Name: rapid scan electron paramagnetic resonance opens new avenues for imaging physiologically important parameters in vivo
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Partial support of this work by NIH grants NIBIB EB002807 and CA177744 (GRE and SSE) and P41 EB002034 to GRE, Howard J. Halpern, PI, and by the University of Denver is gratefully acknowledged. Mark Tseytlin was supported by NIH R21 EB022775, NIH K25 EB016040, NIH/NIGMS U54GM104942. The authors are grateful to Valery Khramtsov, now at the University of West Virginia, and Illirian Dhimitruka at the Ohio State University for synthesis of the pH sensitive TAM radicals, and to Gerald Rosen and Joseph Kao at the University of Maryland for synthesis of the mHCTPO, proxyl, BMPO and nitronyl radicals.

1. Setup of the Rapid Scan Coil Driver at 250 MHz
<ol>
	<li>Calculation of Rapid Scan Experimental Conditions 
	Note: The most important parameter in RS-EPR is scan rate, &#945;, which is the product of scan frequency and scan width (Equation 3). For narrow scan widths, faster scan rates are used, and for wider sweep widths, slower scan rates are used. The following instructions step through the latter case and show how to arrive at the experimental coil driver parameters of 7 mT sweep width and 6...

<ol>
	<li>Bobko, A. A., 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=In+vivo+monitoring+of+pH,+redox+status,+and+glutathione+using+L-band+EPR+for+assessment+of+therapeutic+effectiveness+in+solid+tumors.">In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors.</a> Magn. Reson. Med. 67 (6), 1827-1836 (2012).</li><li>Utsumi, H., 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=Simultaneous+molecular+imaging+of+redox+reactions+monitored+by+overhauser-enhanced+MRI+with+14N-and+15N-labeled+nitroxyl+radicals.">Simultaneous molecular imaging of redox reactions monitored by overhauser-enhanced MRI with 14N-and 15N-labeled nitroxyl radicals.</a> Proc. Nat. Acad. Sci. U.S.A. 103 (5), 1463-1468 (2006).</li><li>Khramtsov, V. V., Grigor'ev, I. A., Foster, M. A., Lurie, D. J., Nicholson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+applications+of+spin+pH+probes.">Biological applications of spin pH probes.</a> Cell. Mol. Bio. 46 (8), 1361-1374 (2000).</li><li>Halpern, H. J., 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=Oxymetry+Deep+in+Tissues+with+Low-Frequency+Electron-Paramagnetic+Resonance.">Oxymetry Deep in Tissues with Low-Frequency Electron-Paramagnetic Resonance.</a> Proc. Nat. Acad. Sci. U.S.A. 91 (26), 13047-13051 (1994).</li><li>Matsumoto, 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=Low-field+paramagnetic+resonance+imaging+of+tumor+oxygenation+and+glycolytic+activity+in+mice.">Low-field paramagnetic resonance imaging of tumor oxygenation and glycolytic activity in mice.</a> J. Clin. Invest. 118 (5), 1965-1973 (2008).</li><li>Velan, S. S., Spencer, R. G. S., Zweier, J. L., Kuppusamy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+paramagnetic+resonance+oxygen+mapping+(EPROM):+Direct+visualization+of+oxygen+concentration+in+tissue.">Electron paramagnetic resonance oxygen mapping (EPROM): Direct visualization of oxygen concentration in tissue.</a> Magn. Reson. Med. 43 (6), 804-809 (2000).</li><li>Elas, M., 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=Electron+paramagnetic+resonance+oxygen+image+hypoxic+fraction+plus+radiation+dose+strongly+correlates+with+tumor+cure+in+FSA+fibrosarcomas.">Electron paramagnetic resonance oxygen image hypoxic fraction plus radiation dose strongly correlates with tumor cure in FSA fibrosarcomas.</a> Int. J. Radiat. Oncol. 71 (2), 542-549 (2008).</li><li>Dreher, M. R., 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=Nitroxide+conjugate+of+a+thermally+responsive+elastin-like+polypeptide+for+noninvasive+thermometry.">Nitroxide conjugate of a thermally responsive elastin-like polypeptide for noninvasive thermometry.</a> Med. Phys. 31 (10), 2755-2762 (2004).</li><li>Gallez, B., Mader, K., 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=Noninvasive+measurement+of+the+pH+inside+the+gut+by+using+pH-sensitive+nitroxides.+An+in+vivo+EPR+study.">Noninvasive measurement of the pH inside the gut by using pH-sensitive nitroxides. An in vivo EPR study.</a> Magn. Reson. Med. 36 (5), 694-697 (1996).</li><li>Halpern, H. J., 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=Diminished+aqueous+microviscosity+of+tumors+in+murine+models+measured+with+in+vivo+radiofrequency+electron+paramagnetic+resonance.">Diminished aqueous microviscosity of tumors in murine models measured with in vivo radiofrequency electron paramagnetic resonance.</a> Cancer Res. 59 (22), 5836-5841 (1999).</li><li>Elas, M., Ichikawa, K., Halpern, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+Stress+Imaging+in+Live+Animals+with+Techniques+Based+on+Electron+Paramagnetic+Resonance.">Oxidative Stress Imaging in Live Animals with Techniques Based on Electron Paramagnetic Resonance.</a> Radiat. Res. 177 (4), 514-523 (2012).</li><li>Kuppusamy, P., 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=Noninvasive+imaging+of+tumor+redox+status+and+its+modification+by+tissue+glutathione+levels.">Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels.</a> Cancer Res. 62 (1), 307-312 (2002).</li><li>Khramtsov, V. V., Yelinova, V. I., Glazachev, Y. I., Reznikov, V. A., Zimmer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+and+reversible+modification+of+thiols+using+imidazolidine+biradical+disulfide+label.">Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label.</a> J. Biochem. Biophys. Methods. 35 (2), 115-128 (1997).</li><li>Plonka, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+paramagnetic+resonance+as+a+unique+tool+or+skin+and+hair+research.">Electron paramagnetic resonance as a unique tool or skin and hair research.</a> Exp. Dermatol. 18, 472-484 (2009).</li><li>Halevy, R., Shtirberg, L., Shklyar, M., 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=Electron+Spin+Resonance+Micro-Imaging+of+Live+Species+for+Oxygen+Mapping.">Electron Spin Resonance Micro-Imaging of Live Species for Oxygen Mapping.</a> J. Vis. Exp. (42), e122 (2010).</li><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=Microimaging+of+oxygen+concentration+near+live+photosynthetic+cells+by+electron+spin+resonance.">Microimaging of oxygen concentration near live photosynthetic cells by electron spin resonance.</a> Biophys J. 99 (3), 971-978 (2010).</li><li>Eaton, G. R., Eaton, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Concepts Magn. Reson. 7, 49-67 (1995).</li><li>Maltempo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiaon+of+spectral+and+spatial+components+in+EPR+imaging+using+2-D+image+reconstruction+algorithms.">Differentiaon of spectral and spatial components in EPR imaging using 2-D image reconstruction algorithms.</a> J. Magn. Reson. 69, 156-161 (1986).</li><li>Tseitlin, M., 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=New+spectral-spatial+imaging+algorithm+for+full+EPR+spectra+of+multiline+nitroxides+and+pH+sensitive+trityl+radicals.">New spectral-spatial imaging algorithm for full EPR spectra of multiline nitroxides and pH sensitive trityl radicals.</a> J. Magn. Reson. 245, 150-155 (2014).</li><li>Mitchell, D. G., Radu, N., Koch, 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=.">.</a> Abstracts of Papers of the American Chemical Society. 242, (2011).</li><li>Stoner, J. W., 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=Direct-detected+rapid-scan+EPR+at+250+MHz.">Direct-detected rapid-scan EPR at 250 MHz.</a> J. Magn. Reson. 170 (1), 127-135 (2004).</li><li>Tseytlin, M., Biller, J. R., Mitchell, D. G., Yu, Z., Quine, R. W., Rinard, G. A., Eaton, S. S., Eaton, G. R. <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 Newsletter. 23, 8-9 (2014).</li><li>Biller, J. R., 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=Imaging+of+nitroxides+at+250+MHz+using+rapid-scan+electron+paramagnetic+resonance.">Imaging of nitroxides at 250 MHz using rapid-scan electron paramagnetic resonance.</a> J. Magn. Reson. 242, 162-168 (2014).</li><li>Biller, J. R., 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=Improved+Sensitivity+for+Imaging+Spin+Trapped+Hydroxyl+Radical+at+250+MHz.">Improved Sensitivity for Imaging Spin Trapped Hydroxyl Radical at 250 MHz.</a> Chem. Phys. Chem. 16 (3), 528-531 (2015).</li><li>Burks, S. R., Bakhshai, M. A., Makowsky, M. A., Muralidharan, S., Tsai, P., Rosen, G. M., Kao, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2H,+15N-Substituted+nitroxides+as+sensitive+probes+for+electron+paramagnetic+resonance+imaging.">2H, 15N-Substituted nitroxides as sensitive probes for electron paramagnetic resonance imaging.</a> J. Org. Chem. 75, 6463-6467 (2010).</li><li>Dhimitruka, I., Bobko, A. A., Hadad, C. M., Zweier, J. L., Khramtsov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+amino+derivatives+of+persistent+trityl+radicals+as+dual+function+pH+and+oxygen+paramagnetic+probes.">Synthesis and characterization of amino derivatives of persistent trityl radicals as dual function pH and oxygen paramagnetic probes.</a> J. Am. Chem. Soc. 130 (32), 10780-10787 (2008).</li><li>Elajaili, H. B., 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=Electron+spin+relaxation+times+and+rapid+scan+EPR+imaging+of+pH-sensitive+amino-substituted+trityl+radicals.">Electron spin relaxation times and rapid scan EPR imaging of pH-sensitive amino-substituted trityl radicals.</a> Magn. Reson. Chem. 53 (4), 280-284 (2015).</li><li>Elajaili, H., Biller, J. R., Rosen, G. M., Kao, J. P. Y., Tseytlin, M., Buchanan, L. B., Rinard, G. A., Quine, R. W., McPeak, J., Shi, Y., Eaton, S. S., Eaton, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Disulfides+at+250+MHz+to+Monitor+Redox.">Imaging Disulfides at 250 MHz to Monitor Redox.</a> J. Magn. Reson. , (2015).</li><li>Tseitlin, M., Rinard, G. A., Quine, R. W., Eaton, S. S., Eaton, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvolution+of+sinusoidal+rapid+EPR+scans.">Deconvolution of sinusoidal rapid EPR scans.</a> J. Magn. Reson. 208 (2), 279-283 (2011).</li><li>Halpern, H. J., Peric, M., Nguyen, T. D., Spencer, D. P., Teicher, B. A., Lin, Y. J., Bowman, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isotopic+labeling+of+a+nitroxide+spin+label+to+enhance+sensitivity+for+T2+oxymetry.">Selective isotopic labeling of a nitroxide spin label to enhance sensitivity for T2 oxymetry.</a> J. Magn. Reson. 90, 40-51 (1990).</li><li>Tsai, P., 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=Esters+of+5-carboxyl-5-methyl-1-pyrroline+N-oxide:+A+family+of+spin+traps+for+superoxide.">Esters of 5-carboxyl-5-methyl-1-pyrroline N-oxide: A family of spin traps for superoxide.</a> J. Org. Chem. 68 (20), 7811-7817 (2003).</li><li>Biller, J. R., 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=Frequency+dependence+of+electron+spin+relaxation+times+in+aqueous+solution+for+a+nitronyl+nitroxide+radical+and+perdeuterated-tempone+between+250+MHz+and+34+GHz.">Frequency dependence of electron spin relaxation times in aqueous solution for a nitronyl nitroxide radical and perdeuterated-tempone between 250 MHz and 34 GHz.</a> J. Magn. Reson. 225, 52-57 (2012).</li><li>Rosen, G. M., 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=Dendrimeric-containing+nitronyl+nitroxides+as+spin+traps+for+nitric+oxide:+Ssynthesis,+kinetic,+and+stability+studies.">Dendrimeric-containing nitronyl nitroxides as spin traps for nitric oxide: Ssynthesis, kinetic, and stability studies.</a> Macromolecules. 36 (4), 1021-1027 (2003).</li><li>Bobko, A. A., 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=Redox-sensitive+mechanism+of+no+scavenging+by+nitronyl+nitroxides.">Redox-sensitive mechanism of no scavenging by nitronyl nitroxides.</a> Free Radical Biol. Med. 36 (2), 248-258 (2004).</li><li>Roshchupkina, G. I., 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=In+vivo+EPR+measurement+of+glutathione+in+tumor-bearing+mice+using+improved+disulfide+biradical.">In vivo EPR measurement of glutathione in tumor-bearing mice using improved disulfide biradical.</a> Free Radical Bio. Med. 45 (3), 312-320 (2008).</li><li>Mitchell, D. G., 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=Use+of+Rapid-Scan+EPR+to+Improve+Detection+Sensitivity+for+Spin-Trapped+Radicals.">Use of Rapid-Scan EPR to Improve Detection Sensitivity for Spin-Trapped Radicals.</a> Biophysical Journal. 105 (2), 338-342 (2013).</li><li>Bobko, A. A., Dhimitruka, I., Zweier, J. L., Khramtsov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trityl+radicals+as+persistent+dual+function+pH+and+oxygen+probes+for+in+vivo+electron+paramagnetic+resonance+spectroscopy+and+imaging:+Concept+and+experiment.">Trityl radicals as persistent dual function pH and oxygen probes for in vivo electron paramagnetic resonance spectroscopy and imaging: Concept and experiment.</a> J. Am. Chem. Soc. 129 (23), (2007).</li><li>Biller, J. R., 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=Electron+spin-lattice+relaxation+mechanisms+of+rapidly-tumbling+nitroxide+radicals.">Electron spin-lattice relaxation mechanisms of rapidly-tumbling nitroxide radicals.</a> J. Magn. Reson. 236, 47-56 (2013).</li><li>Redler, G., Barth, E. D., Bauer, K. S., Kao, J. P. Y., Rosen, G. M., Halpern, H. J. <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+electron+paramagnetic+resonance+imaging+of+differential+tumor+targeting+using+cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.">In vivo electron paramagnetic resonance imaging of differential tumor targeting using cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.</a> Magn. Reson. Med. 71 (4), 1650-1656 (2013).</li></ol>

Rapid-scan signals have higher frequency components than CW, and require a larger resonator bandwidth depending on linewidths, relaxation times, and the speed of the rapid-scans. The bandwidth required for a given experiment is based upon the linewidth and the scan rate of the magnetic field (Equation 2). Depending on the relaxation times of the probe under study (T2 and T2*), and the scan rate, oscillations can appear on the trailing edge of the signal. For nitroxide radicals with T2 ~50...

Relative to other medical imaging modalities, electron paramagnetic resonance imaging (EPRI) is uniquely able to quantitatively image physiological properties including pH1-3, pO24-7, temperature8, perfusion and viability of tissues9, microviscosity and ease of diffusion of small molecules10 and oxidative stress11. Estimation of the ease of disulfide cleavage by glutathione (GSH) in tissue and cells12,13 can report on redox status. For in vivo imaging, EPR in the frequency range between 250 MHz and 1 GHz is chosen because these frequencies provide sufficient depth of tissue penetration (up to several cm) to generate images for small animals in which intensities are not diminished by dielectric loss effects. Higher frequencies, such as 9.5 GHz14 (X-band) and 17 GHz (Ku-band)15,16 can be used for imaging of skin and hair or single cells, respectively. The success of EPRI at all frequencies depends on paramagnetic spin probes that are specific for tissues so that their location and fate may be imaged.
If the environment of an electron spin probe is spatially heterogeneous, the EPR spectrum is the sum of contributions from all locations. Spectral-spatial imaging divides the sample&#39;s volume into an array of small spatial segments and calculates the EPR spectrum for each of these segments17. This allows mapping of the local environment by measuring the spatial variation in the EPR spectrum. Magnetic field gradients are used to encode spatial information into EPR spectra, which are called projections. The spectral-spatial image is reconstructed from these projections18,19.
In RS-EPR the magnetic field is scanned through resonance in a time that is short relative to electron spin relaxation times (Figure 2)20,21. Deconvolution of the rapid-scan signal gives the absorption spectrum, which is equivalent to the first integral of the conventional first-derivative CW spectrum. The rapid-scan signal is detected in quadrature, so that both absorption and dispersion components of the spin system response are measured. This is essentially collecting twice the amount of data per unit time. Saturation of the signal in a rapid scan experiment happens at higher powers than for CW, so higher powers can be used without concern for saturation.20,22 Many more averages can be done per unit time in comparison to CW. Higher power, direct quadrature detection and more averages per unit time combine to give rapid scan a better signal-to-noise ratio (SNR), especially at high gradient projections that define spatial separation, leading to higher quality images. To achieve about the same SNR for an image of a phantom required about 10 times as long for CW as for rapid scan23.
The increased SNR also allows experiments at 250 MHz with low concentration spin trap adducts formed by the reaction of OH with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO-OH) which would be invisible to the CW method24. Dinitroxides connected with a disulfide linker are sensitive to cleavage by glutathione, and so can report on cellular redox status. Equilibrium exists, dependent on the concentration of glutathione present, between the di- and mono-radical forms. Observing these changes requires capture of the entire 5 mT wide spectrum, and can be achieved much faster with rapid scan EPR compared to stepping the magnetic field in a CW experiment.
A complete rapid scan system consists of four parts: the spectrometer, the main field magnet, the rapid scan coil driver, and the rapid scan cross-loop resonator. The spectrometer and the main field magnet function the same as in a CW experiment, setting the main Zeeman field and collecting the data from the resonator. The rapid scan coil driver generates the sinusoidal scan current that goes into specially designed rapid scan coils on the rapid scan cross-loop resonator. The rapid scan coils on the rapid scan cross-loop resonator generate a large homogeneous magnetic field, which is swept at frequencies between 3 and 15 kHz.

<table border="0" cellpadding="0" cellspacing="0" width="1763">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4,1-15N)piperdinyloxyl (15N PDT)</td>
			<td style="width:152px;">CDN Isotopes&#160;</td>
			<td style="width:135px;">M-2327</td>
			<td style="width:964px;">98% atom 15N, 98 % atom D, Quebec Canada</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">4-1H-3-carbamoyl-2,2,5,5-tetra(2H3)methyl-3-pyrrolinyloxyl (15N mHCTPO)</td>
			<td style="width:152px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in Reference 29</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">3-carboxy-2,2,5,5-tetra(2H3)methyl-1-(3,4,4-2H3,1-15N)pyrrolidinyloxyl (15N Proxyl)</td>
			<td style="width:152px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in reference 25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">4 mm Quartz EPR Tubes</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">707-SQ-100M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">4-oxo-2,2,6,6-tetra(2H3)methyl-1-(3,3,5,5-2H4)piperdinyloxyl (14N PDT)</td>
			<td style="width:152px;">CDN Isotopes</td>
			<td style="width:135px;">D-2328</td>
			<td>98% atom D, Quebec Canada</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">pH sensitive trityl radical (aTAM4)</td>
			<td style="width:152px;">Ohio State University</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at Ohio State University and described in reference 26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Potassum Phosphate, Monobasic</td>
			<td style="width:152px;">J.T. Baker Chemicals</td>
			<td style="width:135px;">1-3246</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">6 mm Quartz EPR Tubes</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">Q-5M-6M-0-250/RB</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:513px;">8 mm Quartz EPR Tubes</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">Q-7M-8M-0-250/RB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO)&#160;</td>
			<td style="width:152px;">N/A</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in reference 30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Hydrogen Peroxide</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:135px;">H1009 SIGMA</td>
			<td>30%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">16 mm Quartz EPR tube</td>
			<td style="width:152px;">Wilmad Glass</td>
			<td style="width:135px;">16-7PP-11QTZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Medium Pressure 450 W UV lamp</td>
			<td style="width:152px;">Hanovia</td>
			<td style="width:135px;">679-A36</td>
			<td>Fairfield, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">L-Glutathione, reduced</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:135px;">G470-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Nitronyl</td>
			<td style="width:152px;">NA</td>
			<td style="width:135px;">N/A</td>
			<td>Synthesized at U.Maryland and described in reference 31</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:513px;">Sodium Hydroxide&#160;</td>
			<td style="width:152px;">J.T. Baker Chemicals</td>
			<td style="width:135px;">1-3146</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The product of the experiment is a set of projections that are reconstructed into two-dimensional (one spectral, one spatial) images with a false color scale to represent signal amplitude. Deep blue denotes baseline where no signal is present, green is low amplitude and red is highest. Slices along the x-axis (spectral dimension) depict the EPR signal (EPR transition) on a magnetic field axis. Along the y-axis (spatial dimension), separation between signals corresponds to the physical spa...

Watch this Scientific Journal Video about Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo at JoVE.com

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.

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 ...

The overall goal of Rapid Scan Electron Paramagnetic Imaging, or RSEPRI, is to provide quantitative information on oxygen concentration, pH, redox status and concentration of signaling molecules that are useful for biomedical research. EPRI is a tool that can be used to answer key questions in cancer research regarding the tumor environment. The main advantage of rapid-scan EPR imaging is that you can acquire more information in a much faster time, with a wider variety of probe molecules as compared with continuous wave or CW EPR imaging.The molecules which are sensitive to redox status or pH are a good example of where rapid-scan EPRI really shines. Begin this procedure with the calculation of rapid-scan experimental conditions as determined in the text protocol. One important piece of rapid-scan is to understand the dependence of the signal on the resonator bandwidth and experimental conditions such as scan frequency sweep width.To really optimize the experiment you need to understand all three. The rapid-scan coil driver has two amplifiers. When selecting a capacitor, the capacitor box needs to be balanced with an equal capacitance on each side of the box.The two sides are in series. Unscrew the top cover of the capacitor box and insert capacitors on both sides that are equal to the determined value. Replace the top of the capacitor box and screw it down to ensure it stays on.Using the front panel of the resonated coil driver, adjust the output frequency until the sinusoidal waveform has the maximum amplitude. To prepare the radicals, remove N-15 PDT from the freezer and allow the container to come to room temperature. Weight out 1.4 milligrams of N-15 PDT using an analytical balance.Add 1.4 milligrams of N-15 PDT to 15 milliliters of deionized water for a final concentration of 0.5 millimolar. Next, combine 50 milligrams of BMPO with five milliliters of water in a 16 millimeter quartz irradiation tube. Add 100 microliters of 300 millimolar hydrogen peroxide.Irradiate the mixture in the 16 millimeter quartz irradiation tube with a medium-pressure 450 watt ultraviolet lamp for five minutes. Using a glass transfer pipette, transfer two point five milliliters of the irradiated BMPO-OH solution out of the quartz irradiation tube and into one side of a 16 millimiter quartz sample tube with a three millimeter divider. Transfer the remaining two point five milliliters of irradiated BMPO-OH into the other side of the quartz sample tube with the divider.A second critical step is to understand the power saturation curve. As scan rate increases, you access higher powers and larger signal amplitudes before your signal saturates. Larger signal amplitudes is another way to decrease your acquisition time.First, tune the resonator with an aqueous sample of the nitroxide radical by inserting the 15-milliliter sample of 0.5 millimolar N-15 PDT in water into a 16-millimeter quartz electron paramagnetic resonance tube. Insert the quartz tube into the detection side of the cross-loop rapid scan electron paramagnetic resonance or RSEPR resonator. Change the frequency of the instrument source until it matches the frequency of the detection side that contains the sample.Now, change the frequency of the excitation side to match the frequencies of the experiment source and detection side of the resonator. Change the frequency of the excitation side by turning a variable capacitor within the resonator cavity. To set up the instrument console and main magnet turn on the spectrometer and choose an experiment which records transient data with time on the abscissa.Perform a power saturation curve on a standard nitroxide radical sample under the same experimental conditions that will be used to look at radicals sensitive to pH or redox status. Once the system is set up, gradients are applied manually or through a computer program for the imaging experiment. EPR signal intensity is directly proportional to the microwave field in the resonator that causes the spin to go from one energy level to the next.This microwave field is named B1.B1 is proportional to the square root of microwave power. As microwave power is quadrupled, B1 is doubled. In this figure the power is quadrupled from eight to 32 milliwatts, and B1 is doubled from 18 to 36 milliGauss.A power saturation curve can be constructed by plotting relative amplitude as a function of the square root of microwave power, or if the resonator efficiency is known, B1.The region of this curve that is linear shows the power region where the EPR signal is not saturated or distorted. One of the advantages of a rapid-scan experiment is that the linear power range is extended, represented by the colored dots, in comparison to the regular CW experiment represented by the black dots. Shown here is a two-dimensional spectral spatial image of phantoms consisting of a BMPO-OH adduct separated by three millimeters at a concentration of five micromolar.A slice through the image shows the spectral shape at 250 megahertz. This image shows the N-14 nitranyl radical which can be used for trapping nitric oxide in vivo in a phantom, where a one point five millimeter thick wall separates two sample chambers. The spectral shape at 250 megahertz is shown here.The two-dimensional image of pH-sensitive triarylmethyl radicals reflects the difference in spectral features when the phosphate buffer pH equals seven point zero, or the pH equals seven point four. This image shows N-15 dinitroxide in a two-compartment phantom with a 10-millimeter spacer. Initially, both compartments contain 0.5 millimolar probe.Addition of glutathione to the dinitroxide breaks the reduced linker region and produces two mononitroxides, a change that is reflected in the two-dimensional image. The development of rapid-scan EPR is a total paradigm shift in our ability to study molecules with unpaired electrons. You saw today its application to in vivo EPR, but it also has wonderful possibilities for low-temperature studies of compounds with metals in them, and many other kinds of systems.For all of the samples that we've studied so far, we've gotten improvements in signal to noise of at least an order of magnitude and sometimes even more.

rapid-scan-electron-paramagnetic-resonance-opens-new-avenues-for

Research

JoVE Journal

Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...