4. Methods used to study glycolipids at the water-air interface

Given the significance of glycolipids in cellular processes and their association with chronic diseases, it is important to seek a clear understanding of the biophysical and biological properties of these glycolipids. In this section, we attempt to address some of the techniques which might be helpful and a survey of studies done using these methods will be discussed. Each method has pros and cons associated with it and therefore two or more complimentary methods are often employed together for the complete assurance of the result observed.

#### 4.1. Surface pressure isotherms

The plot of surface pressure, Π, versus molecular area, A, at constant temperature as the monolayer film is compressed, by closing the barrier at constant rate, after initial deposition and solvent evaporation is known as an isotherm. Surface pressure-area isotherms provide information about molecular packing, molecular stability, phase transitions and compressibility of phases. Isotherms recorded at various temperatures helps to obtain phase diagrams [21]. The data from Π-A isotherms can also be used for various thermodynamic calculations.

Water molecules on the surface are under tension due to imbalance of the force compared to the bulk where each molecule is attracted by equal force from all direction. The surface tension decreases when a monolayer is deposited at the air-water interface. The difference in surface tension before and after monolayer deposition is known as surface pressure, Π, which is given by the relation

$$
\Pi = \mathcal{V}\_0 - \mathcal{V} \tag{1}
$$

phases and phase transitions upon compression [58]. At high molecular areas there can be a gas-like phase in which molecules are widely separated and the surface pressure is very low. Upon compression, a liquid-like phase can be entered that is known as the liquid-expanded (LE) phase in which molecules are closer together and ordered like a two-dimensional liquid. A liquid-condensed (LC) phase in which molecules are well-ordered as in a two-dimensional liquid crystal, and may be oriented vertically or tilted, is entered upon further compression. Other phases are possible and the sequence of phases seen on compression depends on temperature and other conditions. Coexistence regimes between phases, such as LE + LC, are often encountered. Kinks or flat regions in an isotherm signal these phase transitions for single component monolayers. At very high surface pressures, two-dimensional solid-like phases can be observed for some compounds. Compression of the monolayer beyond its stability limit

Figure 4. Schematic showing a pressure area, Π-A isotherm with the major phases and transitions shown for a single

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Π-A isotherms mainly provide information about later stages of monolayer compression when the molecules are closer together. Surface-potential measurement allows one to probe a Langmuir monolayer at higher surface area before it has been significantly compressed. Surface potential vs. area, ΔV-A isotherms, have a region in the higher range of molecular area which marks the initial rise of the surface potential and is attributed to aggregation of microdomains present in the monolayer [59]. In addition to these advantages, surface potential measurements are used to gain information about molecular orientation, to calculate dipole moment, surface

results in collapse into three-dimensional structures.

4.2. Surface potential measurement

component lipid monolayer.

charge density and interfacial thickness.

where γ<sup>0</sup> and γ are surface tension in the absence and presence of monolayer, respectively. Surface pressure is measured by the Wilhelmy plate method in which a plate made of platinum or thin filter paper is contacted with the water and changes in downward force are measured. A schematic Π-A isotherm is shown in Figure 4. Monolayers can exhibit a range of

Figure 4. Schematic showing a pressure area, Π-A isotherm with the major phases and transitions shown for a single component lipid monolayer.

phases and phase transitions upon compression [58]. At high molecular areas there can be a gas-like phase in which molecules are widely separated and the surface pressure is very low. Upon compression, a liquid-like phase can be entered that is known as the liquid-expanded (LE) phase in which molecules are closer together and ordered like a two-dimensional liquid. A liquid-condensed (LC) phase in which molecules are well-ordered as in a two-dimensional liquid crystal, and may be oriented vertically or tilted, is entered upon further compression. Other phases are possible and the sequence of phases seen on compression depends on temperature and other conditions. Coexistence regimes between phases, such as LE + LC, are often encountered. Kinks or flat regions in an isotherm signal these phase transitions for single component monolayers. At very high surface pressures, two-dimensional solid-like phases can be observed for some compounds. Compression of the monolayer beyond its stability limit results in collapse into three-dimensional structures.

#### 4.2. Surface potential measurement

3.4. Lipopolysaccharides

220 Cell Culture

4.1. Surface pressure isotherms

by the relation

Lipopolysaccharides (LPS), also referred to as endotoxins, are a major component of the outer membrane of gram negative bacteria and are essential for maintaining the structural integrity of the membrane. LPS have three components: lipid A, core oligosaccharide and O-antigen polysaccharide. Lipid A is the active component and under normal conditions consists of β(1–6) linked glucosamine disaccharides. The diglucosamine backbone is phosphorylated and decorated with multiple fatty acids anchoring them in the outer leaflet of the bacterial cell membrane. Lipid A, when released from the cell, is recognized by pattern recognition receptor TLR4 triggering cytokine synthesis. At low level of endotoxins, the innate immune system

Given the significance of glycolipids in cellular processes and their association with chronic diseases, it is important to seek a clear understanding of the biophysical and biological properties of these glycolipids. In this section, we attempt to address some of the techniques which might be helpful and a survey of studies done using these methods will be discussed. Each method has pros and cons associated with it and therefore two or more complimentary methods are often employed together for the complete assurance of the result observed.

The plot of surface pressure, Π, versus molecular area, A, at constant temperature as the monolayer film is compressed, by closing the barrier at constant rate, after initial deposition and solvent evaporation is known as an isotherm. Surface pressure-area isotherms provide information about molecular packing, molecular stability, phase transitions and compressibility of phases. Isotherms recorded at various temperatures helps to obtain phase diagrams [21]. The data from Π-A isotherms can also be used for various thermodynamic calculations.

Water molecules on the surface are under tension due to imbalance of the force compared to the bulk where each molecule is attracted by equal force from all direction. The surface tension decreases when a monolayer is deposited at the air-water interface. The difference in surface tension before and after monolayer deposition is known as surface pressure, Π, which is given

where γ<sup>0</sup> and γ are surface tension in the absence and presence of monolayer, respectively. Surface pressure is measured by the Wilhelmy plate method in which a plate made of platinum or thin filter paper is contacted with the water and changes in downward force are measured. A schematic Π-A isotherm is shown in Figure 4. Monolayers can exhibit a range of

Π ¼ γ<sup>0</sup> � γ (1)

eliminates it, but at high concentrations it can prove fatal [57].

4. Methods used to study glycolipids at the water-air interface

Π-A isotherms mainly provide information about later stages of monolayer compression when the molecules are closer together. Surface-potential measurement allows one to probe a Langmuir monolayer at higher surface area before it has been significantly compressed. Surface potential vs. area, ΔV-A isotherms, have a region in the higher range of molecular area which marks the initial rise of the surface potential and is attributed to aggregation of microdomains present in the monolayer [59]. In addition to these advantages, surface potential measurements are used to gain information about molecular orientation, to calculate dipole moment, surface charge density and interfacial thickness.

Two common methods applied to measure surface potential are the vibrating electrode and the ionizing electrode. The vibrating electrode method also known as Kelvin probe method uses a plate-like electrode placed at a certain distance above air-water interface. The electrode is connected to the reference electrode placed in the subphase. The electrode above the interface is periodically vibrated during measurements. The ionizing electrode method employs the same measurement setup as the vibrating plate method. In this method, the electrodes are coated with α-emitters such as 241Am or 210Po to increase the conductivity of the air gap [60].

is doped with an amphiphilic fluorescent probe that has different solubility in two surface phases resulting in contrast when excited using light from an arc lamp or a laser. Despite the usefulness of fluorescence microscopy, it has a few drawbacks. First the fluorophores added, although in trace amount, can alter the original monolayer if their concentration is too high. Secondly, observation of more highly ordered phases is difficult because they reject the fluorescent molecular probe [59]. Also, problems may occur due to dissolution of the probe into

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The problems associated with fluorescence microscopy due to the addition of fluorescent dye can be avoided with Brewster angle microscopy (BAM) as it does not require any probe molecule. When a light beam is directed onto a water surface a portion of it is reflected, and the reflected light intensity depends upon the angle of incidence. For plane polarized light, there exists a certain angle of incidence at which no reflection occurs known as the Brewster angle. For water at room temperature the Brewster angle is 53�. For monolayer study at the airwater interface, light is directed onto the surface at the Brewster angle and the reflected light is observed using a CCD camera. The monolayer on the water surface has a different refractive index and this leads to violation of the Brewster angle condition and increased reflectivity so

X-rays provide a very sensitive tool for the study of monolayers by virtue of their small wavelength of few Å. Unlike visible light, reflection of X-rays from a denser medium coming from rarer (gas) medium at a certain critical angle are totally reflected without any diffraction into the denser medium (solid or liquid) [60]. Grazing incident X-ray diffraction (GIXD) has been an important technique used for investigating in situ structural arrangement of amphiphiles for more than two decades. In GIXD the incident angle, α is slightly below the critical angle. The small incident angle is desirable for larger penetration of X-rays. Two commonly used X-ray techniques are X-ray reflectivity (XRR) and

XRR provides data that can be used to estimate the thickness of the monolayer, d calculated

Besides thickness of monolayer, XRR also provides information about the electron density distribution perpendicular to the interface and the roughness of the monolayer. XRR data can be used to model thickness of different segments of the monolayer. XD provides information about the two-dimensional arrangement of the molecules on the surface and the tilt angles of

nλ ¼ 2dsinθ (2)

the subphase.

4.5. Brewster angle microscopy

the monolayer domains will appear bright [61].

4.6. X-ray reflectivity and scattering

diffraction (XD).

using Bragg's law:

the molecules.

### 4.3. Transfer as Langmuir-Blodgett film for study by atomic force microscopy

The deposited monolayer can be transferred to a solid support. For example, CaF2 plates are used for transmission infrared spectroscopy while germanium silicon and ZnSe plates are used for internal reflection infrared spectroscopy [21]. The substrate most commonly used for atomic force microscopy (AFM) studies is freshly cleaved mica, because of its atomically flat surface. AFM is helpful for the visualization of coexisting phases with great resolution, which can be down to a few nanometers, and not readily visualized by other methods. Care must be taken while analyzing the micrograph that the structures visualized are a true representation of what was on the deposited monolayer and are not artifacts created during the transfer process.

The monolayer can be transferred to a solid support by Langmuir-Blodgett deposition which is carried out as a vertical transfer or by Langmuir-Schaefer transfer which is carried out as a horizontal transfer. The Langmuir-Blodgett transfer method can be performed in a number of ways depending upon the nature of the substrate (hydrophobic or hydrophilic) and the number of layers desired. For a hydrophilic substrate, the transfer is performed by immersing the substrate into the subphase prior to spreading the monolayer. The monolayer is then compressed to a desired surface pressure and the substrate is lifted out of the subphase at a suitable speed while maintaining a constant surface pressure. A series of immersion and emersion cycle can be performed to generate multilayers. In the horizontal transfer method, the substrate is held horizontally above the compressed monolayer, lowered until it makes contact with the water surface and is then lifted gently thus transferring the monolayer onto the substrate.

#### 4.4. Fluorescence microscopy

Fluorescence microscopy is another important method that is often used for studying interfacial behavior of monolayers. Even though the images from AFM help visualize the structure of monolayers at a near molecular level resolution they only provide snapshots of the continuous process and require transfer to a substrate. Fluorescence microscopy has an edge over AFM or any other static visualization method, as it provides a real time picture of the events as they occur.

Fluorescence microscopy provided the first evidence of coexisting domains in the plateau region of Π-A isotherms of Langmuir monolayers. In fluorescence microscopy, the monolayer is doped with an amphiphilic fluorescent probe that has different solubility in two surface phases resulting in contrast when excited using light from an arc lamp or a laser. Despite the usefulness of fluorescence microscopy, it has a few drawbacks. First the fluorophores added, although in trace amount, can alter the original monolayer if their concentration is too high. Secondly, observation of more highly ordered phases is difficult because they reject the fluorescent molecular probe [59]. Also, problems may occur due to dissolution of the probe into the subphase.

#### 4.5. Brewster angle microscopy

Two common methods applied to measure surface potential are the vibrating electrode and the ionizing electrode. The vibrating electrode method also known as Kelvin probe method uses a plate-like electrode placed at a certain distance above air-water interface. The electrode is connected to the reference electrode placed in the subphase. The electrode above the interface is periodically vibrated during measurements. The ionizing electrode method employs the same measurement setup as the vibrating plate method. In this method, the electrodes are coated with α-emitters such as 241Am or 210Po to increase the conductivity

The deposited monolayer can be transferred to a solid support. For example, CaF2 plates are used for transmission infrared spectroscopy while germanium silicon and ZnSe plates are used for internal reflection infrared spectroscopy [21]. The substrate most commonly used for atomic force microscopy (AFM) studies is freshly cleaved mica, because of its atomically flat surface. AFM is helpful for the visualization of coexisting phases with great resolution, which can be down to a few nanometers, and not readily visualized by other methods. Care must be taken while analyzing the micrograph that the structures visualized are a true representation of what was on the deposited monolayer and are not artifacts created during the transfer

The monolayer can be transferred to a solid support by Langmuir-Blodgett deposition which is carried out as a vertical transfer or by Langmuir-Schaefer transfer which is carried out as a horizontal transfer. The Langmuir-Blodgett transfer method can be performed in a number of ways depending upon the nature of the substrate (hydrophobic or hydrophilic) and the number of layers desired. For a hydrophilic substrate, the transfer is performed by immersing the substrate into the subphase prior to spreading the monolayer. The monolayer is then compressed to a desired surface pressure and the substrate is lifted out of the subphase at a suitable speed while maintaining a constant surface pressure. A series of immersion and emersion cycle can be performed to generate multilayers. In the horizontal transfer method, the substrate is held horizontally above the compressed monolayer, lowered until it makes contact with the water

Fluorescence microscopy is another important method that is often used for studying interfacial behavior of monolayers. Even though the images from AFM help visualize the structure of monolayers at a near molecular level resolution they only provide snapshots of the continuous process and require transfer to a substrate. Fluorescence microscopy has an edge over AFM or any other static visualization method, as it provides a real time picture of the events as

Fluorescence microscopy provided the first evidence of coexisting domains in the plateau region of Π-A isotherms of Langmuir monolayers. In fluorescence microscopy, the monolayer

surface and is then lifted gently thus transferring the monolayer onto the substrate.

4.3. Transfer as Langmuir-Blodgett film for study by atomic force microscopy

of the air gap [60].

222 Cell Culture

process.

4.4. Fluorescence microscopy

they occur.

The problems associated with fluorescence microscopy due to the addition of fluorescent dye can be avoided with Brewster angle microscopy (BAM) as it does not require any probe molecule. When a light beam is directed onto a water surface a portion of it is reflected, and the reflected light intensity depends upon the angle of incidence. For plane polarized light, there exists a certain angle of incidence at which no reflection occurs known as the Brewster angle. For water at room temperature the Brewster angle is 53�. For monolayer study at the airwater interface, light is directed onto the surface at the Brewster angle and the reflected light is observed using a CCD camera. The monolayer on the water surface has a different refractive index and this leads to violation of the Brewster angle condition and increased reflectivity so the monolayer domains will appear bright [61].

### 4.6. X-ray reflectivity and scattering

X-rays provide a very sensitive tool for the study of monolayers by virtue of their small wavelength of few Å. Unlike visible light, reflection of X-rays from a denser medium coming from rarer (gas) medium at a certain critical angle are totally reflected without any diffraction into the denser medium (solid or liquid) [60]. Grazing incident X-ray diffraction (GIXD) has been an important technique used for investigating in situ structural arrangement of amphiphiles for more than two decades. In GIXD the incident angle, α is slightly below the critical angle. The small incident angle is desirable for larger penetration of X-rays. Two commonly used X-ray techniques are X-ray reflectivity (XRR) and diffraction (XD).

XRR provides data that can be used to estimate the thickness of the monolayer, d calculated using Bragg's law:

$$n\lambda = 2d \sin \theta \tag{2}$$

Besides thickness of monolayer, XRR also provides information about the electron density distribution perpendicular to the interface and the roughness of the monolayer. XRR data can be used to model thickness of different segments of the monolayer. XD provides information about the two-dimensional arrangement of the molecules on the surface and the tilt angles of the molecules.

#### 4.7. Infrared spectroscopy

Infrared Spectroscopy is another important technique widely used to characterize the conformation and orientation of monolayers transferred onto the solid support or in situ at the airwater interface. It is desirable to carry out the experiments in situ to avoid problems due to artifacts during transfer. Infrared reflection absorption spectroscopy (IRRAS) and polarization modulated (PM)-IRRAS are two versions of Fourier transform infrared spectroscopy (FTIR) frequently employed for the study of Langmuir monolayers [62].

term inside parenthesis is the excess surface area. Negative values of ΔGexc indicate favorable interactions resulting in a more stable monolayer and positive ΔGexc indicates unfavorable interactions and possible phase separation. Furthermore, one could calculate a free energy of

where R and T are gas constant and temperature respectively. Phase separation may also occur upon compression in mixed monolayers, resulting in coexisting phases of different composi-

Gangliosides have been the subject of many studies as monolayers and we survey some of the reported results. In an early study using surface pressure isotherms of gangliosides GM1, GM2, GM3, GD1a, GD3 and GT1, it was found that increasing the number of sialic acid residues caused an increase in the prevalence of the LE phase and that the surface pressure isotherms shifted to higher molecular areas and the monolayers became more compressible [64]. This trend was attributed to increasing electrostatic repulsions with introduction of additional negatively charged sialic acids in the structures. In a related study using surface pressure and surface potential measurements, it was found that the strength of interaction of Ca2+ ions in the subphase with gangliosides depended on the number and arrangement of the sialic acid units [65]. A study using surface pressure isotherms of mixed monolayers of GM1, GD1a, and GT1 with phospholipids showed positive deviations from ideality at 30 mN m�<sup>1</sup> for DPPE, a phosphatidylinositol and a phosphatidylserine, but negative deviations from ideality for mixtures with DPPC [66]. In contrast, the interaction of gangliosides with neutral glycosphingolipids was found to be favorable. Mixed monolayers of ceramide or of glucosyl ceramide with gangliosides were found to show favorable interactions, while those of lactosyl ceramide and gangliosides showed immiscibility [67]. The activity of the enzyme phospholipase A2 injected into the subphase against mixed monolayers of dilauroylphosphatidylcholine and gangliosides GM1, GD1a, and GT1b was found to be strongly inhibited [68]. Gangliosides were also found to inhibit the activity of phospholipase C against model membrane systems including monolayers [69]. The sensitivity of monolayer parameters to trace impurity of peptide materials in isolated gangliosides has been

tion. Occurrence of critical points and azeotropes in two-dimensions is also possible.

6. Survey of studies of monolayers containing glycolipids

emphasized along with provision of methods for rigorous purification [70].

Gangliosides are known for their ability to form microdomains in biological membranes. The partitioning of ganglioside, GM1 in phase separated DOPC/DPPC LB films transferred to

<sup>Δ</sup>Gmix <sup>¼</sup> <sup>Δ</sup>Gid <sup>þ</sup> <sup>Δ</sup>Gexc (4)

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<sup>Δ</sup>Gid <sup>¼</sup> RT xð Þ 1ln <sup>x</sup><sup>1</sup> <sup>þ</sup> <sup>x</sup>2ln <sup>x</sup><sup>2</sup> (5)

mixing, ΔGmix using the following equations:

6.1. Gangliosides

ΔGid is the ideal value that can be calculated using

In IRRAS, the sample is irradiated with an IR beam and the intensity of the reflected beam is measured as a function of wavelength. The measurements can be carried out with p- and spolarized light at various angles of incidence above or below the Brewster angle. If the samples are on metal substrates, a "surface selection" rule is followed which states that only vibrational dipole moments oriented perpendicular to the substrate are observed. IRRAS has a disadvantage while studying Langmuir films, the strong absorption of water vapor conceals the spectral regions with desired molecular information. PM-IRRAS, which is insensitive to IR absorption of water vapor, was introduced to avoid such problems. In PM-IRRAS, the incident beam is alternated between s- and p-polarization at a frequency of tens of kHz [63] and differential reflectivity is calculated. Besides these two IR spectroscopic techniques, there are others such as surface-enhanced infrared-absorption spectroscopy (SEIRA) and attenuated total reflection ATR-FTIR spectroscopy.
