Lipid bilayer characterization

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Lipid bilayer characterization is the use of various optical, chemical and physical probing methods to study the properties of lipid bilayers. Many of these techniques are elaborate and require expensive equipment because the fundamental nature of the lipid bilayer makes it a very difficult structure to study. An individual bilayer, since it is only a few nanometers thick, is invisible in traditional light microscopy. The bilayer is also a relatively fragile structure since it is held together entirely by non-covalent bonds and is irreversibly destroyed if removed from water. In spite of these limitations dozens of techniques have been developed over the last seventy years to allow investigations of the structure and function of bilayers. The first general approach was to utilize non-destructive in situ measurements such as x-ray diffraction and electrical resistance which measured bilayer properties but did not actually image the bilayer. Later, protocols were developed to modify the bilayer and allow its direct visualization at first in the electron microscope and, more recently, with fluorescence microscopy. Over the past two decades, a new generation of characterization tools including AFM has allowed the direct probing and imaging of membranes in situ with little to no chemical or physical modification. More recently, dual polarisation interferometry has been used to measure the optical birefringence of lipid bilayers to characterise order and disruption associated with interactions or environmental effects.

Contents

Fluorescence Microscopy

Human red blood cells viewed through a microscope. The cell membrane has been stained with a fluorescent dye. Scale bar is 20 mm. Sedimented red blood cells.jpg
Human red blood cells viewed through a microscope. The cell membrane has been stained with a fluorescent dye. Scale bar is 20 μm.

Fluorescence microscopy is a technique whereby certain molecules can be excited with one wavelength of light and will emit another longer wavelength of light. Because each fluorescent molecule has a unique spectrum of absorption and emission, the location of particular types of molecules can be determined. Natural lipids do not fluoresce, so it is always necessary to include a dye molecule in order to study lipid bilayers with fluorescence microscopy. To some extent, the addition of the dye molecule always changes the system, and in some cases it can be difficult to say whether the observed effect is due to the lipids, the dye or, most commonly, some combination of the two. The dye is usually attached either to a lipid or a molecule that closely resembles a lipid, but since the dye domain is relatively large it can alter the behavior of this other molecule. This is a particularly contentious issue when studying the diffusion or phase separation of lipids, as both processes are very sensitive to the size and shape of the molecules involved.

This potential complication has been given an argument against the use of one of fluorescence recovery after photobleaching (FRAP) to determine bilayer diffusion coefficients. In a typical FRAP experiment a small (~30 μm diameter) area is photobleached by exposure to an intense light source. This area is then monitored over time as the “dead” dye molecules diffuse out and are replaced by intact dye molecules from the surrounding bilayer. By fitting this recovery curve it is possible to calculate the diffusion coefficient of the bilayer. [1] [2] An argument against the use of this technique is that what is actually being studied is the diffusion of the dye, not the lipid. [3] While correct, this distinction is not always important, since the mobility of the dye is often dominated by the mobility of the bilayer.

In traditional fluorescence microscopy the resolution has been limited to approximately half the wavelength of the light used. Through the use of confocal microscopy and image processing this limit can be extended, but typically not much below 100 nanometers, which is much smaller than a typical cell but much larger than the thickness of a lipid bilayer. More recently, advanced microscopy methods have allowed much greater resolution under certain circumstances, even down to sub-nm. One of the first of these methods to be developed was Förster resonance energy transfer (FRET). In FRET, two dye molecules are chosen such that the emission spectrum of one overlaps the absorption spectrum of the other. This energy transfer is extremely distance dependent, so it is possible to tell with angstrom resolution how far apart the two dyes are. This can be used for instance to determine when two bilayers fuse and their components mix. [4] Another high resolution microscopy technique is fluorescence interference contrast microscopy (FLIC). This method requires that the sample be mounted on a precisely micromachined reflective surface. By studying the destructive interference patterns formed it is possible to individually resolve the two leaflets of a supported bilayer and determine the distribution of a fluorescent dye in each. [5]

Electrical

Patch clamp recordings of changes in conductivity associated with the opening and closing of an ion channel. V-clamp-GlyR.jpg
Patch clamp recordings of changes in conductivity associated with the opening and closing of an ion channel.

Electrical measurements are the most straightforward way to characterize one of the more important functions of a bilayer, namely its ability to segregate and prevent the flow of ions in solution. Accordingly, electrical characterization was one of the first tools used to study the properties of model systems such as black membranes. It was already known that the cell membrane was capable of supporting an ionic gradient and that this gradient is responsible for the ability of neurons to send signals via an action potential. Demonstrating that similar phenomena could be replicated in vitro was an important verification of the utility of model systems. [6]

Fundamentally, all electrical measurements of bilayers involve the placement of an electrode on either side of the membrane. By applying a bias across these electrodes and measuring the resulting current, it is possible to determine the resistance of the bilayer. This resistance is typically quite high for intact bilayers, often exceeding 100 GΩ since the hydrophobic core is impermeable to charged hydrated species. Because this resistance is so large, the presence of even a few nanometer-scale holes results in a dramatic increase in current and can be easily determined. [7] The sensitivity of this system is such that even the activity of single ion channels can be resolved. [8] In such DC measurements, it is necessary to use electrochemically active electrodes to provide the necessary positive charges on one side and negative charges on the other. The most common system is the silver/silver chloride electrode since this reaction is stable, reversible, involves a single electron transfer and can produce large currents. [9] In addition to simple DC current measurements it is also possible to perform AC electrical characterization to extract information about the capacitance and complex impedance of a bilayer. Because capacitance is inversely proportional to thickness and bilayers are very thin they typically have a very large capacitance, on the order of 2 μF/cm2. Capacitance measurements are particularly useful when dealing with black lipid membranes, as they can be used to determine when the solvent/lipid plug thins down to a single bilayer.

Optical

Lipids are highly polar molecules which when self assembled into bilayers creates a highly birefringent layer [10] where the optical properties parallel are very different from those perpendicular. This effect, studied by dual polarisation interferometry has been used to measure dynamic reorganisation of the layer due to temperature, ionic strength, and molecular interactions with e.g. antimicrobial peptides.

Hydrated bilayers show rich vibrational dynamics and are good media for efficient vibrational energy transfer. Vibrational properties of lipid monolayers and bilayers has been investigated by ultrafast spectroscopic techniques [11] and recently developed computational methods. [12]

AFM

Illustration of a typical AFM scan of a supported lipid bilayer. The pits are defects in the bilayer, exposing the smooth surface of the substrate underneath. Bilayer AFM schematic.png
Illustration of a typical AFM scan of a supported lipid bilayer. The pits are defects in the bilayer, exposing the smooth surface of the substrate underneath.

Atomic force microscopy (AFM) has been used in recent years to image and probe the physical properties of lipid bilayers. AFM is a promising technique because it has the potential to image with nanometer resolution at room temperature and even underwater, conditions necessary for natural bilayer behavior. These capabilities have allowed direct imaging of the subtle ripple phase transition in a supported bilayer. [13] Another AFM experiment performed in a tapping mode under aqueous buffer medium allowed (1) to determine the formation of transmembrane pores (holes) around nanoparticles of approximately 1.2 to 22 nm diameter via subtraction of AFM images from series recorded during the lipid bilayer formation and (2) to observe adsorption of single insulin molecules onto exposed nanoparticles. [14] Another advantage is that AFM does not require fluorescent or isotopic labeling of the lipids, as the probe tip interacts mechanically with the bilayer surface. Because of this, the same scan can reveal information about both the bilayer and any associated structures, even to the extent of resolving individual membrane proteins. [15] In addition to imaging, AFM can also probe the mechanical nature of small delicate structures such as lipid bilayers. One study demonstrated the possibility of measuring the elastic modulus of individual nano-scale membranes suspended over porous anodic alumina. [16]

Although AFM is a powerful and versatile tool for studying lipid bilayers, there are some practical limitations and difficulties. Because of the fragile nature of the bilayer, extremely low scanning forces (typically 50pN or less [13] [17] ) must be used to avoid damage. This consideration is particularly important when studying metastable systems such as vesicles adsorbed on a substrate, since the AFM tip can induce rupture and other structural changes. [18] Care must also be taken to choose an appropriate material and surface preparation for the AFM tip, as hydrophobic surfaces can interact strongly with lipids and disrupt the bilayer structure. [19]

Electron microscopy

Image from a Transmission Electron Microscope of a lipid vesicle. The two dark bands around the edge are the two leaflets of the bilayer. Similar electron micrographs confirmed the bilayer nature of the cell membrane in the 1950s Annular Gap Junction Vesicle.jpg
Image from a Transmission Electron Microscope of a lipid vesicle. The two dark bands around the edge are the two leaflets of the bilayer. Similar electron micrographs confirmed the bilayer nature of the cell membrane in the 1950s

In electron microscopy a beam of focused electrons interacts with the sample rather than a beam of light as in traditional microscopy. Electrons have a much shorter wavelength than light so electron microscopy has much higher resolution than light microscopy, potentially down to the atomic scale. Because lipid bilayers are arranged on the molecular level, this higher resolution has been invaluable. In 1960, when the structure of the bilayer was still debated, it was electron microscopy that offered the first direct visualization of the two apposing leaflets. [20] In conjunction with rapid freezing techniques, electron microscopy has also been used to study the mechanisms of inter- and intracellular transport, for instance in demonstrating that exocytotic vesicles are the means of chemical release at synapses. [21] Often, electron microscopy is the only probe technique with sufficient resolution to determine complex nanometer-scale morphologies.

The limitations of electron microscopy in the study of lipid structures deal primarily with sample preparation. Most electron microscopes require the sample to be under vacuum, which is incompatible with hydration at room temperature. To surmount this problem, samples can be imaged under cryogenic conditions with the associated water frozen, or a metallic negative can be made from a frozen sample. It is also typically necessary to stain the bilayer with a heavy metal compound such as osmium tetroxide or uranyl acetate because the low atomic weight constituents of lipids (carbon, nitrogen, phosphorus, etc.) offer little contrast compared to water. If a Transmission electron microscope (TEM) is being used, it is also necessary to cut or polish the sample into a very thin (<1 micrometre) sheet, which can be difficult and time-consuming. Scanning Electron Microscopy (SEM) does not require this step, but cannot offer the same resolution as TEM. Both methods are surface-sensitive techniques and cannot reveal information about deeply buried structures.

Neutron and X-ray scattering

Both X-rays and high-energy neutrons are used to probe the structure and periodicity of biological structures including bilayers because they can be tuned to interact with matter at the relevant (angstrom-nm) length scales. Often, these two classes of experiment provide complementary information because each has different advantages and disadvantages. X-rays interact only weakly with water, so bulk samples can be probed with relatively easy sample preparation. This is one of the reasons that x-ray scattering was the technique first used to systematically study inter-bilayer spacing. [22] X-ray scattering can also yield information on the average spacing between individual lipid molecules, which has led to its use in characterizing phase transitions. [23] One limitation of x-ray techniques is that x-rays are relatively insensitive to light elements such as hydrogen. This effect is a consequence of the fact that x-rays interact with matter by scattering off of electron density which decreases with decreasing atomic number. In contrast, neutrons scatter off of nuclear density and nuclear magnetic fields so sensitivity does not decrease monotonically with z. This mechanism also provides strong isotopic contrast in some cases, notably between hydrogen and deuterium, allowing researchers to tune the experimental baseline by mixing water and deuterated water. Using reflectometry rather than scattering with neutrons or x-rays allow experimenters to probe supported bilayers or multilayer stacks. These measurements are more complicated to perform an analyze, but allow determination of cross sectional composition, including the location and concentration of water within the bilayer. [24] In the case of both neutron and x-ray scattering measurements, the information provided is an ensemble average of the system and is therefore subject to uncertainty based on thermal fluctuations in these highly mobile structures. [25]

Related Research Articles

<span class="mw-page-title-main">Microscopy</span> Viewing of objects which are too small to be seen with the naked eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

<span class="mw-page-title-main">Surface science</span> Study of physical and chemical phenomena that occur at the interface of two phases

Surface science is the study of physical and chemical phenomena that occur at the interface of two phases, including solid–liquid interfaces, solid–gas interfaces, solid–vacuum interfaces, and liquid–gas interfaces. It includes the fields of surface chemistry and surface physics. Some related practical applications are classed as surface engineering. The science encompasses concepts such as heterogeneous catalysis, semiconductor device fabrication, fuel cells, self-assembled monolayers, and adhesives. Surface science is closely related to interface and colloid science. Interfacial chemistry and physics are common subjects for both. The methods are different. In addition, interface and colloid science studies macroscopic phenomena that occur in heterogeneous systems due to peculiarities of interfaces.

<span class="mw-page-title-main">Optical microscope</span> Microscope that uses visible light

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.

<span class="mw-page-title-main">Lipid bilayer</span> Membrane of two layers of lipid molecules

The lipid bilayer is a thin polar membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are made of a lipid bilayer, as are the nuclear membrane surrounding the cell nucleus, and membranes of the membrane-bound organelles in the cell. The lipid bilayer is the barrier that keeps ions, proteins and other molecules where they are needed and prevents them from diffusing into areas where they should not be. Lipid bilayers are ideally suited to this role, even though they are only a few nanometers in width, because they are impermeable to most water-soluble (hydrophilic) molecules. Bilayers are particularly impermeable to ions, which allows cells to regulate salt concentrations and pH by transporting ions across their membranes using proteins called ion pumps.

<span class="mw-page-title-main">Atomic force microscopy</span> Type of microscopy

Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

<span class="mw-page-title-main">Single-molecule experiment</span>

A single-molecule experiment is an experiment that investigates the properties of individual molecules. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior of molecules cannot be distinguished, and only average characteristics can be measured. Since many measurement techniques in biology, chemistry, and physics are not sensitive enough to observe single molecules, single-molecule fluorescence techniques caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past. Indeed, since the 1990s, many techniques for probing individual molecules have been developed.

<span class="mw-page-title-main">Near-field scanning optical microscope</span>

Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves. In SNOM, the excitation laser light is focused through an aperture with a diameter smaller than the excitation wavelength, resulting in an evanescent field on the far side of the aperture. When the sample is scanned at a small distance below the aperture, the optical resolution of transmitted or reflected light is limited only by the diameter of the aperture. In particular, lateral resolution of 6 nm and vertical resolution of 2–5 nm have been demonstrated.

<span class="mw-page-title-main">STED microscopy</span> Technique in fluorescence microscopy

Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. It was developed by Stefan W. Hell and Jan Wichmann in 1994, and was first experimentally demonstrated by Hell and Thomas Klar in 1999. Hell was awarded the Nobel Prize in Chemistry in 2014 for its development. In 1986, V.A. Okhonin had patented the STED idea. This patent was unknown to Hell and Wichmann in 1994.

<span class="mw-page-title-main">Characterization (materials science)</span> Study of material structure and properties

Characterization, when used in materials science, refers to the broad and general process by which a material's structure and properties are probed and measured. It is a fundamental process in the field of materials science, without which no scientific understanding of engineering materials could be ascertained. The scope of the term often differs; some definitions limit the term's use to techniques which study the microscopic structure and properties of materials, while others use the term to refer to any materials analysis process including macroscopic techniques such as mechanical testing, thermal analysis and density calculation. The scale of the structures observed in materials characterization ranges from angstroms, such as in the imaging of individual atoms and chemical bonds, up to centimeters, such as in the imaging of coarse grain structures in metals.

In biology, membrane fluidity refers to the viscosity of the lipid bilayer of a cell membrane or a synthetic lipid membrane. Lipid packing can influence the fluidity of the membrane. Viscosity of the membrane can affect the rotation and diffusion of proteins and other bio-molecules within the membrane, there-by affecting the functions of these things.

<span class="mw-page-title-main">Lipid polymorphism</span>

Polymorphism in biophysics is the ability of lipids to aggregate in a variety of ways, giving rise to structures of different shapes, known as "phases". This can be in the form of spheres of lipid molecules (micelles), pairs of layers that face one another, a tubular arrangement (hexagonal), or various cubic phases. More complicated aggregations have also been observed, such as rhombohedral, tetragonal and orthorhombic phases.

<span class="mw-page-title-main">Nanometrology</span> Metrology of nanomaterials

Nanometrology is a subfield of metrology, concerned with the science of measurement at the nanoscale level. Nanometrology has a crucial role in order to produce nanomaterials and devices with a high degree of accuracy and reliability in nanomanufacturing.

A model lipid bilayer is any bilayer assembled in vitro, as opposed to the bilayer of natural cell membranes or covering various sub-cellular structures like the nucleus. They are used to study the fundamental properties of biological membranes in a simplified and well-controlled environment, and increasingly in bottom-up synthetic biology for the construction of artificial cells. A model bilayer can be made with either synthetic or natural lipids. The simplest model systems contain only a single pure synthetic lipid. More physiologically relevant model bilayers can be made with mixtures of several synthetic or natural lipids.

<span class="mw-page-title-main">Raman microscope</span> Laser microscope used for Raman spectroscopy

The Raman microscope is a laser-based microscopic device used to perform Raman spectroscopy. The term MOLE is used to refer to the Raman-based microprobe. The technique used is named after C. V. Raman, who discovered the scattering properties in liquids.

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field or on the far-field. Among techniques that rely on the latter are those that improve the resolution only modestly beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment, the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.

The technique of vibrational analysis with scanning probe microscopy allows probing vibrational properties of materials at the submicrometer scale, and even of individual molecules. This is accomplished by integrating scanning probe microscopy (SPM) and vibrational spectroscopy. This combination allows for much higher spatial resolution than can be achieved with conventional Raman/FTIR instrumentation. The technique is also nondestructive, requires non-extensive sample preparation, and provides more contrast such as intensity contrast, polarization contrast and wavelength contrast, as well as providing specific chemical information and topography images simultaneously.

The following outline is provided as an overview of and topical guide to biophysics:

<span class="mw-page-title-main">Laurdan</span> Chemical compound

Laurdan is an organic compound which is used as a fluorescent dye when applied to fluorescence microscopy. It is used to investigate membrane qualities of the phospholipid bilayers of cell membranes. One of its most important characteristics is its sensitivity to membrane phase transitions as well as other alterations to membrane fluidity such as the penetration of water.

Super-resolution dipole orientation mapping (SDOM) is a form of fluorescence polarization microscopy (FPM) that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization (FP) is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.

References

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