Name: electron spin resonance micro-imaging of live species for oxygen mapping
Uploaded: 2010-08-26T17:00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping at JoVE.com

इस काम आंशिक रूप से कोई अनुदान द्वारा समर्थित किया गया था. 213/09 इजराइली विज्ञान फाउंडेशन से कोई अनुदान. 2005258 बीएसएफ नींव से कोई अनुदान. यूरोपीय अनुसंधान परिषद (ईआरसी) से, 201665 और प्रणालियों पर रसेल Berrie नैनो संस्थान द्वारा. हम Schulich और cyanobacteria की आपूर्ति से निपटने के बारे में प्रणालियों में रसायन विज्ञान के संकाय के प्रोफेसर नोम Adir और Faris Salame की मदद से स्वीकार करते हैं. मदद और प्रणालियों लघु नैनो निर्माण यूनिट से स्वेतलाना Yoffis के समर्थन काफी सराहना की है.

<ol>
	<li>Halevy, R., Tormyshev, V., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-imaging+of+Oxygen+Concentration+near+Live+Photosynthetic+Cells+by+Electron+Spin+Resonance.">Micro-imaging of Oxygen Concentration near Live Photosynthetic Cells by Electron Spin Resonance.</a> Biophysical Journal. , (2010).</li><li>Blank, A., Suhovoy, E., Halevy, R., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESR+imaging+in+solid+phase+down+to+sub-micron+resolution:+methodology+and+applications.">ESR imaging in solid phase down to sub-micron resolution: methodology and applications.</a> J Phys Chem. 11, 6689-6699 (2009).</li><li>Gallez, B., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+EPR:+when,+how+and+why?.">In vivo EPR: when, how and why?.</a> NMR Biomed. 17, 223-225 (2004).</li><li>Eaton, G. R., Eaton, S. S., Ohno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EPR imaging and in vivo EPR. , (1991).</li><li>Matsumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+tumor+oxygenation+and+microvascular+permeability+using+Overhauser+enhanced+MRI.">Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.</a> Proc Natl Acad Sci USA. 106, 17898-17903 (2009).</li><li>Talmon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diffusion+in+Porous+Media+by+PGSE.">Molecular Diffusion in Porous Media by PGSE.</a> ESR. J Phys Chem. , (2010).</li><li>Halevy, R., Talmon, Y., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+production+of+glass+sample+holders+for+improved+sensitivity+and+resolution+in+ESR+microscopy.">Photolithographic production of glass sample holders for improved sensitivity and resolution in ESR microscopy.</a> Applied Magnetic Resonance. 31, 591-598 (2007).</li><li>Allen, M. M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+Division+of+Some+Unicellular+Blue-Green+Algae.">Growth and Division of Some Unicellular Blue-Green Algae.</a> J Gen Microbiol. 51, 199-199 (1968).</li><li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+Assignments,+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria.">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> J Gen Microbiol. 111, 1-61 (1979).</li><li>Blank, A., Talmon, Y., Shklyar, M., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+diffusion+in+liquid+phase+by+electron+spin+resonance.">Direct measurement of diffusion in liquid phase by electron spin resonance.</a> Chem Phys Lett. 465, 147-152 (2008).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

इस प्रोटोकॉल से पता चलता है कैसे सूक्ष्म इमेजिंग ESR रहते छोटे नमूनों के निकट ऑक्सीजन एकाग्रता नक्शा करने के लिए लागू किया जा सकता है. ~ 30 x 30 के एक स्थानिक संकल्प x 100 सुक्ष्ममापी, के पास micromolar ऑक्सीजन एकाग्रता संवेदनशीलता और उप femtomole voxel के प्रति पूर्ण ऑक्सीजन संवेदनशीलता के साथ प्रदर्शन किया है. कोशिकाओं के पास ऑक्सीजन के मानचित्रण के लिए सूक्ष्म इमेजिंग ESR का उपयोग वर्तमान में उपलब्ध सूक्ष्म इलेक्ट्रोड phosphorescence / या प्रतिदीप्ति पर आधारित तकनीक का पूरक है. इसके अलावा, उचित paramagnetic जांच के साथ, यह आसानी से intracellular ऑक्सीजन सूक्ष्म इमेजिंग, एक क्षमता है जो अन्य तरीकों को खोजने के लिए बहुत मुश्किल से हासिल के लिए लागू हो जाएगा. निकट भविष्य में हम आगे इस पद्धति में सुधार के कुछ microns के एक संकल्प के साथ रहते हैं नमूना छवियों को प्रदान करने पर योजना है, ऐसे सुपर ऑक्साइड एकाग्रता, अम्लता (पीएच), जांच प्रसार गुणांक और पाठ्यक्रम के, ऑक्सीजन एकाग्रता के रूप में इसके विपरीत मापदंडों प्रदान. दोनों विपरीत प्रकार के मामले में और भी नमूने विशेषताओं (जैसे गैर पारदर्शी मोटी नमूने और कुछ मामलों में, intracellular बनाम कोशिकी माप) के मौजूदा ऑप्टिकल आधारित तरीके के लिए इन क्षमताओं के पूरक हैं.

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Centrifuge </td>
			<td>&nbsp;</td>
			<td>Kendro</td>
			<td>Heraus, 75003235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Perdeuterated triarylmethyl (trityl) radical</td>
			<td>&nbsp;</td>
			<td>Synthesized at Novosibirsk using the method described in reference 6.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BG-11 buffer</td>
			<td>&nbsp;</td>
			<td>For instruction preparation, see Scheme 1 and references 8, 9. </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>&nbsp;</td>
			<td>Hamilton</td>
			<td>Microliter 7000.5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultraviolet Curing</td>
			<td>&nbsp;</td>
			<td>Norland Products, Inc.</td>
			<td>NOA63, or NOA61.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping at JoVE.com

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

इस प्रोटोकॉल माइक्रोन पैमाने इलेक्ट्रॉन स्पिन गूंज माइक्रोस्कोपी द्वारा जीवित कोशिकाओं के तत्काल वातावरण में ऑक्सीजन एकाग्रता के तीन आयामी इमेजिंग के लिए एक विधि का वर्णन करता है.

इलेक्ट्रॉन स्पिन आक्सीजन मानचित्रण के लिए लाइव प्रजाति की माइक्रो इमेजिंग अनुनाद

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

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