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Method Article

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

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DOI:

10.3791/56812

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January 3rd, 2018

 ,  ,  ,  ,  , 
1Laboratory of Food Process Engineering, ETH Zurich, 2MAX IV Laboratory, Lund University

In This Article

Summary

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' magnetic alignability and temperature-induced structural changes are monitored by birefringence measurements, a complimentary technique to nuclear magnetic resonance and small angle neutron scattering.

Abstract

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' dimensions by extrusion through membrane filters with defined pore sizes. The magnetically alignable bicelles are regenerated by cooling to 5 °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.

Introduction

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'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,

Static equilibrium equation E_mag=-nΔχB²/2μ₀Nₐ, diagram for educational use. (1)

where B is the magnetic field strength, Magnetic permeability equations μ₀=4π×10⁻⁷; key formula in a physics concept chart. the magnetic constant, n the aggregation number and Δχ in static equilibrium equation; ΣFx=0; method diagram; educational use. 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 Δχ. 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 Δχ 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 °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

Lipid film hydration and extrusion diagram; DMPC, cholesterol; temperature effects; vesicle formation.
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&C). H&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. Please click here to view a larger version of this figure.

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 Δn′ 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 δ. The degree of retardation δ is measured and converted into a birefringence signal Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram. to quantify the degree of anisotropy in the material using

Refractive index change equation Δn'=-δλ/2πd; formula for optical measurement analysis. (2)

where λ 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 Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram. 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 Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram., while those aligned along the z-axis result in a positive Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram.. No birefringence is observed when the optical axis coincides with the direction of light propagation as the phospholipid aligns parallel to the y-axis.

Static equilibrium diagram with light source, magnetic field (B), Brownian motion, Δn' equations.
Figure 2: Alignment of the phospholipids and corresponding sign of the magnetically induced birefringence Δn' symbol, refractive index change concept in optical studies.. The sign of the measured Δn' symbol, refractive index change concept in optical studies. 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° and propagates in the y direction. The magnetic field B is in the z direction. This figure has been modified from M. Liebi.11 Please click here to view a larger version of this figure.

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 δ. The bicelles must also align in order to orientate the optically active phospholipids in their bilayers, causing a retardation δ 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 Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram., while those aligned parallel will yield a negative Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram.. The sign depends on the alignment of the setup and may be checked with a reference sample.

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Protocol

1. Fabrication procedure for DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) and DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) polymolecular assemblies

  1. Preliminary preparations
    1. Wash all the glassware by flushing once with ethanol stabilized chloroform (>99% chloroform) and dry with compressed air.
    2. Produce 2 distinct 10 mg/mL stock solutions of DMPC and DMPE-DTPA in ethanol-stabilized chloroform (>99% chloroform), a 10 mM stock solution of Chol-OH in ethanol-stabilized chloroform (>99% chloroform) and a 10 mM stock solution of TmCl3 in methanol.
    3. Prepare a 50 mM phosphate buffer at a pH value of 7.4 by mixing 0.121 g of sodium dihydrogen phosphate dihydrate and 0.599 g of anhydrous di-sodium hydrogen phosphate in 100 mL of ultrapure H2O.
  2. Preparation of the dry lipid film
    1. Weigh the required amounts of amphiphiles (DMPC, DMPE-DTPA, and optionally Chol-OH) and Ln3+ stock solutions in separate 3 mL glass snap-cups with a 2.5 mL glass syringe.
      1. For a 3 mL sample volume of DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1, total lipid concentration of 15 mM), weigh in 3.6435 g of the DMPC stock solution, 1.4731 g of the DMPE-DTPA stock solution and 0.7126 g of the TmCl3 stock solution.
      2. For a 3 mL sample volume of DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5, total lipid concentration of 15 mM), weigh in 2.9148 g of the DMPC stock solution, 1.4731 g of the DMPE-DTPA stock solution, 1.0749 g of Chol-OH stock solution and 0.7126 g of the TmCl3 stock solution.
        CAUTION: Chloroform and methanol are toxic and volatile at room temperature. Work under a fume hood and promptly proceed with the mass measurements.
    2. Transfer the content of the snap-cups to a 25 mL round bottom flask. Flush each snap-cup into the round bottom flask with about 2.5 mL of the corresponding solvent.
    3. Remove the solvent under vacuum in a rotary evaporator at 40 °C. Set the initial pressure to 30 000 Pa until most of the solvent is removed. Reduce the pressure to 100 Pa and dry the sample under rotation for a minimum of 2 h. Obtain a uniform dry lipid film on the glass walls of the flask.
    4. Place the dry lipid film for 1 minute under a steady stream of argon to avoid lipid oxidation in air and store the sample in the freezer prior to rehydration.
  3. Hydration of the dry lipid film
    1. Add the 3 mL of phosphate buffer to the round bottom flask to reach a total lipid concentration of 15 mM.
    2. Carry out a freeze-thawing (FT) cycle by plunging the flask under rotation in liquid nitrogen until it is thoroughly frozen (the liquid nitrogen stops boiling), then heat back up to 60 °C by placing the sample for 5 min in a water bath, swirling the flask continuously to aid the melting process. Apply 30 s of vortexing before each freezing cycle when the sample is liquid to aid the hydration of the lipid film.
      NOTE: No lipid film should be visible on the flask walls after the second freeze thawing cycle.
    3. Repeat 1.3.2 a total of five times. Close the flask with a cap to avoid unnecessary evaporation of the phosphate buffer when the sample is hot. The protocol may be paused when the sample is frozen.
    4. Proceed to two heating and cooling (H&C) cycles to stabilize the sample coming out of the last freezing step, or keep frozen for up to two months. Heat the sample to 40 or 60 °C for DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) or DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5), respectively, before cooling to 5 °C at 1 °C/min. Maintain the sample 5 min at the maximum and minimum temperatures of the cycle.
    5. Now, either determine the birefringence signal of the sample in an external magnetic field (step 2) or further extrude the sample to tailor the bicelle dimensions and magnetic alignability (step 1.4).
      Note: DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) samples are mainly composed of bicelles with a mean hydrodynamic diameter DH of 70 nm as revealed by a number distribution obtained from dynamic light scattering (DLS) measurements at 5 °C. These samples also contain larger polymolecular assemblies with a mean DH of 500 nm as revealed by an intensity distribution. DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) samples are highly polydisperse in size with typical intensity distributions revealing a mean DH of 700 nm, while the number distributions reveal a population dominated by smaller bicelles in the size range of 200 nm. More detailed size distributions and cryo transmission electron microscopy images of these samples have been reported by Isabettini et al.23
  4. Extrusion of the polymolecular assemblies.
    1. Assemble the extruder as shown in Figure 3. Use gloves and tweezers with protecting silica tubes for handling. Wet the filter paper (5) with a few drops of buffer to allow for an optimal placement of the membrane filter (6). Make sure the paper has no folds after placing the o-ring (7) on top.
      NOTE: The extrusion process was tested on membrane filters (6) with a pore diameter of 800, 400, 200 and 100 nm; see Figure 7.
    2. Set the water bath to 40 °C for DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) samples or 60 °C for DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) samples to guarantee the formation of extrudable vesicles.
    3. Connect the extruder to a pressurized nitrogen bottle using a high-pressure PVC tube (> 4 MPa) equipped with serto adaptors and extrude the liquid material through the membrane. 1 MPa of pressure is usually required for extrusion through membrane filters (6) with a pore diameter of 200 nm and above. 1.5-2.5 MPa are required for the smaller membrane filters (6) with a pore diameter of 100 nm.
      NOTE: Change the membrane filter if abnormally high pressures (>2.5 MPa) are required to extrude the sample (this is the first sign of clogging).
    4. Open the cover (10) and insert the sample using a 2 mL glass pipette. Then close the cover (10) and open the pressure valve (12) while holding the sample outlet tube (2). Close the pressure valve (12) after the extrusion cycle is completed, vent and continue with the next cycle.
      NOTE: Do not leave the sample in contact too long with the hot jacketed vessel (8) to avoid excessive sample loss by evaporation. 30-60 s is enough time for a 3 mL sample to equilibrate in the extruder before opening the pressure valve (12).
    5. Proceed to 10 extrusion cycles for a given membrane pore dimension as shown in Figure 3. Most of the bicelle systems are extruded 10 times through membranes with a pore diameter of 200 and another 10 times through membranes with a pore diameter of 100 nm, guaranteeing sample comparability.
    6. Now, determine the birefringence signal of the sample in an external magnetic field (step 2).

Filter press setup for solid-liquid separation; includes filter discs, valves; schematic diagram.
Figure 3: Laboratory extruder used for bicelle and vesicle preparations. The extruder is assembled from the bottom up: (1) mount, (2) sample collecting space with a 2.4 mm (inner diameter) plastic outlet tube and o-ring, (3) and (4) large and small stabilizing mesh, (5) filter paper, (6) membrane filters, (7) o-ring, (8) jacketed vessel, (9) top cover with inlet and pressure connection, (10) cover, (11) butterfly screws, (12) pressure valve. A sketch of the assembled extruder is shown of the right-hand-side. The nitrogen gas (N2) is supplied by a pressure vessel and the jacketed vessel (9) is connected to a water bath for temperature control. The sample undergoes 10 extrusion cycles for any given membrane filter pore diameter (sample path shown in blue). Please click here to view a larger version of this figure.

2. Birefringence measurements of DMPC/DMPE-DTPA/Tm3+ (molar ratio 4:1:1) and DMPC/Chol-OH/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) polymolecular assemblies.

  1. Build and connect the birefringence setup as presented in Figure 4 and supply power to the respective electronic elements. Do not place the PEM, the sample, and the second polarizer in the laser path at this stage. Avoid detecting back-scattered laser light by covering reflecting surfaces, for example aluminum mirror holders, with black paper.
  2. Adjust the mirrors to maximize the laser intensity at the detector, which is represented by the intensity of the direct current Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis. obtained from the low pass filter in Figure 4B.
    CAUTION: Wear appropriate eye protection when adjusting the mirrors and consult with a laser safety instructor if manipulating lasers for the first time.
  3. Turn the first crossed linear polarizer (maintained perpendicular to the incident laser beam) to maximize the Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis..

Polarimetry setup; optical diagram A, electronic flowchart B, equipment photo C; studies birefringence.
Figure 4: Schematic representation of the birefringence setup and connections for the optical signals. A) A superconducting electromagnet supplies a 5.5 T magnetic field. The light from a diode laser at 635 nm is polarized by two crossed polarizers. A photoelastic modulator PEM-90 operates at 50 KHz with an amplitude A0 of 2.405 rad and placed between the two polarizers. The sample lies in the magnet between the PEM and the second polarizer. Non-polarizing mirrors guide the light through the different elements and is finally detected by a photo detector. The first and second harmonic Optical excitation formula \(I_{1\omega}\) in photonics equation diagram for spectroscopy analysis. and Optical excitation process, I<sub>2ω</sub> formula, related to photonic studies, diagram. of the AC signal are monitored, permitting the calculation of the birefringence signal giving information on the magnetic alignability of the Ln3+ chelating polymolecular assemblies. The sample cuvette is connected to an external water bath for temperature control (blue). The temperature of the sample is monitored with a temperature probe (red). B) The signal from the photo detector is fed into a second order Sallen-Key low pass filter (24 V AC power supply) with a cut-off frequency of 360 Hz through a ±12 V DC power supply braded cable (3). The low pass filter extracts the DC component Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis. and delivers it to the PC-interface (4) through a BNC 50 Ω cable. The signal from the photo detector is delivered to the two lock-in amplifiers (which extract the first and second harmonic Optical excitation formula \(I_{1\omega}\) in photonics equation diagram for spectroscopy analysis. and Optical excitation process, I<sub>2ω</sub> formula, related to photonic studies, diagram.) through a BNC 50 Ω cable (1) & (2). The harmonic intensities are detected by a phase-sensitive detection. Consequently, the PEM signal is used as reference signal for the lock-in amplifiers (1f-output of the PEM into the first lock-in amplifier and 2f-output into the second, connected with BNC 50 Ω cables). The output signals are delivered to the PC-interface unit through BNC 50 Ω cables. Analog acquisition units cFP-AI-110 and cFP-CB-1 digitalize the signal that is transferred to the computer through a RS 232 cable for monitoring. The type K temperature probe is also connected to the PC-interface unit where analog acquisition units cFP-CB-3 and cFP-TC-120 digitalize the signal before transferring it to the computer through a RS 232 cable for monitoring. C) Picture of the schematic setup presented in B. Key elements are identified with corresponding numbers from 1 to 4. Please click here to view a larger version of this figure.

  1. Place the second crossed linear polarizer perpendicular to the incident laser beam, as shown in Figure 4A. Minimize the Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis. by setting the second polarizer at a 90° angle with respect to the first.
  2. Place the photoelastic modulator (PEM) at 0° between the two crossed linear polarizers and perpendicular to the incident laser beam as shown in Figure 4A. Adjust the PEM to a frequency of 50 kHz and amplitude A0 to 2.405 rad as shown in Figure 5A. This makes the DC component independent of birefringence and boosts the Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis..
    NOTE: The optical axis of the PEM may be tuned by a few degrees to maintain a constant Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis. in air before measuring any sample.
  3. Wait for 1 h after turning on the laser and electronic equipment to stabilize the signal. The signal is stable once the auto-phasing of the lock-in amplifiers remains constant.
  4. Place the sample in a temperature controlled quartz cuvette with a path-length of 10 mm and connect it to an external water bath initially set at 5 °C.
  5. Place a 0.5 mm thick type K thermocouple (temperature probe) directly in the sample to monitor the temperature of the sample. Verify that the probe does not interfere with the laser light by placing a white paper in the laser path (after the cuvette) and looking for shadows caused by the probe.
    NOTE: There is a 2-3 °C difference between the temperature recording of the water bath and the temperature of the sample.
  6. Place the cuvette in the bore of the magnet, as shown in Figure 4A. The laser light propagates horizontally through the sample, is deflected by non-polarizing mirrors and detected by a photo detector.
    NOTE: The laser is directed down, through the sample, and back up the same path to account for Faraday effects (i.e. the rotation of the polarization plane of light caused by the magnetic field when going down is canceled when coming back out in the opposite direction).
  7. Apply a steady air flow of compressed air at room temperature and 10000 Pa on the cuvette to avoid condensation of water on the cell walls, which would reduce the intensity of the signal and increase noise. This is especially important when measuring at 5 °C.
  8. Detect the first and second harmonic Optical excitation formula \(I_{1\omega}\) in photonics equation diagram for spectroscopy analysis. and Optical excitation process, I<sub>2ω</sub> formula, related to photonic studies, diagram. of the AC signal with two lock-in amplifiers. Auto phase the lock-in amplifiers by pressing the button (2) shown in Figure 5B and adjust the sensitivity as shown in Figure 5B (1). Make sure there are not more than four red bars on the amplifiers as shown in Figure 5B (3) to avoid signal overload. Note down the employed sensitivity for both lock-in amplifiers in the program Tesla_Magnet_Const_V092 as shown in Figure 5C (8). The program is provided as supplementary information.
  9. Ramp the magnetic field up to 5.5 T by supplying current to the magnet through the program Tesla_Magnet_Const_V092 as shown in Figure 5C (5).
  10. Obtain the birefringence Δn', refractive index change, optical study, formula, photonics, spectral fitting diagram. using equation 2, where the retardation is calculated with
    Static equilibrium; formula δ=arctan(...) in polarimetry method; equation for optical analysis. (3)
    where static equilibrium example, ΣFx=0, diagram, illustrating force balance principles and Static equilibrium diagram; ΣFx=0, ΣFy=0; force and torque balance illustration. are Bessel functions of the first kind, with Bessel function equation J₁(2.405)=0.5191, mathematical analysis, formula. and Bessel function equation, J2(2.405) = 0.4317, mathematical analysis, formula illustration..11,13,18,33,34 Plot the retardation in the program Tesla_Magnet_Const_V092, as shown in Figure 5C (4).
    NOTE: The retardation provided by the program should not be used to calculate the birefringence signal if the two lock-in amplifiers are not operating at the same sensitivity (see step 2.12). The logged harmonic intensities Optical excitation formula \(I_{1\omega}\) in photonics equation diagram for spectroscopy analysis. and Optical excitation process, I<sub>2ω</sub> formula, related to photonic studies, diagram. have to be multiplied by the sensitivity of the lock-in amplifiers to get the correct dimensions. Moreover, the birefringence signal measured under a magnet field must be normalized by subtracting the mean birefringence signal obtained at 0 T.
  11. Monitor the sample's birefringence signal at constant or changing temperature (1 °C/min) by regulating the temperature of the water bath connected to the cuvette shown in Figure 4.
  12. Log the experimental data by filling in the experimental description in Figure 5C (8), providing a file name in (9), and pressing the "START log" button (10).

Photelastic modulator setup, Tesla magnet control interface, measuring optical properties, data display.
Figure 5: Illustrations of the employed settings and program screenshots. A) PEM settings: retardation 2.405 rad, wavelength 635 nm, frequency 50 Hz. White circles indicate what settings to be activated (USR = user defined retardation, LOC = local mode of operation). B) Lock-in amplifier settings. The sensitivity (1) has to be selected before each measurement as required in step 2.11. There should not be more than four red bars on the display (3) to avoid a signal overload. An overload occurs when the red led in (1) turns on, making a measurement impossible. Press the auto phase button (2) before every measurement. C) Screenshots of the program Tesla_Magnet_Const_V092 provided as supplementary information. The program allows control of the magnetic field and recording of all the signal outputs as a function of time. The magnetic field strength and sample temperature are plotted in (1). The first and second harmonic Optical excitation formula \(I_{1\omega}\) in photonics equation diagram for spectroscopy analysis. and Optical excitation process, I<sub>2ω</sub> formula, related to photonic studies, diagram. of the AC signal measured by the two lock-in amplifiers are plotted in (2). The intensity of the direct current Static equilibrium formula, ΣFx=0, digital diagram for educational reference and analysis. is plotted in (3). The retardation is calculated as described in step 2.13 and plotted in (4). The magnetic field strength is set in (5). The direct measurement of the temperature recorded by the Type K thermocouple is presented in (6) and the output signals (Static equilibrium equations ΣFx=0, MA=0, diagram, educational keywords, stress analysis. and Nonlinear optics concept, symbol \(I_{2\omega}\), relating to frequency doubling processes.) in (7). Additional sample information may be inserted in (8) such as the employed sensitivity of the amplifiers, sample name, etc. The data may be logged and exported to a .txt file provided in (9). Start and stop the data acquisition with the "START log" button (10). Please click here to view a larger version of this figure.

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Results

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 °C and back at a rate of 1 °C/min (Figure 6). The birefringence results confirmed high magnetic alignments at 5 °C with a value of 1.5 x 10-5, twice as strong as for the reported extruded systems.6,7

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Discussion

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,

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)Avanti Polar Lipids850345P>99%
1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexammonium salt (DMPE-DTPA)Avanti Polar Lipids790535P>99%
Thulium(III) chlorideSigma-Aldrich439649anhydrous, powder, 99.9% trace metals basis
Dysprosium(III) chlorideSigma-Aldrich325546anhydrous, powder, 99.9% trace metals basis
Ytterbium(III) chlorideSigma-Aldrich439614anhydrous, powder, 99.9% trace metals basis
ChloroformSigma-Aldrich319988contains ethanol as stabilizer, ACS reagent, ≥99.8%
MethanolSigma-Aldrich34860≥99.9%
CholesterolAmresco433Ultra pure grade
D2OARMAR chemicals141099.8 atom % D
Ultrapure waterMilliporeSynergy pak2 (SYPK0SIX2), Millipack GP (MPGP02001)
electronic pH meterMetrohm17440010
Whatmann Nuclepore 25 mm 100nm membrane filterVWR515-2028
Whatmann Nuclepore 25 mm 200nm membrane filterVWR515-2029
Whatmann Nuclepore 25 mm 400nm membrane filterVWR515-2030
Whatmann Nuclepore 25 mm 800nm membrane filterVWR515-2032
Whatmann Filter paperVWR230600
25 ml round bottom flaskVWR201-135214/23 NS
3 ml glass snap-cupVWR548-0554ND18, 18x30mm
2.5 ml glass syringeHamilton
Sodium dihydrogen phosphate dihydrateMerk1.06342Salt used to make phosphate buffer
di-Sodium hydrogen phosphateMerk1.06586Salt used to make phosphate buffer
Liquid NitrogenCarbagas-
Pressurized Nitrogen gasCarbagas-200 bar bottle
Lipid Extruder 10 mlLipex-Fully equipped with thermobarrel
High-pressure PVC tubeGR NETUM-must resist more than 4 MPa
Serto adaptorsSertot-
Nitrile glovesVWR-
2 ml glass pipettesVWR612-1702230 mm long
Diode LaserNewportLPM635-25C
DSP Dual Phase Lock-in AmplifierSRSSR830
Photodiode DetectorSilonex Inc.SLSD-71N55mm2, Silicon, photo-conductive
5.5 T Cryogenic MagneticCryogenic/Oerlikon AG-12 bar He-cooled. RW4000/6000 compressor, RGD 5/100 TA cryo-head
Second order low pass filterhome-built-Linear power supply 24V DC, second order, Sallen Key, cut-off frequency 360 Hz, +/- 12V, max 10 mA
Photoelastic modulatorHinds instrumentsPEM-90
Glan-Thompson Calcite PolarizerNewport10GT0425.4mm diameter
Quartz sample cuvetteHellma165-10-40temperature controlled cell, 0.8 ml, 10mm path length
Temperature probeThermocontrol-Type K, 0.5mm diameter, Thermocoax
Non-polarizing mirrorsNewport50326-100225.4mm
RS 232 cablesNational Instruments189284-02For Connecting to the RS-232 Port on the front of Compact FieldPoint Controllers
BNC 50 Ω cable and connectorsNational Instruments763389-01
cFP-AI-110National Instruments777318-1108-Channel Analog Voltage and Current Input Module for Compact FieldPoint
cFP-CB-1National Instruments778618-01Integrated Connector Block for Wiring to Compact FieldPoint I/O
cFP-CB-3National Instruments778618-03Integrated Isothermal Connector Block for Wiring Thermocouples to the cFP-TC-120 Module
cFP-TC-120National Instruments777318-1208-Channel Thermocouple Input Module for Compact FieldPoint
cFP-1804National Instruments779490-01Ethernet/Serial Interface for NI Compact FieldPoint
LabView 2010National Instruments-
Industrial power supplyTraco PowerTCL 060-124100-240V AC
WaterbathJulaboFP40-HErefrigerated/Heating Circulator

References

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Magnetic AssembliesLipid ExtrusionDMPC DMPE DTPACholesterol Doped BicellesMagnetic Field AlignmentVesicle Precursor FormationTemperature Controlled AssemblyPolymolecular Assembly Design
Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies
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