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

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 (....

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

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).......

Materials

List of materials used in this article

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