Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Summary

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Abstract

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force.

The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

Introduction

Atomic Force Microscopy (AFM) generates an image of a surface by scanning across an area of the sample using a cantilever with a very sharp tip¹. The movement of the cantilever probes the surface topology of the sample. AFM has been widely applied to biological molecules — including proteins, DNA, and membranes, owing to its versatility in analyzing fixed samples in air or near-native state in liquid^2-5.

Apart from its high-resolution imaging capability in the nanometer range, the AFM cantilever acts as a spring to probe interaction forces (adhesion and repulsion) and mechanical properties of the sample^5,6. This is known as force spectroscopy. In this mode, the probe first approaches the sample and exerts a force on it, then is retracted until it loses contact with the sample (Figure 1A). The generated curves show force as a function of distance of the cantilever for both the approach and retraction. Several properties including the elastic modulus to measure the stiffness of a material, and adhesion forces can be derived.

Supported lipid bilayers are biological model membranes lying on top of a solid support — usually mica, borosilicate glass, fused silica, or oxidized silicon⁷. They are prepared using various techniques like vesicle deposition, Langmuir-Blodgett method and spin-coating^8,9. AFM imaging has been used to follow the formation of these supported bilayers¹⁰, and probe different structures formed by membranes of different compositions^11-15.

Performing force spectroscopy on supported bilayers results in a peak in the approach curve. This peak indicates the force needed to pierce the bilayer, and is called breakthrough force. The bilayer thickness can also be measured using the force curve⁶ . The typical breakthrough force of bilayers range between 1-50 nN⁶. These properties depend on lipid packing (liquid or gel phase) and structure (acyl chain length and degree of unsaturation) and altered by membrane-active agents¹⁶. The theory behind the rupture has been explained¹⁷ and other experimental parameters such as cantilever softness, tip radius and approach speed also affect the breakthrough force^15,16,18. Force spectroscopy has been used to analyze properties of different lipid phases^11,19, composition-dependent changes^12,20, as well as effects of other biomolecules, like peptides, on the stability of the membrane²¹.

The flat orientation of supported bilayers is advantageous for combining AFM with other methods such as surface plasmon resonance²² and fluorescence microscopy^11,19 to better characterize structure and properties of membranes.

This detailed video protocol is intended to prepare supported lipid bilayers using vesicle deposition and analyze them with AFM and force spectroscopy. While vesicles of various sizes may be used to prepare bilayers, this protocol focuses on small and large unilamellar vesicles. Supported bilayers that phase separate into liquid ordered (L_o) and liquid disordered (L_d) phases were characterized^11,15. The membrane is composed of di-oleoyl-phosphatidylcholine (DOPC), sphingomyelin (SM), and cholesterol (Chol) at 2:2:1 ratio. This composition models the lipid rafts, which are proposed to behave as platforms important for protein trafficking and sorting, cell signaling and other cellular processes^23,24.

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Protocol

1. Preparation of Supported Lipid Bilayers (SLB)^11,12,21

1. Preparation of Lipid Mixture and Multilamellar Vesicle Suspensions
   1. Prepare the following buffers beforehand.
      1. Prepare PBS buffer at concentrations of 2.7 mM KCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, and 137 mM NaCl, pH 7.2.
      2. Prepare SLB (supported lipid bilayer) buffer at concentrations of 150 mM NaCl, 10 mM HEPES, pH 7.4.
      3. Prepare a solution of 1 M CaCl₂.
   2. Dissolve the following lipids in chloroform at a desired concentration: for example, di-oleoyl-phosphatidylcholine (DOPC) at 25 mg/ml, sphingomyelin (SM) at 25 mg/ml and cholesterol (Chol) at 10 mg/ml.
   3. Take 18.38 µl of DOPC, 17.10 µl of SM and 11.30 µl of Chol to make a solution with 1 mg of total lipids in a 2:2:1 DOPC:SM:Chol molar ratio. Vary the volumes accordingly based on the concentrations of the lipids prepared in 1.1.2. However, maintain the molar ratio at 2:2:1 DOPC:SM:Chol, as changing the molar ratio will change the phase characteristics of the membrane²⁵.
   4. Mix the solution completely by vortexing. Add a fluorescent lipidic dye at 0.05 mol % if one wishes to visualize the bilayer by a fluorescence microscope.
      Note: The family of long-chain dialkylcarbocyanines, like DiD, DiI and DiO span a wide range of wavelengths and can be ideally used to label lipid membranes. Alternatively, fluorescently labeled lipids, like rhodamine-phosphatidylethanolamine or BODIPY-cholesterol can be used. In any case, the concentration of the dye should be optimized according to the microscopy setting, keeping in mind that high concentrations of the fluorophore bound to the lipid can change the lipid behavior¹⁹.
   5. Dry off the chloroform using N₂ gas (or any available inert gas such as Ar and He).
   6. Further dry the lipid mixture under vacuum for at least 1 hr.
      Note: If storing this lipid mixture, purge air with inert gas (N₂, Ar or He) and store at -20 °C. Aliquot excess lipids-in-chloroform into small brown vials. Dry them in a similar manner as the lipid mixture, displace air with inert gas (N₂, Ar or He) and store at -20 °C.
   7. Dissolve the dried lipids in PBS buffer to a concentration of 10 mg/ml.
   8. Vortex the mixture vigorously for about 20 sec to 1 min to get a cloudy suspension, containing multilamellar vesicles. Make sure that the there are no lipid films sticking to the sides of the vial.
   9. Distribute this suspension into 10 µl aliquots (1 mg of lipid makes 10 aliquots).
   10. Store at -20 °C until further use.
2. Preparation of Unilamellar Vesicles
   Note: Various sizes of unilamellar vesicles can be prepared using the sonication method or the extrusion method. Sonication uses sound energy to agitate the mixture and separate the layers of the multilamellar suspension. This is indicated by clearing up of the hazy suspension. Extrusion forces the multilamellar vesicles to pass through a membrane filter with a definite pore size.
   1. Sonication Method
      1. Take the 10 µl of multilamellar lipid suspensions and add 150 µl of SLB buffer.
      2. Transfer the mixture into small (2 ml) capped glass vials and seal with Parafilm.
      3. Sonicate the mixture at room temperature in a bath sonicator for 10 min or until it becomes clear to yield small unilamellar vesicles (SUVs, below 50 nm in diameter).
   2. Extrusion Method
      1. Take the 10 µl of multilamellar lipid suspensions and add 150 µl of SLB buffer.
      2. Transfer the suspension into a cryogenic tube.
      3. Freeze the lipid suspension by submerging the cryogenic tube for a few seconds in liquid nitrogen.
      4. Thaw the lipid suspension by submerging the cryogenic tube in a water bath at room temperature or 37 °C for a few minutes. Repeat the freeze-thaw steps at least 10 times.
      5. In order to extrude the lipid suspension, use a polycarbonate membrane with 200 nm pore size (other pore sizes are available like, 100 nm or 400 nm) and a commercial extrusion device²⁶.
         Note: Currently, there are two extruder devices commercially available: a manual extruder is available for small volumes (200 µl to a few ml). In this case, the sample is extruded through the membrane by manually pushing the suspension back and forth between two syringes. Depending on the manufacturer, lipid aliquots would need to be combined to reach minimum volume of the set up. For bigger volumes (up to 50 ml) more sophisticated devices are used in which the suspension is forced through the polycarbonate membrane by using compressed gas. Set-up and extrusion conditions can change according to the model. Refer to the instructions from the manufacturer before use. This protocol refers to the manual extruder for small volumes.
      6. Equilibrate the extruder in water by passing water through the extruder. Immerse the membrane in water.
      7. Place the membrane between the two pistons, assemble the device and equilibrate in buffer, by passing the buffer through the extruder, from one syringe to the other. This step allows additionally testing the leakiness of the system. If it leaks, disassemble and reassemble it again changing the membrane.
      8. Take the lipid suspension using one syringe of the extruder ('dirty side').
      9. Attach the syringe containing the lipid suspension on one chamber and the empty syringe on the opposing chamber.
      10. Push the syringe on one end. Pass the solution through the membrane and into the opposing syringe. This is the 1^st pass.
      11. Pass it through the membrane for a total of 31 times such that the solution ends up on the opposing syringe ('clean side').
          Note: Check the membrane after extruding. If it was broken, then extrusion process was not successful and needs to be repeated.
      12. Empty the syringe into a small tube. Large unilamellar vesicles (LUVs) are stable for 1-2 days if kept at 4 °C.
3. Preparation of Mica and Coverslips
   1. Wash coverslips first in water and then in ethanol. Air dry.
   2. Take mica discs and peel off the outer layer using adhesive tape.
   3. Attach the mica disc onto the coverslip. Transparent optical glues are optimal so that one can check the bilayers with a fluorescence microscope.
   4. Attach plastic cylinders (2.5 cm outer diameter, 2 cm inner diameter, and 0.5 cm height) using optical glue/grease. Make sure that everything is sealed and that there are no leaks. Use grease when wanting to re-use the cylinders as they can be easily detached from the coverslip and washed. The dimensions of the plastic cylinders depend on the size of the AFM cantilever holder and should be adjusted accordingly.
4. Deposition of Unilamellar Vesicles on Solid Support
   1. Pipet the whole liposome solution directly onto the mica. Add more SLB buffer to reach a volume of 300 µl.
   2. Add 0.9 µl of 1 M calcium chloride solution to reach a final concentration of 3 mM Ca²⁺.
   3. Incubate at 37 °C for 2 min and then at 65 °C for at least 10 min. Always cover the bilayers with buffer. Contact with air and air bubbles will destroy it. During incubation, add more buffer if necessary, especially if incubating at higher temperatures.
      ​Note: Incubation temperatures vary according to the lipid mixtures and phases required. Incubation time of at least 10 min is needed to form supported bilayers, but it could be longer, depending on the temperature and composition.
   4. Wash the bilayer with hot (65 °C) SLB buffer 15-20 times to remove calcium and unfused vesicles. Take extra care during the washing steps to keep the bilayer under buffer and gently pipet the washing solution to avoid destroying the bilayer.
   5. Cool supported bilayers to room temperature before AFM measurements. This is needed to form the different phases of the membrane as well as minimize thermal drift in the instrument.

2. Atomic Force Microscopy Measurements^11,12

1. Imaging
   Note: Perform atomic force microscopy imaging in contact mode or tapping mode. Image supported bilayers in solution; do not allow solution to evaporate completely as this will destroy the bilayers. As one would be working with buffer, please observe safety precautions in making sure that any AFM part is protected from liquid spills.
   1. Select a soft cantilever (below 0.2 N/m spring constant is advised) and mount it to the AFM. The choice of cantilever stiffness is more critical for force spectroscopy (and this will be discussed later).
   2. Mount the sample on the AFM stage and make sure that all vibration-isolation modules are turned on. Wait for at least 15 min to equilibrate and reduce thermal drift of the system. Depending on the AFM set-up, tuning and imaging specifications may vary. Consult the manufacturer’s handbook.
   3. Approach the sample with the AFM tip until contact.
   4. Since lipid bilayers are usually 4 nm in height, use the Z-range of the instrument that will give the highest z-resolution (for example 1.5 µm).
   5. Select a region to image. To get an overview of the lipid bilayer, a 50 µm x 50 µm or 25 µm x 25 µm region will be a good starting point to image. If smaller features appear, one can image in smaller areas (10 µm x 10 µm, or 2 µm x 2 µm). The choice of the imaging area also depends on the working range of the piezoelectric scanner. Please consult the instrument manual for this.
   6. As bilayers are very fragile, adjust the set point of the AFM tip during imaging to keep the force at minimum and correct for thermal drift, while maintaining contact with the sample. In contact mode, minimize the set point. In tapping mode, maximize the set point.
   7. Process images by fitting each scan line to polynomial leveling functions. Perform line removal for scans that lose contact with the membrane using the proprietary software of the AFM manufacturing company.
2. Force Spectroscopy
   1. Calibrate the cantilever following the instructions according to manufacturer’s protocol. Calibration allows the conversion of the electrical signal of the photodiodes to force (Figure 1B).
      1. Calibrate the tip’s sensitivity to measure deflection as z-piezo movement (in units of length) instead of electrical signal in volts.
         Note: AFM detects changes in the deflection of the cantilever using a laser reflected at the back of the cantilever. The laser reaches a photodiode that can spatially detect the position of the laser spot. A change in the deflection of the cantilever as it bends upon contact with the sample is detected by this photodiode as “vertical deflection”, and this is in the units of voltage. Since the cantilever is moved by the z-piezo, the vertical deflection is related to the movement in the z-piezo. The ratio between vertical deflection and z-movement is called sensitivity and converts voltage to distance.
      2. Calculate the cantilever spring constant using the thermal noise frequency method, usually incorporated in the AFM software. In this method, plot the amplitude of vibration from noise with respect to frequency of oscillation, creating a power spectral density plot. This plot will show a peak around the resonance frequency. The frequency where the amplitude is the greatest is the resonance frequency. Fit a Lorentzian function around the peak to automatically calculate the spring constant by the software. Some useful resources for the theory of the thermal noise method are given in references^27-30.
   2. After imaging an area of the bilayer, select a 5 µm x 5 µm region in the bilayer to perform force spectroscopy.
   3. Set up the AFM so that it takes force curves in a 16 x 16 grid of that region. This results in 256 force curves per region. The space between two neighboring points should be enough to avoid that the membrane area being probed by one point would coincide with the next point. This is dependent on the tip radius. A 100 nm distance between two points would be ideal. Divide the 5 µm x 5 µm region into 16 x 16 grid. Depending on the size of the phase, one can select a smaller or bigger region, and then adjust the grid size accordingly.
   4. Set z-piezo displacement to 400 nm. This determines the maximum range of movement of the z-piezo. It should be big enough to make sure that the cantilever has fully retracted away from the sample in between measurements.
   5. Set approach speed to 800 nm/sec and retract speed to 200 nm/sec. Use an approach speed between 400 to 6,000 nm/sec depending on the bilayer composition and lipid phase to be studied^6,16.
   6. After setting up all the parameters, acquire force curves, by pressing the “run” button of the AFM software.
   7. Save force curves as force vs. height curves (a measure of z-piezo movement). This does not take into account the bending of the cantilever when in contact with the sample. During the data processing step, the AFM software allows correction for cantilever bending to get force vs. tip-sample separation curves (Figure 1C), which will give proper values for bilayer thickness. The correction values are usually incorporated in the calibration, which is saved with each force curve. Calculate tip-sample separation by subtracting the vertical deflection from the piezo movement at a given point.
      Note: The peak in the approach force curve (the force value goes down even if the cantilever movement is still on the approach cycle) is the breakthrough force of the membrane, while the difference between the position of this peak and the position of the point where the force begins to rise up again provides the thickness of the membrane.
   8. Acquire at least 500 force curves for each condition to get good statistics. Plot a histogram of the values using programs like Origin or MatLab, and fit the histograms to a Gaussian distribution to get the peak and width of the distribution.

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Results

Supported lipid bilayers composed of DOPC:SM:Chol (2:2:1) were imaged in AFM (Figure 2 A-C). Because of the lipid composition, SM/Chol-rich L_o and DOPC-rich L_d phases were observed. The height profile from the AFM imaging can provide important information on the membrane structure. By looking at the height profile, the bilayer thickness can be measured in presence of defects in the membrane (Figure 2B), or the difference in height between the L_o/L_d

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Discussion

SLBs composed of DOPC:SM:Chol (2:2:1) were formed on mica after vesicle adsorption and rupture induced by calcium chloride. This lipid composition separated into L_d and L_o phases. The L_o phase is enriched in sphingomyelin and cholesterol and is less fluid/more viscous (Figure 1A) than the L_d phase¹¹. The separation of L_o from L_d phase manifests as circular structures elevated above the surrounding (Figure 1B, C)...

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Materials

Name	Company	Catalog Number	Comments
1,2-dioleoyl-sn-glycero-3-phosphocholine	Avanti Polar Lipids, Inc.	850375P	Comes as lyophilized powder in sealed vials. Dissolve all powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20 °C. Visit here for more information on storage and handling.
Sphingomyelin (Brain, Porcine)	Avanti Polar Lipids, Inc.	860062P	Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20 °C. Visit here for more information on storage and handling.
Cholesterol	Avanti Polar Lipids, Inc.	700000P	Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20 °C. Visit here for more information on storage and handling.
Sodium chloride (NaCl), 99.8%	Carl Roth GmbH + Co. KG	9265.1
Potassium chloride (KCl), 88%	Sigma	P9541
Sodium hydrogenphosphate (Na2HPO4), >99%	AppliChem GmbH	A1046
Potassium dihydrogenphosphate (KH2PO4), 99%	Carl Roth GmbH + Co. KG	3904.1
Calcium chloride dihydrate (CaCl2), molecular biology grade	AppliChem GmbH	A4689
HEPES, molecular biology grade	AppliChem GmbH	A3724
Glass coverslip, 24 x 60 mm, 1 mm thickness	Duran Group	2355036
Punch and Die Set	Precision Brand Products, Inc	40105
Optical Adhesive	Norland Products, Inc.	NOA 88	Liquid adhesive that hardens when cured under long wavelength UV light.
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Mica blocks	NSC Mica Exports Ltd.		These are mica pieces at least 1 sq. inches in area and thickness ranging from 0.006 inches to 0.016 inches. They are cut to a specific size by the company for shipping. Small mica discs can be punched from the mica blocks using the punch and die set.  Always handle mica with gloves or tweezers.
Laboratory Equipment Grease	Borer Chemie AG	Glisseal N
Liposome Extruder	Avestin	LiposoFast-Basic	As an alternative one can also look at offers from Northern Lipids, Inc.
Adhesive Tape	3M	Scotch(R) Magic (TM) Tape 810 (1-inch)
Bath Sonicator	Bandelin Sonorex Digitec	DT-31	No heating, Frequency: 35 kHz, Ultrasonic Peak Output: 160 W, HF Power: 40 W. (Data sheet)
Silicon Nitride AFM Cantilever	Bruker AFM Probes	DNP-10	Each cantilever has four tips and their nominal tip radius is 20 nm (with possible maximum at 60 nm). Based on the specifications, we use tip D with resonance frequency of 18 kHz, and nominal spring constant of 0.06 N/m.
AFM	JPK	JPK Nanowizard II mounted on Zeiss Axiovert 200