The authors acknowledge the Swiss National Science Foundation for financing SMhardBi (project number 200021_150088/1). The SANS experiments were performed at the Swiss spallation neutron source SINQ, Paul Scherrer Instute, Villigen, Switzerland. The authors warmly thank Dr. Joachim Kohlbrecher for his guidance with the SANS experiments. The birefringence measurement setup under high magnetic fields was inspired from the existing setup at the high-field magnetic laboratory HFML, Nijmegen, The Netherlands. We thank Bruno Pfister for his help in developing the electronics of the birefringence setup, Jan Corsano and Daniel Kiechl for constructing the frameworks permitting fine and facile alignment of the laser, and Dr. Bernhard Koller for ongoing technical support.

<ol><li>Sanders, C. R., Hare, B. J., Howard, K. P., Prestegard, J. H. <a target="_blank" href="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/pii/007965659480012X">Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules.</a> Prog. Nucl. Magn. Reson. Spectrosc. 26, 421-444 (1994).</li>
<li>Glover, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+evaluation+of+phospholipid+bicelles+for+solution-state+studies+of+membrane-associated+biomolecules%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules.</a> Biophys. J. 81 (4), 2163-2171 (2001).</li>
<li>Katsaras, J. H. T. A., Pencer, J., Nieh, M. -P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22Bicellar%22+lipid+mixtures+as+used+in+biochemical+and+biophysical+studies%5BTitle%5D)+AND+%22Naturwissenschaften%22%5BJournal%5D)">"Bicellar" lipid mixtures as used in biochemical and biophysical studies.</a> Naturwissenschaften. 92 (8), 355-366 (2005).</li>
<li>Sanders, C. R., Prosser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicelles%3A+a+model+membrane+system+for+all+seasons%3F%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Bicelles: a model membrane system for all seasons?</a> Structure. 6 (10), 1227-1234 (1998).</li>
<li>Dürr, U. H. N., Soong, R., Ramamoorthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((When+detergent+meets+bilayer%3A+birth+and+coming+of+age+of+lipid+bicelles%5BTitle%5D)+AND+%22Prog.+Nucl.+Magn.+Reson.+Spectrosc%22%5BJournal%5D)">When detergent meets bilayer: birth and coming of age of lipid bicelles.</a> Prog. Nucl. Magn. Reson. Spectrosc. 69, 1-22 (2013).</li>
<li>Dürr, U. H. N., Gildenberg, M., Ramamoorthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+magic+of+bicelles+lights+up+membrane+protein+structure%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">The magic of bicelles lights up membrane protein structure.</a> Chem. Rev. 112, 6054-6074 (2012).</li>
<li>Ujwal, R., Abramson, J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22257923">High-throughput crystallization of membrane proteins using the lepidic bicelle method.</a> J. Vis. Exp. (59), (2012).</li>
<li>Barbosa-Barros, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicelles%3A+lipid+nanostructured+platforms+with+potential+dermal+applications%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">Bicelles: lipid nanostructured platforms with potential dermal applications.</a> Small. 6, 807-818 (2012).</li>
<li>Lin, L., et al. <a target="_blank" href="http://pubs.rsc.org/is/content/articlelanding/2016/ra/c6ra18112k">Hybrid bicelles as a pH-sensitive nanocarrier for hydrophobic drug delivery.</a> RSC Adv. 6, 79811-79821 (2016).</li>
<li>Beck, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+type+of+bicellar+disks+from+a+mixture+of+DMPC+and+DMPE-DTPA+with+complexed+lanthanides%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Novel type of bicellar disks from a mixture of DMPC and DMPE-DTPA with complexed lanthanides.</a> Langmuir. 26 (8), 5382-5387 (2010).</li>
<li>Liebi, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Tailored+phospholipid+bicelles+to+generate+magnetically+switchable+material%22">Tailored phospholipid bicelles to generate magnetically switchable material</a>. , ETH Zürich. Switzerland. PhD Thesis n° 21048, ISBN 978-3-905609-55-4 (2013).</li>
<li>Liebi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetically+enhanced+bicelles+delivering+switchable+anisotropy+in+optical+gels%5BTitle%5D)+AND+%22ACS.+Appl.+Mater.+Interfaces%22%5BJournal%5D)">Magnetically enhanced bicelles delivering switchable anisotropy in optical gels.</a> ACS. Appl. Mater. Interfaces. 6 (2), 1100-1105 (2014).</li>
<li>Liebi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alignment+of+bicelles+studied+with+high-field+magnetic+birefringence+and+small-angle+neutron+scattering+measurements%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Alignment of bicelles studied with high-field magnetic birefringence and small-angle neutron scattering measurements.</a> Langmuir. 29, 3467-3473 (2013).</li>
<li>Prosser, R. S., Hwang, J. S., Vold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetically+aligned+phospholipid+bilayers+with+positive+ordering%3A+a+new+model+membrane+system%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Magnetically aligned phospholipid bilayers with positive ordering: a new model membrane system.</a> Biophys J. 74, 2405-2418 (1998).</li>
<li>Prosser, R. S., Bryant, H., Bryant, R. G., Vold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lanthanide+chelates+as+bilayer+alignment+tools+in+NMR+studies+of+membrane-associated+peptides%5BTitle%5D)+AND+%22J.+Magn.+Reson%22%5BJournal%5D)">Lanthanide chelates as bilayer alignment tools in NMR studies of membrane-associated peptides.</a> J. Magn. Reson. 141, 256-260 (1999).</li>
<li>Liebi, M., Kohlbrecher, J., Ishikawa, T., Fischer, P., Walde, P., Windhab, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholesterol+increases+the+magnetic+aligning+of+bicellar+disks+from+an+aqueous+mixture+of+DMPC+and+DMPE-DTPA+with+complexed+thulium+ions%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Cholesterol increases the magnetic aligning of bicellar disks from an aqueous mixture of DMPC and DMPE-DTPA with complexed thulium ions.</a> Langmuir. 28 (29), 10905-10915 (2012).</li>
<li>Liebi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholesterol-diethylenetriaminepentaacetate+complexed+with+thulium+ions+integrated+into+bicelles+to+increase+their+magnetic+alignability%5BTitle%5D)+AND+%22J.+Phys.+Chem.+B%22%5BJournal%5D)">Cholesterol-diethylenetriaminepentaacetate complexed with thulium ions integrated into bicelles to increase their magnetic alignability.</a> J. Phys. Chem. B. 117 (47), 14743-14748 (2013).</li>
<li>Isabettini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tailoring+bicelle+morphology+and+thermal+stability+with+lanthanide-chelating+cholesterol+conjugates%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Tailoring bicelle morphology and thermal stability with lanthanide-chelating cholesterol conjugates.</a> Langmuir. 32, 9005-9014 (2016).</li>
<li>Isabettini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mastering+the+magnetic+susceptibility+of+magnetically+responsive+bicelles+with+3%CE%B2-Amino-5-Cholestene+and+complexed+lanthanide+ions%5BTitle%5D)+AND+%22Phys.+Chem.+Chem.+Phys%22%5BJournal%5D)">Mastering the magnetic susceptibility of magnetically responsive bicelles with 3β-Amino-5-Cholestene and complexed lanthanide ions.</a> Phys. Chem. Chem. Phys. 19, 10820-10824 (2017).</li>
<li>De Angelis, A. A., Opella, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bicelle+samples+for+solid-state+NMR+of+membrane+proteins%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Bicelle samples for solid-state NMR of membrane proteins.</a> Nat. Protoc. 2 (10), 2332-2338 (2007).</li>
<li>Son, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22Q-Titration%22+of+long-chain+and+short-chain+lipids+differentiates+between+structured+and+mobile+residues+of+membrane+proteins+studied+in+bicelles+by+solution+NMR+spectroscopy%5BTitle%5D)+AND+%22J.+Magn.+Reson%22%5BJournal%5D)">"Q-Titration" of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy.</a> J. Magn. Reson. 214, 111-118 (2012).</li>
<li>Avanti Polar Lipids Inc. Bicelle Preparation. , Available from:
 <a target="_blank" href="https://avantilipids.com/tech-support/liposome-preparation/bicelle-preparation">https://avantilipids.com/tech-support/liposome-preparation/bicelle-preparation</a> (2017).</li>
<li>Isabettini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+Generating+Highly+Magnetically+Responsive+Lanthanide-Chelating+Phospholipid+Polymolecular+Assemblies%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Methods for Generating Highly Magnetically Responsive Lanthanide-Chelating Phospholipid Polymolecular Assemblies.</a> Langmuir. 33, 6363-6371 (2017).</li>
<li>Nieh, M. -P., Glinka, C. J., Krueger, S., Prosser, R. S., Katsaras, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SANS+study+on+the+effect+of+lanthanide+ions+and+charged+lipids+on+the+morphology+of+phospholipid+mixtures%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">SANS study on the effect of lanthanide ions and charged lipids on the morphology of phospholipid mixtures.</a> Biophysical Journal. 82 (5), 2487-2498 (2002).</li>
<li>Watts, A., Spooner, P. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipid+phase+transitions+as+revealed+by+NMR%5BTitle%5D)+AND+%22Chem.+Phys.+Lip%22%5BJournal%5D)">Phospholipid phase transitions as revealed by NMR.</a> Chem. Phys. Lip. 57, 195-211 (1991).</li>
<li>Bleaney, B. <a target="_blank" href="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/pii/0022236472900273">Nuclear magnetic-resonance shifts in solution due to lanthanide ions.</a> J. Magn. Reson. 8, 91-100 (1972).</li>
<li>Prosser, R. S., Volkov, V. B., Shiyanovskaya, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Solid-state+NMR+studies+of+magnetically+aligned+phospholipid+membranes%3A+taming+lanthanides+for+membrane+protein+studies%5BTitle%5D)+AND+%22Biochem.+Cell+Biol%22%5BJournal%5D)">Solid-state NMR studies of magnetically aligned phospholipid membranes: taming lanthanides for membrane protein studies.</a> Biochem. Cell Biol. 76, 443-451 (1998).</li>
<li>Prosser, R. S., Volkov, V. B., Shiyanovskaya, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+chelate-induced+magnetic+alignment+of+biological+membranes%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Novel chelate-induced magnetic alignment of biological membranes.</a> Biophys. J. 75, 2163-2169 (1998).</li>
<li>Shklyarevskiy, I. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+alignment+of+self-assembled+anthracene+organogel+fibers%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Magnetic alignment of self-assembled anthracene organogel fibers.</a> Langmuir. 21, 2108-2112 (2005).</li>
<li>Christianen, P. C. M., Shklyarevskiy, I. O., Boamfa, M. I., Maan, J. C. <a target="_blank" href="https://www-sciencedirect-com.remotexs.ntu.edu.sg/science/article/pii/S0921452604000766">Alignment of molecular materials in high magnetic fields.</a> Physica B: Condens. Matter. 346, 255-261 (2004).</li>
<li>Maret, G., Dransfeld, K. <a target="_blank" href="https://link-springer-com.remotexs.ntu.edu.sg/chapter/10.1007%2F3-540-13504-9_10">Biomolecules and polymers in high steady magnetic fields.</a> Top. App. Phys. 57, 143-204 (1985).</li>
<li>Gielen, J. C., Shklyarevskiy, I. O., Schenning, A. P. H. J., Christianen, P. C. M., Maan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+magnetic+birefringence+to+determine+the+molecular+arrangement+of+supramolecular+nanostructures%5BTitle%5D)+AND+%22Sci.+Tech.+Adv.+Mater%22%5BJournal%5D)">Using magnetic birefringence to determine the molecular arrangement of supramolecular nanostructures.</a> Sci. Tech. Adv. Mater. 10 (1), 014601(2009).</li>
<li>Shklyarevskiy, I. O. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Deformation+and+ordering+of+molecular+assemblies+in+high+magnetic+fields%22">Deformation and ordering of molecular assemblies in high magnetic fields</a>. , Nijmegen University. The Netherlands. PhD Thesis, ISBN 90-9018956-4 (2005).</li>
<li>Fuller, G. G. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFuller%2C+GG+%22Optical+rheometry+of+complex+fluids%22">Optical rheometry of complex fluids</a>. , Oxford University Press. NY. (1995).</li>
<li>Walde, P., Cosentino, K., Engel, H., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Giant+vesicles%3A+preparations+and+applications%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Giant vesicles: preparations and applications.</a> ChemBioChem. 11, 848-865 (2010).</li>
<li>Avanti Polar Lipids Inc. Liposome Preparation. , Available from:
 <a target="_blank" href="https://avantilipids.com/tech-support/liposome-preparation">https://avantilipids.com/tech-support/liposome-preparation</a> (2017).</li>
<li>Avanti Polar Lipids Inc. Preparing Large, Unilamellar Vesicles by Extrusion (LUVET). , Available from:
 <a target="_blank" href="https://avantilipids.com/tech-support/liposome-preparation/luvet">https://avantilipids.com/tech-support/liposome-preparation/luvet</a> (2017).</li>
<li>Isabettini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+engineering+of+lanthanide+ion+chelating+phospholipids+generating+assemblies+with+a+switched+magnetic+susceptibility%5BTitle%5D)+AND+%22Phys.+Chem.+Chem.+Phys%22%5BJournal%5D)">Molecular engineering of lanthanide ion chelating phospholipids generating assemblies with a switched magnetic susceptibility.</a> Phys. Chem. Chem. Phys. 19, 20991-21002 (2017).</li>
<li>Battaglia, M. R., Ritchie, G. L. D. <a target="_blank" href="http://pubs.rsc.org/en/content/articlelanding/1977/f2/f29777300209">Molecular magnetic anisotropies from the Cotton-Mouton effect.</a> J. Chem. Soc., Faraday Trans. 2. 73 (2), 209-221 (1977).</li>
<li>Sprunt, S., Nounesis, G., Litster, J. D., Ratna, B., Shashidhar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-field+magnetic+birefringence+study+of+the+phase+behavior+of+concentrated+solutions+of+phospholipid+tubules%5BTitle%5D)+AND+%22Phys.+Rev.+E%22%5BJournal%5D)">High-field magnetic birefringence study of the phase behavior of concentrated solutions of phospholipid tubules.</a> Phys. Rev. E. 48 (1), 328-339 (1993).</li>
<li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+paranematic+ordering+of+rigid+and+semiflexible+amyloid-Fe3O4+hybrid+fibrils+in+an+external+magnetic+field%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Continuous paranematic ordering of rigid and semiflexible amyloid-Fe3O4 hybrid fibrils in an external magnetic field.</a> Biomacromolecules. 17 (8), 2555-2561 (2016).</li>
</ol>

The authors have nothing to disclose.

A detailed account of how birefringence measurements were used in combination with SANS experiments to evaluate methods for generating highly magnetically responsive Ln3+ chelating phospholipids assemblies is in Isabettini et al.23 The proposed fabrication protocols are also applicable for assemblies composed of the longer DPPC and DPPE-DTPA phospholipids or for those containing chemically engineered steroid derivatives in their bilayer.11,12,17,18,19 The only requirement being that the sample is heated to sufficiently high temperatures in steps 1.3.2, 1.3.3, 1.3.4 and 1.4.2. The temperatures must allow the bilayer lipids to enter a liquid disordered phase, guarantying optimal hydration of the dry lipid film or sample regeneration. DPPC/DPPE-DTPA/Tm3+ (molar ratio 4:1:1) assemblies, for example, need to be heated above the phase transition temperature of DPPC at 42 &#176;C, while the analogous DMPC-based system must be heated above 24 &#176;C. A sufficiently high temperature is also necessary to guarantee the formation of extrudable vesicles occurring when the lipid bilayer is in a disordered state in step 1.4. The freeze-thawing cycles in step 1.3.2 may be fully replaced by H&#38;C cycles.23 However, the sample needs more time to fully hydrate the lipid film with this procedure and must be vortexed 20 min when at 5 &#176;C and 2 min when at 60 &#176;C. Additional H&#38;C cycles are undertaken if elements of the dry lipid film are still observed on the glass walls of the flask.
The Tm3+ chelating bicelles presented in this protocol align perpendicular to the magnetic field direction. This alignment direction originates from the large positive magnetic susceptibility of Tm3+.11,14 Other lanthanide ions such as Dy3+ and Yb3+ may also be applied.11,13,19 The different magnetic anisotropy of the Ln3+ offers additional means of tailoring the magnetic alignment of the bicelles. For example, Dy3+ enhances the intrinsically negative magnetic susceptibility of the bilayer phospholipids, resulting in a high degree of alignment of the bicelles parallel to the magnetic field direction.13 This change in alignment direction is detected by a change in sign of both the birefringence signal and the alignment factors computed from anisotropic 2D SANS patterns. It is important to note that the magnetic susceptibility is not solely dictated by the chemical nature of the Ln3+ but by the chelate geometry of the Ln3+-phospholipid complex.19,38 The magnetic susceptibility may be engineered by synthesizing different Ln3+ chelating phospholipid headgroups, defining the magnetic response of the resulting assemblies.38
Every sample is optically different depending on the nature of the constituting lipids employed. Monitoring the sample&#39;s turbidity as a function of temperature is a complementary method to evaluate temperature-induced structural transformations in the assemblies. Although these measurements are usually conducted in the absence of a magnetic field in a spectrophotometer, monitoring the intensity of the laser&#39;s direct current <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg" /> with the setup proposed herein offers the same information in the presence of a magnetic field.11,16 The DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) samples are usually less turbid than their Chol-OH containing DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) counterparts at 5 &#176;C. Samples resembling water at 5 &#176;C are generally not alignable in a magnetic field. At room temperature, both samples look transparent because the DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) samples are in a transition phase between bicelles and vesicles and large concentric holes appear in the DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles.11,16,23 The transition state of bicelles to vesicles in DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) is also accompanied with an increase in the sample&#39;s viscosity at room temperature. This temperature-dependent change in turbidity makes choosing the right sensitivity in step 2.11 difficult. If the sensitivity is adjusted too high on a turbid sample at 5 &#176;C, the more transparent nature of the sample on heating may cause an overload of the amplifiers. Moreover, highly turbid samples will considerably increase the noise to signal ratio and may not be suitable for birefringence measurements. The laser light must be able to go through the sample in order to be detected.
Non-extruded samples are always more turbid and have the tendency to aggregate upon short term storage in the fridge. Nevertheless, the magnetically responsive samples are readily regenerated with a H&#38;C cycle. Non-extruded samples may also be stored in the frozen state and readily regenerated by H&#38;C cycles. Extruded samples are kept in the fridge and commonly measured in one week following the sample&#39;s preparation. No studies report on the prolonged storage of extruded species in either a liquid or a frozen state. Therefore, the size distribution of the assemblies obtained from extrusion cannot be guaranteed over prolonged storage.
Analogously to any bicelle system, these magnetically alignable planar assemblies only exist in a defined range of lipid composition and concentration. Altering the lipid ratios will result in different assembly architectures, including the formation of micelles, ribbons, and vesicles.5,11,16,18,20 The phosphate buffer concentration and pH in step 1.1.3 plays a crucial role in shaping the bicelles and their magnetic response. The buffer defines the physico-chemical interactions governing the hydrophilic environment surrounding the polymolecular assemblies. Lower buffer concentrations result in different assembly architectures, whilst higher concentrations cause sample aggregation and precipitation due to an excess charge screening. Under acidic conditions with pH values between 3 and 4, the carboxylic acid moieties serving as ligands in the DMPE-DTPA/Ln3+ complex are protonated. This results in the destruction of the magnetically responsive polymolecular assemblies, observed by aggregation and precipitation in the sample. The magnetically responsive Ln3+ polymolecular assemblies have a reasonable resistance towards more basic pH values. However, DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) bicelles were shown to break up into micelles at pH values of 12.9.11 The samples must never be exposed to tap water or other salts. Any other ion will disturb the Ln3+ chelating process or result in aggregation of the assemblies due to charge screening. For SANS measurements, the buffer is prepared as described in step 1.1.3 in D2O instead of ultrapure H2O. Note that the pH meter reading will be 7.0 (corresponding to a pD value of 7.4).
The structural transformations occurring in polymolecular assemblies subject to a heating and cooling cycle are thermo-reversible. Therefore, the final birefringence signal at 5 &#176;C should be the same as before the temperature cycle.11,16 If the birefringence signal is higher after the cycle, the sample was not properly regenerated in step 1.3.4. This commonly occurs in samples stored for a prolonged period of time. A lower birefringence signal after the temperature cycle as observed in Figure 6 indicates a problem in the experimental setup. Most commonly, the laser light path was disturbed by back scattering or another object. This is especially problematic with the temperature probe inserted directly into the sample (see step 2.8) which should be placed as to not interfere with the direct path of the laser light. A disturbed light path causes a fall in the <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg" />, a noisy signal, and/or abnormal peaks in the birefringence-temperature curves. For example, the peak occurring on heating at about 35 &#176;C in Figure 6 was caused by expansion of the water-cooling tubes into the direct path of the laser light. The birefringence signal may not be trusted from that point onwards. Although the general shape of the cooling curve was normal, the lower birefringence signal obtained at 5 &#176;C was caused by the interference.
The birefringence values obtained from following this protocol are not absolute and are used to compare samples amongst themselves. For comparison with literature values, a calibration with a reference system is required. For example, the sign of the measured retardation depends on the alignment of the set-up and may be checked with toluene, which has a Cotton-Mouton constant of 3.27 &#215; 10&#8722;9 T&#8722;2.39,40
The birefringence signal originating from changes in the sample&#39;s magnetic alignment may be decoupled from the signal caused by molecular rearrangements in the bilayer. Alignment factors computed from anisotropic 2D SANS patterns obtained under a magnetic field are only influenced by the bulk alignment of the polymolecular assemblies. The two methods are complementary and allow decoupling of the contributions to the birefringence signal. The proposed birefringence setup could be perfected by splitting the laser beam, allowing for the simultaneous monitoring of samples with and without exposure to the external magnetic field. The birefringence results obtained for the sample in the magnetic field could be normalized by the signal obtained for the sample at 0 T, effectively accounting for the background.
Birefringence measurements are not limited to quantifying the magnetic alignment of bicelles. Numerous soft-materials generate a birefringence signal due to ordering of their internal structure. The proposed setup allows to monitor the birefringence of such materials as a function of temperature with or without an external magnetic field. Anthracene organogel fibers, wormlike micelles under flow, nanocrystalline cellulose and amyloid-Fe3O4 fibrils are a few examples whose birefringence behavior was successfully evaluated with the proposed setup.29,30,32,41

Bicelles are disk-like polymolecular assemblies obtained from numerous lipid mixtures.1,2,3,4,5 They are widely used for the structural characterization of membrane biomolecules by NMR spectroscopy.6,7 However, recent efforts aim to expand the field of possible applications.5,8,9 The most studied bicelle system is composed of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), constituting the planar part of the assembly, and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) phospholipid covering the edge.1,2,3 The molecular geometry of the phospholipids composing the bilayer dictate the architecture of the self-assembled polymolecular structure.4,5 Replacing DHPC with DMPE-DTPA generates highly magnetically responsive and tunable bicelle systems.10,11 DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1) bicelles associate with many more paramagnetic lanthanide ions (Ln3+) on the bilayer&#39;s surface, resulting in an enhanced magnetic response.10 Moreover, replacing the water-soluble DHPC molecules with DMPE-DTPA/Ln3+ enables the formation of dilution-resistant bicelles.11
The magnetic alignability of planar polymolecular assemblies is dictated by their overall magnetic energy,
<img alt="Equation 1" src="/files/ftp_upload/56812/56812eq1v2.jpg" /> (1)
where B is the magnetic field strength, <img alt="Equation 2" src="/files/ftp_upload/56812/56812eq2.jpg" /> the magnetic constant, n the aggregation number and <img alt="Equation 3" src="/files/ftp_upload/56812/56812eq3.jpg" /> the molecular diamagnetic susceptibility anisotropy of the lipids composing the bilayer. Therefore, the response of DMPC/DMPE-DTPA/Ln3+ bicelles to magnetic fields is tailored by their size (aggregate number n) and the molecular diamagnetic susceptibility anisotropy &#916;&#967;. The latter is readily achieved by changing the nature of the chelated Ln3+.12,13,14,15 Introducing cholesterol (Chol-OH) or other steroid derivatives in the bilayer offers the possibility of tuning both the aggregate number n and the magnetic susceptibility &#916;&#967; of the assemblies.11,16,17,18,19 For a given lipid composition, larger assemblies contain more lipids capable of contributing to the Emag (larger aggregate number n), resulting in more alignable species. The size of DMPC/DHPC bicelles, for example, is conventionally controlled through optimization of the composing lipid ratio or total concentration.20,21,22 Although this is possible in DMPC/DMPE-DTPA/Ln3+ bicelles, their thermo-reversible transformation from bicelle to vesicles upon heating offers added tailoring options. Mechanical means such as extrusion through membrane filters allows shaping of the vesicles. The magnetically alignable bicelles are regenerated upon cooling to 5 &#176;C and their dimensions are dictated from the vesicle precursors.11 Herein, we focus on the potential of mechanical fabrication procedures with DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) or DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) as reference systems. The process works analogously when working with other Ln3+ than Tm3+. The wide range of possibilities offered by these techniques are highlighted in Figure 1 and extensively discussed elsewhere.23
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56812/56812fig1.jpg" /> 
Figure 1: Schematic overview of the possible fabrication procedures. The studied magnetically alignable Ln3+ chelating polymolecular assemblies are composed of either DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) or DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5). The dry lipid film is hydrated with a 50 mM phosphate buffer at a pH value of 7.4 and the total lipid concentration is 15 mM. An effective hydration of the lipid film requires either freeze thawing cycles (FT) or heating and cooling cycles (H&#38;C). H&#38;C cycles are necessary to regenerate samples after the last freeze thawing step, or to regenerate samples kept frozen over a prolonged period of time if they are to be used without further extrusion. These steps are extensively discussed by Isabettini et al.23 Maximally alignable polymolecular assemblies are achieved, delivering different assembly architectures based on the lipid composition. The bicelle size and magnetic alignability is tunable by extrusion (Ext) through nanopore membrane filters. The presented alignment factors Af were computed from 2D small angle neutron scattering (SANS) patterns of a DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample extruded through either 800, 400, 200, or 100 nm pores. SANS measurements are a complementary means of quantifying bicelle alignment that will not be covered in more detail herein.11,16 The Af ranges from -1 (parallel neutron scattering or perpendicular alignment of the bicelles with respect to the magnetic field direction) to 0 for isotropic scattering. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56812/56812fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The structure of bicelles has been extensively studied by a wide range of characterization techniques.13 The alignment of bicelles exposed to a magnetic field has been quantified by using either NMR spectroscopy or small angle neutron scattering (SANS) experiments.5,10,11,12,13,16,17,18,19,24,25 However, the shift and broadening of the NMR peaks occurring in the presence of Ln3+ are serious limitations to the method.15,26,27,28 Although SANS experiments do not suffer from this limitation, alternative and more accessible techniques are desirable for routine quantification of magnetically induced alignment of assemblies in solution. Birefringence measurements are a viable and comparatively simple alternative. Analogously to NMR experiments, birefringence measurements reveal valuable information on lipid rearrangements and lipid phases occurring in the bilayer. Moreover, geometric transformations occurring in the polymolecular assembly with changing environmental conditions such as temperature are monitored.11,12,13,16 Magnetically induced birefringence &#916;n&#8242; has been used to study various types of phospholipid systems.13,29,30 Birefringence measurements based on the phase modulation technique in a magnetic field is a viable method to detect orientation of bicelles.12,16,18,29,31,32 The possibility of investigating bicelles with birefringence in high magnetic fields up to 35 T was also demonstrated by M. Liebi et al.13
When polarized light enters an anisotropic material, it will be refracted in an ordinary and extraordinary wave.11 The two waves have different velocities and are shifted in phase by a retardation &#948;. The degree of retardation &#948; is measured and converted into a birefringence signal&#160;<img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg" /> to quantify the degree of anisotropy in the material using
<img alt="Equation 6" src="/files/ftp_upload/56812/56812eq6.jpg" /> (2)
where &#955; is the wavelength of the laser and d is the thickness of the sample. Phospholipids are optically anisotropic and their optical axis coincides with their long molecular axes, parallel to the hydrocarbon tails.11,12 No retardation is measured if the phospholipids are randomly orientated in solution. Retardation is measured when phospholipids are aligned parallel to each other. The magnetically induced birefringence&#160;<img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg" /> can have a positive or negative sign depending on the orientation of the molecules in the magnetic field; see Figure 2. Phospholipids aligned parallel to the x-axis will result in a negative <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg" />, while those aligned along the z-axis result in a positive <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg" />. No birefringence is observed when the optical axis coincides with the direction of light propagation as the phospholipid aligns parallel to the y-axis.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56812/56812fig2.jpg" /> 
Figure 2: Alignment of the phospholipids and corresponding sign of the magnetically induced birefringence <img alt="Equation 12" src="/files/ftp_upload/56812/56812eq12.jpg" />. The sign of the measured <img alt="Equation 12" src="/files/ftp_upload/56812/56812eq12.jpg" /> depends on the orientation of the phospholipid in the magnetic field. Dashed lines indicate the optical axis of the molecule. The light is polarized at 45&#176; and propagates in the y direction. The magnetic field B is in the z direction. This figure has been modified from M. Liebi.11 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56812/56812fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In the case of an isotropic colloidal suspension of bicelles, the orientation induced by the arrangement of the phospholipids in the bilayer will be lost, zeroing the retardation &#948;. The bicelles must also align in order to orientate the optically active phospholipids in their bilayers, causing a retardation &#948; of the polarized light. Consequently, birefringence is a sensitive tool to quantify the magnetic alignability of polymolecular assemblies. Bicelles aligned perpendicular to the magnetic field will yield a positive <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg" />, while those aligned parallel will yield a negative <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg" />. The sign depends on the alignment of the setup and may be checked with a reference sample.

<table><tbody><tr><td>1,2-dimyristoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DMPC)</td><td>Avanti Polar Lipids</td><td>850345P</td><td>&gt;99%</td></tr><tr><td>1,2-dimyristoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexammonium salt (DMPE-DTPA)</td><td>Avanti Polar Lipids</td><td>790535P</td><td>&gt;99%</td></tr><tr><td>Thulium(III) chloride</td><td>Sigma-Aldrich</td><td>439649</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Dysprosium(III) chloride</td><td>Sigma-Aldrich</td><td>325546</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Ytterbium(III) chloride</td><td>Sigma-Aldrich</td><td>439614</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>319988</td><td>contains ethanol as stabilizer, ACS reagent, &amp;ge;99.8%</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>&amp;ge;99.9%</td></tr><tr><td>Cholesterol</td><td>Amresco</td><td>433</td><td>Ultra pure grade</td></tr><tr><td>D2O</td><td>ARMAR chemicals</td><td>1410</td><td>99.8 atom % D</td></tr><tr><td>Ultrapure water</td><td>Millipore</td><td></td><td>Synergy pak2 (SYPK0SIX2), Millipack GP (MPGP02001)</td></tr><tr><td>electronic pH meter</td><td>Metrohm</td><td>17440010</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 100nm membrane filter</td><td>VWR</td><td>515-2028</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 200nm membrane filter</td><td>VWR</td><td>515-2029</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 400nm membrane filter</td><td>VWR</td><td>515-2030</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 800nm membrane filter</td><td>VWR</td><td>515-2032</td><td></td></tr><tr><td>Whatmann Filter paper</td><td>VWR</td><td>230600</td><td></td></tr><tr><td>25 ml round bottom flask</td><td>VWR</td><td>201-1352</td><td>14/23 NS</td></tr><tr><td>3 ml glass snap-cup</td><td>VWR</td><td>548-0554</td><td>ND18, 18x30mm</td></tr><tr><td>2.5 ml glass syringe</td><td>Hamilton</td><td></td><td></td></tr><tr><td>Sodium dihydrogen phosphate dihydrate</td><td>Merk</td><td>1.06342</td><td>Salt used to make phosphate buffer</td></tr><tr><td>di-Sodium hydrogen phosphate</td><td>Merk</td><td>1.06586</td><td>Salt used to make phosphate buffer</td></tr><tr><td>Liquid Nitrogen</td><td>Carbagas</td><td>-</td><td></td></tr><tr><td>Pressurized Nitrogen gas</td><td>Carbagas</td><td>-</td><td>200 bar bottle</td></tr><tr><td>Lipid Extruder 10 ml</td><td>Lipex</td><td>-</td><td>Fully equipped with thermobarrel</td></tr><tr><td>High-pressure PVC tube</td><td>GR NETUM</td><td>-</td><td>must resist more than 4 MPa</td></tr><tr><td>Serto adaptors</td><td>Sertot</td><td>-</td><td></td></tr><tr><td>Nitrile gloves</td><td>VWR</td><td>-</td><td></td></tr><tr><td>2 ml glass pipettes</td><td>VWR</td><td>612-1702</td><td>230 mm long</td></tr><tr><td>Diode Laser</td><td>Newport</td><td>LPM635-25C</td><td></td></tr><tr><td>DSP Dual Phase Lock-in Amplifier</td><td>SRS</td><td>SR830</td><td></td></tr><tr><td>Photodiode Detector</td><td>Silonex Inc.</td><td>SLSD-71N5</td><td>5mm2, Silicon, photo-conductive</td></tr><tr><td>5.5 T Cryogenic Magnetic</td><td>Cryogenic/Oerlikon AG</td><td>-</td><td>12 bar He-cooled. RW4000/6000 compressor, RGD 5/100 TA cryo-head</td></tr><tr><td>Second order low pass filter</td><td>home-built</td><td>-</td><td>Linear power supply 24V DC, second order, Sallen Key, cut-off frequency 360 Hz, +/- 12V, max 10 mA</td></tr><tr><td>Photoelastic modulator</td><td>Hinds instruments</td><td>PEM-90</td><td></td></tr><tr><td>Glan-Thompson Calcite Polarizer</td><td>Newport</td><td>10GT04</td><td>25.4mm diameter</td></tr><tr><td>Quartz sample cuvette</td><td>Hellma</td><td>165-10-40</td><td>temperature controlled cell, 0.8 ml, 10mm path length</td></tr><tr><td>Temperature probe</td><td>Thermocontrol</td><td>-</td><td>Type K, 0.5mm diameter, Thermocoax</td></tr><tr><td>Non-polarizing mirrors</td><td>Newport</td><td>50326-1002</td><td>25.4mm</td></tr><tr><td>RS 232 cables</td><td>National Instruments</td><td>189284-02</td><td>For Connecting to the RS-232 Port on the front of Compact FieldPoint Controllers</td></tr><tr><td>BNC 50 &amp;Omega; cable and connectors</td><td>National Instruments</td><td>763389-01</td><td></td></tr><tr><td>cFP-AI-110</td><td>National Instruments</td><td>777318-110</td><td>8-Channel Analog Voltage and Current Input Module for Compact FieldPoint</td></tr><tr><td>cFP-CB-1</td><td>National Instruments</td><td>778618-01</td><td>Integrated Connector Block for Wiring to Compact FieldPoint I/O</td></tr><tr><td>cFP-CB-3</td><td>National Instruments</td><td>778618-03</td><td>Integrated Isothermal Connector Block for Wiring Thermocouples to the cFP-TC-120 Module</td></tr><tr><td>cFP-TC-120</td><td>National Instruments</td><td>777318-120</td><td>8-Channel Thermocouple Input Module for Compact FieldPoint</td></tr><tr><td>cFP-1804</td><td>National Instruments</td><td>779490-01</td><td>Ethernet/Serial Interface for NI Compact FieldPoint</td></tr><tr><td>LabView 2010</td><td>National Instruments</td><td>-</td><td></td></tr><tr><td>Industrial power supply</td><td>Traco Power</td><td>TCL 060-124</td><td>100-240V AC</td></tr><tr><td>Waterbath</td><td>Julabo</td><td>FP40-HE</td><td>refrigerated/Heating Circulator</td></tr></tbody></table>

fabrication procedures and birefringence measurements for designing magnetically responsive lanthanide ion chelating phospholipid assemblies

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural studies by nuclear magnetic resonance (NMR) to nanotechnological developments including the formation of optically active and magnetically switchable gels. Such technologies require high control of the assembly size, magnetic response and thermal resistance. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its lanthanide ion (Ln3+) chelating phospholipid conjugate, 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA), assemble into highly magnetically responsive assemblies such as DMPC/DMPE-DTPA/Ln3+ (molar ratio 4:1:1) bicelles. Introduction of cholesterol (Chol-OH) and steroid derivatives in the bilayer results in another set of assemblies offering unique physico-chemical properties. For a given lipid composition, the magnetic alignability is proportional to the bicelle size. The complexation of Ln3+ results in unprecedented magnetic responses in terms of both magnitude and alignment direction. The thermo-reversible collapse of the disk-like structures into vesicles upon heating allows tailoring of the assemblies&#39; dimensions by extrusion through membrane filters with defined pore sizes. The magnetically alignable bicelles are regenerated by cooling to 5 &#176;C, resulting in assembly dimensions defined by the vesicle precursors. Herein, this fabrication procedure is explained and the magnetic alignability of the assemblies is quantified by birefringence measurements under a 5.5 T magnetic field. The birefringence signal, originating from the phospholipid bilayer, further enables monitoring of polymolecular changes occurring in the bilayer. This simple technique is complementary to NMR experiments that are commonly employed to characterize bicelles.

The birefringence signal of a non-extruded DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) sample was monitored under a 5.5 T magnetic field during a heating and cooling cycle from 5 to 40 &#176;C and back at a rate of 1 &#176;C/min (Figure 6). The birefringence results confirmed high magnetic alignments at 5 &#176;C with a value of 1.5 x 10-5, twice as strong as for the reported extruded systems.6,7,23 The zeroing of the birefringence signal above the Tm of DMPC at 24 &#176;C was caused by the formation of non-alignable vesicles. The movement appearance of the liquid disordered phase triggered major rearrangements in the polymolecular assemblies. These rearrangements are thermo-reversible. Alignable species were regenerated upon cooling below Tm and the birefringence signal followed the same trend as on heating. The distinct peaks occurring around Tm mark the replacement of the alignable assemblies by non-alignable vesicles.23 The slow kinetics of the molecular rearrangements with respect to the applied heating and cooling rate of 1 &#176;C/min explain why the peaks were not overlapping. Instead, both peaks started at the Tm of DMPC, suggesting that the bilayer lipids must have a certain degree of order to favor the formation of alignable species.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56812/56812fig6.jpg" /> 
Figure 6: Birefringence signal as a function of temperature for a non-extruded DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) sample upon heating (red) and cooling (blue) at 1 &#176;C/min. The sample was prepared following the protocol steps 1 to 1.3.5. The birefringence measurements were conducted following the protocol step 2. The magnetic field strength was ramped up to 5.5 T and the sample was maintained at 5&#176;, achieving a birefringence signal of 1.5 x 10-5 before proceeding to the heating and cooling cycle. The birefringence signal flatlines at temperatures above 35 &#176;C where no alignment was observed as the sample was solely composed of vesicles. Upon cooling, the bicelles were regenerated and a final birefringence signal of 7.2 x 10-6 was achieved at 5.5 T and 5 &#176;C. The magnetic field strength was ramped down to 0 T and the sample was maintained at 5 &#176;C. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56812/56812fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample was hydrated following the heating and cooling procedure in step 1.3.4 and subsequently extruded 10 times at 60 &#176;C through membrane filters of different pore sizes, see step 1.4. At 60 &#176;C, the lipid mixture assembles into vesicles, which are shaped by the extrusion process.16,35,36,37 After completing the extrusion, the bicelles were regenerated by cooling to 5 &#176;C and the hydrodynamic diameter DH was measured by DLS. The magnetic alignability of the bicelles was evaluated at 5 &#176;C by computing the Af with SANS at 8 T and by measuring the birefringence signal at 5.5 T; see Figure 7. The birefringence signal was obtained by ramping the field up to 5.5 T and back down to 0 T as shown in Figure 7A. The peak birefringence occurred at 5.5 T where the highest degree of alignment was expected according to equation 1. The hydrodynamic diameter DH of the bicelles was reduced to 220, 190, 106, and 91 nm by successive extrusions through membranes with pore sizes of 800, 400, 200, and 100 nm respectively. The corresponding decrease in magnetic alignment was confirmed by the decreasing birefringence signal and the reduction in absolute Af as it approached zero in Figure 7B. The results confirmed the possibility of controlling bicelle size and magnetic alignment through tailoring of the vesicles by extrusion at 60 &#176;C and cooling back to 5 &#176;C.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56812/56812fig7.jpg" /> 
Figure 7: Magnetic alignment of a DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample extruded through membrane filters of various pore dimensions. A) Birefringence signal &#916;n&#8242; as a function of the magnetic field strength B when ramping up and down for the sample extruded through 800 nm pores. The peak birefringence was reached at 5.5 T in accordance with equation 1. This maximal birefringence value is reported in B). The same sample was extruded through 400 nm pores. The magnetic alignment was evaluated by both birefringence measurements (black squares) at 5.5 T (analogously to what was done for the previous extrusion step in A) and by computation of alignment factors Af (red circles) at 8 T plotted as a function of the hydrodynamic diameter DH obtained by DLS. The magnetic alignment was evaluated analogously on the same sample extruded through 200 nm pores, and one last time after extruding through 100 nm pores. All measurements were performed at 5 &#176;C. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56812/56812fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies at JoVE.com

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Fabrication procedures for highly magnetically responsive lanthanide ion chelating polymolecular assemblies are presented. The magnetic response is dictated by the assembly size, which is tailored by extrusion through nanopore membranes. The assemblies&#39; magnetic alignability and temperature-induced structural changes are monitored by birefringence measurements, a complimentary technique to nuclear magnetic resonance and small angle neutron scattering.

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural ...

fabrication-procedures-birefringence-measurements-for-designing

Nanyang Technological University

Research

JoVE Journal

Engineering

7.2K Views.  ETH Zurich. Fabricage procedures voor zeer magnetisch responsieve lanthanide ion chelaat polymolecular assembly's worden gepresenteerd. De magnetische reactie wordt bepaald door de grootte van de vergadering, die is afgestemd door extrusie via nanopore membranen. De assemblages magnetische alignability en temperatuur-geïnduceerde structurele veranderingen worden gecontroleerd door de dubbele breking metingen, een gratis techniek om nucleaire magnetische resonantie en kleine hoek neu...

Video: Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Biochemistry

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for the purpose of investigating phase change of those liquid segments. The magnetic beads were externally controlled using a magnet, allowing for the beads to bridge the air valve between the adjacent liquid segments. A hydrophobic coating was applied to the inner surface of the tube to enhance the separation between two liquid segments. The applied magnetic field formed an aggregate cluster of magnetic beads, capturing a certain liquid amount within the cluster that is referred to as carry-over volume. A fluorescent dye was added to one liquid segment, followed by a series of liquid transfers, which then changed the fluorescence intensity in the neighboring liquid segment. Based on the numerical analysis of the measured fluorescence intensity change, the carry-over volume per mass of magnetic beads has been found to be ~2 to 3 &#181;l/mg. This small amount of liquid allowed for the use of comparatively small liquid segments of a couple hundred microliters, enhancing the feasibility of the device for a lab-in-tube approach. This technique of applying small compositional variation in a liquid volume was applied to analyzing the binary phase diagram between water and the surfactant C12E5 (pentaethylene glycol monododecyl ether), leading to quicker analysis with smaller sample volumes than conventional methods.

Here, we present a protocol to investigate multi-component phase diagrams using externally controlled magnetic beads as liquid carriers in a lab-in-tube approach. This approach can aid in applications that seek to gather further information on phase change in complex liquid systems.

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Chemistry

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengineering

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 ...

Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals

Materials showing coupling phenomena between magnetism and (ferro)electricity, i.e., magnetoelectric effects, have attracted a great deal of attention due to their potential applications for future device technologies such as sensors and storage. However, conventional approaches, which usually utilize materials containing magnetic metal ions (or radicals), have a major problem: only a few materials have been found to show the coupling phenomena at room temperature. Recently, we proposed a new approach to achieve room-temperature magnetoelectrics. In contrast to conventional approaches, our alternative proposal focuses on a completely different material, a &#34;liquid crystal&#34;, free from magnetic metal ions. In such liquid crystals, a magnetic field can be utilized to control the orientational state of constituent molecules and the corresponding electric polarization through magnetic anisotropy of the molecules; it is an unprecedented mechanism of the magnetoelectric effect. In this context, this paper provides a protocol to measure ferroelectric properties induced by a magnetic field, that is, the direct magnetoelectric effect, in a liquid crystal. With the method detailed here, we successfully detected magnetically-tuned electric polarization in the chiral smectic C phase of a liquid crystal at room temperature. Taken together with the flexibility of constituent molecules, which directly affects the magnetoelectric responses, the introduced method will serve to allow liquid crystal cells to acquire more functions as room-temperature magnetoelectrics and associated optical materials.

In this report, we present a protocol to examine direct magnetoelectric effects, i.e., induction of ferroelectric polarization by applying magnetic fields, in liquid crystals. This protocol provides a unique approach, supported by the softness of liquid crystals, to achieve room-temperature magnetoelectrics.

Materials showing coupling phenomena between magnetism and (ferro)electricity, i.e., magnetoelectric effects, have attracted a great deal of attention due ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Samenvatting

Meer video's verkennen