SPP 1570 is gratefully acknowledged.

<ol>
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החוקרים אין לי מה לחשוף.

הפרוטוקול מאפשר ניטור של דיפוזיה של מולקולות אורח פאראמגנטיים. גישה הדמיה 1D נבחרה משום שהיא מאפשרת על מנת להגיע לפתרון זמן גבוה לעומת הדמיה 2D או 3D. גישת 1D דורשת שטח חתך קבוע של המדגם בגלל האינטנסיביות של תמונת 1D השיגה תלויה לא רק הריכוז אלא גם על אזור החתך של המדגם. השי...

חומרים נקבוביים לשחק תפקיד מרכזי יישומים מעשיים כגון קטליזה כרומטוגרפיה 1. על ידי הוספת קבוצות משטח והתאמת מאפייני גודל משטח הנקבוביות, החומרים ניתן להתאים את היישום הרצוי 2,3. הפונקציונליות של חומר נקבובי תלוי באופן מכריע על המאפיינים דיפוזיה של מולקולות אורח בתוך הנקבוביות. חומרים נקבוביים, הבחנה צריכה להיעשות בין המיקרו D הדיפוזיה translational המיקרוסקופי, אשר מתאר דיפוזיה בסולם אורך מולקולרי מחד ואת המאקרו D הדיפוזיה translational מקרוסקופית מצד השני, אשר מושפע דיפוזיה דרך הנקבוביות מרובות, גבולות תבואה, tortuosity ו הומגניות של החומר. ישנן מספר שיטות תהודה המגנטית זמינות ללמוד דיפוזיה, כל מתאים חלקבקנה מידה אורך icular. על הסקאלה המילימטר, תהודה מגנטית גרעינית (NMR) הדמיה 4 ו תהודה מגנטית אלקטרוני (EPR) הדמיה (כפי שהוצג בפרוטוקול זה) יכול לשמש. הקשקשים קטנים הופכים נגישים על ידי השימוש הדרגתי שדה פעם ב NMR וכן ניסויי EPR 5,6. על בקנה מידה ננומטרי, ספקטרוסקופיה EPR יכול להיות בשימוש על ידי התבוננות שינויים של אינטראקציה החליפין הייזנברג בין בדיקות ספין 7,8. מחקרים של דיפוזיה translational באמצעות מגוון הדמיה EPR מ זרז תעשייתי תומך, למשל, תחמוצת 9 אלומיניום, כדי איזוטרופי נוזלים 10,11, מערכות שחרור התרופה עשוי ג&#39;ל פולימר 12 - 14 ודגם ממברנות 15. פרוטוקול זה מציג בגישה באתרו באמצעות הדמיה EPR לפקח דיפוזיה translational מקרוסקופית של בדיקות ספין גלילי, בתווך נקבובי. זה מודגם על מערכת מארחת-אורח המורכב הדואר ספין nitroxide חללית 3- (2-Iodoacetamido) -2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) כאורח בתוך organosilica mesoporous התקופתי (PMO) airgel UKON1-GEL כמארח ואתנול כמו מֵמֵס. פרוטוקול זה בהצלחה כבר בשימוש 16 בעבר להשוות מאקרו D כפי שנקבע עם הדמיה EPR עם מיקרו D עבור החומרים מארח UKON1-GEL וסיליקה-GEL ו IPSL מינים אירוח טריס (8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo [1,2-D : 4,5-D &#39;] bis (1,3) dithiole מתיל) (Trityl), ראה איור 1. בשיטות אחרות המבוססות על הגל רציף (CW) EPR הדמיה 17, דיפוזיה מתרחשת מחוץ ספקטרומטר. לעומת זאת, השיטה המוצגת כאן משתמשת בגישה באתרו. סדרת תמונות של 1D ρ הפצת צפיפות ספין 1D (t, γ) היאנרשם על פני תקופה של מספר שעות. במהלך תקופה זו, תמונת מצב אחד הוא נלקח אחרת ומספקת דפוס דיפוזיה בזמן אמת עם רזולוצית זמן של כ 5 דקות. UKON1-GEL וסיליקה-GEL כבר מסונתזים דוגמיות בקוטר פנימי של 3 מ&quot;מ כמתואר בספרות. 16,18,19 סינתזת UKON1-GEL וסיליקה-GEL מובילה בהתכווצות המדגמת. הדגימות ממוקמות בתוך צינור לכווץ למנוע מולקולות אורחים נעו בין airgel לבין הקיר של הצינור המדגם. צעד נוסף זה אינו הכרחי עבור דגימות שניתן מסונתז ישירות בצינור מדגם מבלי לשנות את הגודל שלהם. קריסת דגימות airgel כאשר הם מתייבשים, ולכן הם חייבים להיות שקועים בתוך ממס בכל העת. הטמפרטורה מה שצריך עבור צינורות החום להתכווץ היא גבוהה יותר מאשר נקודת הרתיחה של אתנול בלחץ הסביבה. לכן הפרוטוקול מתאר את השימוש בסיר לחץ להעלות את<html> נקודת רתיחה של אתנול. הפרוטוקול מכסה את הכנת המדגם של-GEL UKON1 המסונתז מראש לצורך ניסוי הדמית EPR ואת ההגדרות ספקטרומטר המשמשות לניטור דיפוזיה של חללית ספין IPSL. לניתוח נתונים, תוכנות שנכתבו מקומית מסופקות והשימוש בו מתואר. הנתונים הגולמיים מן ספקטרומטר ניתן לטעון ישירות. התוכנה מחשבת את הפריסה המרחבית 1D ספין צפיפות ρ 1D (t, γ) ולוקחת בחשבון את פרופיל רגישות מהוד. המשתמש יכול לבחור אזור של airgel וחלון זמן, שעליו הדיפוזיה הוא שיקבע. התוכנה ואז קובעת את תנאי הגבול של משוואת הדיפוזיה מבוסס על כי הבחירה פותר את משוואת הדיפוזיה. הוא תומך הולם מרובע לפחות כדי למצוא את הערך של מאקרו D שבו הפתרון המספרי המתאים ביותר את נתוני הניסוי. תחת = &quot;jove_content&quot;&gt; הפרוטוקול ניתן להשתמש בהתאמות לחומרי אורח ומארח שונים כל עוד שטח החתך של המדגם אינו משתנה לאורך כל המדגם, כלומר ρ 1D (t, γ) מעניק גישה ישירה אל הריכוז אינו מושפע משינוי חתך מדגם. טווח הערכים נגישים עבור מאקרו D ההערכה היא 16 בין 10 -12 מ &#39;2 / sec ו -7 · 10 -9 מ&#39; 2 / sec.

<table><tbody><tr><td>X-Band spectrometer</td><td>Bruker</td><td>E580</td><td></td></tr><tr><td>Spectrometer software</td><td>Bruker</td><td>Xepr 2.6b.108</td><td></td></tr><tr><td>gradient coil system</td><td>Bruker</td><td>E540 GCX2</td><td></td></tr><tr><td>imaging resonator</td><td>Bruker</td><td>TMHS 1007</td><td></td></tr><tr><td>micro-classic pipette controller</td><td>Brand</td><td>25900</td><td></td></tr><tr><td>microcapillary ringcaps 50&amp;nbsp;&amp;micro;l</td><td>Hirschmann</td><td>9600150</td><td>inner diameter 0.5 mm</td></tr><tr><td>EPR sample tube 2 mm inner diameter</td><td>Bruker</td><td>ER 221TUB/2</td><td></td></tr><tr><td>EPR sample tube 4 mm inner diameter</td><td>Bruker</td><td>ER 221TUB/4</td><td></td></tr><tr><td>heat-shrink tubing DERAY-IB</td><td>DSG-Canusa</td><td>2210048952</td><td>4.8 mm/2.4 mm, 2:1, 95&amp;nbsp;&amp;deg;C - 200&amp;nbsp;&amp;deg;C</td></tr><tr><td>heat gun</td><td>Bosch</td><td>PHG 600-3</td><td></td></tr><tr><td>PTFE&amp;nbsp; band</td><td>VWR</td><td>332362S</td><td>width 12 mm</td></tr><tr><td>test tube</td><td></td><td></td><td>length 16 cm, diameter 1.5 cm</td></tr><tr><td>beaker</td><td></td><td></td><td>250 ml, height 9 cm, diameter 7 cm</td></tr><tr><td>capillary tube sealing</td><td>Fisher Scientific</td><td>02-678</td><td></td></tr><tr><td>pressure cooker, 3 L with trivet</td><td>Beem</td><td>Vital-X-Press V2, F1000675</td><td></td></tr><tr><td>magnetic stirrer with heating element</td><td></td><td></td><td></td></tr><tr><td>ethanol (p.a.)</td><td></td><td></td><td></td></tr><tr><td>ethanol (techn.)</td><td></td><td></td><td></td></tr><tr><td>syringe</td><td>Hamilton</td><td>1705</td><td>0.05 ml, custom length: 20 cm</td></tr><tr><td>Pasteur capillary pipette</td><td></td><td></td><td>length 23 cm</td></tr><tr><td>data analysis software</td><td>homemade</td><td></td><td>Available for download at http://www.uni-konstanz.de/drescher/software. Requires Matlab.</td></tr><tr><td>UKON1-GEL</td><td>kindly provided by Prof. Sebastian Polarz, Martin Wessig and Andreas Schachtschneider&amp;nbsp;</td><td></td><td>See references 16, 18, 19 for the synthesis</td></tr></tbody></table>

in situ monitoring of diffusion of guest molecules in porous media using electron paramagnetic resonance imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

תצלום סכימטי של airgel בתוך הצינור מתכווץ מוצג 2a דמויות 2b. התמונה EPR 2d ב איור 2 ג מראה בבירור את הקצה העליון של airgel. עוצמת 1D ρ בתוך הצינור מדגם מעל airgel נמוכה למרות הריכוז של חללית הספין הוא לפחות גבוה ככל בתוך airgel. עם ז...

Watch this Scientific Journal Video about In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging at JoVE.com

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

ניטור באתרו של דיפוזיה של מולקולות צימרים נקבוביות מדיה באמצעות הדמית תהודה מגנטית אלקטרונית

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

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Research

JoVE Journal

Chemistry

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

6.3K Views.  Universität Konstanz. A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

Video: In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Bioimaging רשום של ננו לניטור האבחון והטיפול

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date back to the early 1980s the focus of these early papers primarily characterized the changes to the structures under extremely harsh conditions (pH or temperature)1-3. It is also known that paramagnetic molecular oxygen undergoes a Heisenberg spin exchange interaction with stable radicals that extremely broadens the EPR signal4-6. Recently, we reported interesting results where this interaction of molecular oxygen with a certain part of the existing stable radical structure can be reversibly affected simply by flowing a diamagnetic gas through the carbon samples at STP7. As flows of He, CO2, and N2 had a similar effect these interactions occur at the surface area of the macropore system.
This manuscript highlights the experimental techniques, work-up, and analysis towards affecting the existing stable radical nature in the carbon structures. It is hoped that it will help towards further development and understanding of these interactions in the community at large.

Stable radicals that are present in carbon substrates interact with paramagnetic oxygen through a Heisenberg spin exchange. This interaction can be significantly reduced under STP conditions by flowing a diamagnetic gas over the carbon system. This manuscript describes a simple method to characterize the nature of those radicals.

היכרות עם הטבע הרדיקלי של משטח פחמן על ידי אלקטרונים פאראמגנטיים תהודה ומכוילת גז Flow

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

אין ויוו הערכת EPR pH, pO2, מצב חמצון-חיזור, ואת ריכוזים של פוספט גלוטתיון ב Microenvironment הגידול

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Cancer Research

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

שימוש פנטום אנזים יחיד דינמי רב-תא ללימודים של סוכני תהודה מגנטית Hyperpolarized

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Engineering

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

מהדמיה מהירה פלואורסצנטי לחוק דיפוזיה מולקולרי בקרום תא חי במיקרוסקופ מסחרי

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids with adsorbates. Vibrational spectra provide information on the chemical nature of adsorbed species and their structure. Thus, they are very useful for obtaining molecular level understanding of surface species. The IR spectrum of the sample itself gives some direct information about the material. General conclusions can be drawn concerning hydroxyl groups, some stable surface species and impurities. However, the spectrum of the sample is &#34;blind&#34; with respect to the presence of coordinatively unsaturated ions and gives rather poor information about the acidity of surface hydroxyls, species decisive for the adsorption and catalytic properties of the materials. Furthermore, no discrimination between bulk and surface species can be made. These problems are solved by the use of probe molecules, substances that interact specifically with the surface; the alteration of some spectral features of these molecules as a result of adsorption provides valuable information about the nature, properties, location, concentration, etc., of the surface sites.
The experimental protocol for in-situ IR studies of gas/sample interaction includes preparation of a sample pellet, activation of the material, initial spectral characterization through the analysis of the background spectra, characterization by probe molecules, and study of the interaction with a particular set of gas mixtures. In this paper we investigate a zirconium terephthalate metal organic framework, Zr6O4(OH)4(BDC)6 (BDC = benzene-1,4-dicarboxylate), namely UiO-66 (UiO refers to University of Oslo). The acid sites of the UiO-66 sample are determined by using CO and CD3CN as molecular probes. Furthermore, we have demonstrated that CO2 is adsorbed on basic sites exposed on dehydroxylated UiO-66. Introduction of water to the system produces hydroxyl groups acting as additional CO2 adsorption sites. As a result, CO2 adsorption capacity of the sample is strongly enhanced.

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

The use of FTIR spectroscopy for investigation of the surface properties of polycrystalline solids is described. Preparation of sample pellets, activation procedures, characterization with probe molecules and model studies of CO2 adsorption are discussed.

באתרו FTIR ספקטרוסקופיית ככלי לחקירת גז/אינטראקציה מוצקה: שיתוף משופר מים2 Adsorption ב uio-66 מתכת-אורגני המסגרת

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

ראפיד סריקת אלקטרונים פאראמגנטיים תהודה פותחת אפיקים חדשים עבור פרמטרים חשובים מבחינת פיזיולוגית ההדמיה<em&gt; In vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

ביצוע באתרו תגובות גז תא סגור במיקרוסקופ אלקטרונים ההולכה

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

אלקטרונים ספין תהודה מיקרו הדמיה המינים Live עבור מיפוי חמצן

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Biology

<meta charset="utf8"></head><body>הדמיה פרה-קלינית של רמות החמצן בגידול באמצעות EPRI ואולטרסאונד

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

<meta charset="utf8"></head><body>הערכת היפוקסיה של גידול: הדמיית חמצן תלת מימדית in vivo באמצעות תהודה פרמגנטית אלקטרונית

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

הערכת היפוקסיה של גידול: הדמיית חמצן תלת מימדית in vivo באמצעות תהודה פרמגנטית אלקטרונית 

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

ניטור באתרו של דיפוזיה של מולקולות צימרים נקבוביות מדיה באמצעות הדמית תהודה מגנטית אלקטרונית

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