Nanostructure and Dynamics of Biocompatible Surfactant ... - CiteSeerX

CHAPTER K

Nanostructure and Dynamics of Biocompatible Surfactant Monolayers and Bilayers Heidi E. Warriner Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

An Thien Ngo Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Joseph A. Zasadzinski Department of Chemical Engineering, University of California—Santa Barbara, Santa Barbara, California 93106-5080

Junqi Ding Unilever Research U.S., Edgewater, New Jersey 07020 CONTENTS 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. A Short History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Contemporary Topics and Questions . . . . . . . . . . . . . . .

2.

Methods . . . . . . . . . . . . . . . . . . . . 2.1. Isotherms . . . . . . . . . . . . . . 2.2. Epi-fluorescence Microscopy . 2.3. Brewster Angle Microscopy . 2.4. Atomic Force Microscopy . . . 2.5. Surface Shear Rheometry . . .

3.

Nanoscale Studies of Lung Surfactant . . . . . . . . . . . . . . . . . . 3.1. Clinical Background . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Functional Requirements . . . . . . . . . . . . . . . . . . . . . .

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Handbook of Nanostructured Biomaterials and Their Applications in Nanobiotechnology Edited by H. S. Nalwa Volume 1: Pages (1–57)

ISBN: 1-58883-033-0/$00.00 Copyright © 2005 by American Scientific Publishers All rights of reproduction in any form reserved.

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Nanostructure and Dynamics of Biocompatible Surfactant Monolayers and Bilayers

3.3. 3.4.

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Lung Surfactant Compositions . . . . . . . . . . . . . . . . . . Composition, Phase Behavior and Monolayer Structure . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Structure and Transport . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. INTRODUCTION 1.1. A Short History The study and use of surfactant films has a long and interesting history, some of which we recount here to motivate the current focus on nanometer-scale phenomena. A more complete, and highly enjoyable, account of the historical development of the science of interfacial films can be found in Charles Tanford’s book, Ben Franklin Stilled the Waves [2]. The first credible mention of using a thin film to alter interfacial dynamics occurs in Pliny’s Natural History, an “encyclopedia” written in the first century A.D. Pliny recommends spreading a small amount of oil on the ocean prior to diving in order to calm surface waves which would otherwise reduce sunlight transmission, limiting visibility. Benjamin Franklin read Pliny’s account as a young boy, and later astounded his contemporaries by demonstrating the dramatic calming action of a teaspoon of oil upon the surface of a pond on a windy day at Clapham Common, England. After reporting this feat in Philosophical Transactions of the Royal Society [1], Franklin tried to find a practical use for this effect, proposing that thin oil films might be used to facilitate shipping in turbulent waters. Sadly, too much oil is required to satisfactorily quiet large tracts of stormy sea. Unable to explain the calming action of the oil within the scientific framework of his time and called upon to serve as ambassador to France for the newly created United States of America, Franklin soon abandoned the theoretical and practical problems associated with thin films on a water surface [2]. More than 100 years would pass before Laplace and Young would create the theoretical framework required to understand Franklin’s observations as betraying a reduction in surface tension brought about by a surfactant. Laplace–Young theory stimulated one of the most prominent scientists of the time to reconsider Franklin’s experiment. Lord Rayleigh realized that the spreading of oil to a uniform thickness on a water surface of known area could be exploited to answer a key question: what is the size of a molecule? Although modern instruments have been developed to render surface tension measurement routine, in Rayleigh’s time surface tension measurement was in its infancy. Rayleigh thus used the motion of camphor chips, which jiggle vigorously on a water surface but are at rest on oil, to measure the spreading of an olive oil film added dropwise to the surface of a circular dish of fixed diameter. Dividing the volume of oil that was just enough to arrest the motion of the camphor chips by the dish area, he found a minimum film thickness of about 1.6 nm [2]. Today, Rayleigh’s result is recognized as the first credible measurement of a molecular dimension. His work inspired a general interest in experiments at the air–water interface as a source of molecular information; the most successful of these earned the 1932 Nobel Prize in chemistry for Dr. Irving Langmuir. Although Rayleigh could correctly assign a length of 1.6 nm to triolein, he had no idea of its geometry—i.e., 1.6 nm could be the diameter of a molecular sphere, the side of a molecular cube or rectangle, the height of a molecular cone, etc. At the time of Rayleigh’s experiment, molecular formulas were just becoming available; no one had any idea what conformation the bonds specified in these formulas would yield. However, when Langmuir began his studies of fatty acid monolayers, he had two crucial advantages over Rayleigh: a device to continuously vary the surface area available to the monolayer and simultaneously quantify resulting changes in surface tension (see discussion of Langmuir troughs under Methods) and accurate knowledge of molecular weight. These advancements permitted Langmuir to determine exactly how many molecules of fatty acid, oil, or lipid he had deposited on a water surface and so to simultaneously measure changes in molecular area, length, and surface tension. Langmuir’s work proved that these molecules


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were in fact highly asymmetric. Further, he used his results to argue that the variation in affinity for water of different molecular domains (groups of atoms) dictates its orientation at an interface, creating the monolayer geometry that we accept today: surfactants oriented such that polar or “hydrophilic” groups are immersed in water, while “hydrophobic” groups extend into the air (Fig. 1) [3]. The Langmuir trough remains a standard method of estimating molecular dimensions and surface activity. Some years prior to Langmuir’s work, Dr. Edward Overton, a professor of pharmacology in Sweden, had reported a systematic correlation between solubility in olive oil and the ability of a molecule to permeate the membranes of a variety of plant and animal cells. He suggested therefore that cell membranes must employ molecules similar to olive oil—either fats or lipids. Lipids were, at this point, widely known to be ubiquitous in living matter. One would think therefore that biology would have enthusiastically seized on Langmuir’s result and methodology to further explore the spontaneous ordering adopted by lipids at an interface between two media. However, this did not happen. As is largely true today, biologists were interested in a different class of molecules, proteins, of which some subsets, notably enzymes, had clearly demonstrated physiological and economic value. Biochemistry’s quest to understand the structure, function, and regulatory mechanisms operating in these macromolecules left little energy to spend on “fats” [2]. There were exceptions to this trend; especially notable was the work of two Dutch scientists. Gorter and Grendel used the Langmuir trough to demonstrate that, when spread as a monolayer at the air–water interface, all the lipids extracted from a red blood cell occupy twice the exterior surface area of an intact cell. They argued, in a 1925 publication, that this result established the cell membrane as a bilayer composed of two opposing monolayers of lipids (Fig. 2) [4]. Their work was ignored for nearly 50 years, while electron microscopy and X-ray crystallography slowly demonstrated that the lipid bilayer, with membrane proteins inserted, is indeed the universal architecture of the cell membrane. The fluid mosaic model proposed by Singer and Nicholson in 1972 is now firmly accepted as an essentially correct model of the cell membrane [5] (Fig. 3). Gorter and Grendel’s paper is nonetheless important as the logical foundation for much of the modern work on surfactant monolayers and bilayers, and the first example of using the experimentally tractable monolayer geometry to answer questions about a physiologically important bilayer.

air

water

hydrophobic hydrophilic

O H+ O–

An amphiphilic molecule: Palmitic acid Figure 1. Cartoon of amphiphilic molecules oriented at the air–water interface with nonpolar hydrocarbon “tails” in the air and polar “head”-groups in the water. An example of an amphiphilic molecule (palmitic acid) is also shown.

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air

monolayer

water

bilayer

Figure 2. Goerter and Grenden observed that the lipids extracted from a red blood cell occupy twice the exterior surface area of an intact cell. They surmised from this that the cell membrane is a bilayer containing two opposed monolayers.

1.2. Contemporary Topics and Questions In the Introduction, we have attempted to give some flavor for the development of the science of biocompatible films, particularly to explain why, when a comparable field, biochemistry, focused on exact protein structure essentially from its beginnings, nanometer scale studies of biologically relevant surfactant films have barely begun. Essentially, recognition of the scientific interest of biocompatible surfactant films is fairly recent (the importance of bulk surfactant—shampoos, detergents, creams—was recognized much earlier, so a large body of scientific research exists on bulk surfactant physical properties [6, 7]). We should note that bilayers and monolayers are in general physically larger than and more complex compositionally than enzymes; moreover, they must operate in the same challenging

Figure 3. Fluid mosaic model of the cell membrane showing a mixed composition membrane containing membranespanning proteins (bulky, light groups), glycolipids, and glycoproteins (sugar groups represented by branching structures) and different species of lipids in different gray scales. Reprinted with permission from [258], M. Edidin, Nat. Rev. Mol. Cell Biol. 4, 414 (2003). © 2003, Kibron Inc., Finland.


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environment that has made studying the action of enzymes such an enduring challenge. It seems reasonable therefore to anticipate that a similar effort to that devoted to enzyme structure and function will be called for to understand biocompatible surfactant films. However, whereas biochemistry developed in the early days of modern scientific practice as its own, self-sufficient specialty, nanoscale studies of biocompatible films are developing in the context of many already “mature” scientific fields which can be brought to bear on the questions surrounding these films. Thus we may expect that detailed studies of biocompatible surfactant films will remain interdisciplinary, requiring collaboration between specialists of various training (Table 1). Progress should be somewhat faster than that experienced in the development of biochemistry: interfacial surfactant studies will obviously benefit from advances made during the development of these contributing specialties. For example, various optical and higher resolution microscopies, scattering techniques, and biochemical assays can be adapted to surfactant film studies. Under Methods, we give detailed explanations of the five characterization techniques emphasized in our own research: Langmuir isotherms, epifluorescence microscopy, Brewster angle microscopy, atomic force microscopy, and surface viscometry. The reader is encouraged to consult additional references for clarification of other techniques. The study of biocompatible surfactant films raises specific questions; an obvious one is what is meant by “biocompatibility”. A widely accepted definition of biocompatibility is “the ability…to perform with an appropriate host response in a specific application” [8]. This definition is accurate and encompasses a broad range of applications, even those in which host rejection of the biomaterial is a desired response, but it offers little guidance in how to evaluate or improve biocompatibility. To our knowledge, clear rules of biocompatible material design have yet to be formulated [9]. Not surprisingly, given the confusion surrounding the very meaning of biocompatibility, there is also little consensus on which measurements are necessary in order to understand and predict the fate of a surfactant film in a physiological environment. However, it is clear that classifications resulting from traditional macroscale surface characterization, such as degree of hydrophobicity and hydrophilicity established through contact angle measurement, are useful but insufficient [8, 10, 11]. Nanoscale structure of surfactant films is thus an extremely active area of current research, particularly the nature and effect of fluidity, phase segregation, and defects within surfactant films. The desirability of interfacial charge, film dynamics, such as mechanisms of film degradation (desired and undesired), and timescale—how quickly a functional film can and should react to environmental changes—are also open questions. Some of the modern interest in bilayers and monolayers stems from our tardy acknowledgement that nature has chosen this architecture for the cell membrane and thus bilayers have physiologically important roles. Indeed, between 45% and 60% of all current drug targets are integral membrane proteins with membrane-dependent physiological activity [12, 13]. There is also interest due to the recognition that important physiological reactions occur at a well-defined interface between two media; surfactants, which naturally congregate Table 1. Subjects pertinent to the design of biocompatible interfaces and corresponding specialistsa . Theme

Practitioner

New or modified biomaterials Mechanics of materials, defects Surface modification, surface analysis Proteins at interfaces, immobilization of biomolecules Assessing the toxicological potential of materials Design of biological reactions at the material interface Understanding cellular reaction to biomaterials Testing biological reaction in animals Evaluation and implantation in humans Compliance with legislation to protect the consumer Final packaging

Polymer chemist, materials scientist Materials scientist, mechanical engineer Surface scientist Biochemist toxicologist Molecular biologist, computational chemist Cell biologist Veterinary surgeon Physician Regulatory and legislative specialist Sterilization and packaging specialist

a

Reprinted with permission from [8], B. D. Ratner, Macromol. Symp. 130, 327 (1998), © 1998, Wiley-VCH.

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at interfaces, are logical candidates to use in promoting or discouraging these reactions. However, attempts to characterize the cell membrane, to control the activity of membrane proteins, and to otherwise influence physiologically relevant interfaces have revealed the profound influence of nanometer-scale structures. Particularly good examples of this arise in research aimed at controlling the “foreign body reaction”—i.e., inflammation, scarring, and collagenous encapsulation—triggered by implants such as pacemakers, heart valves, artificial hips, etc. A more ambitious goal than mere elimination of the foreign body reaction, forcefully advocated by Dr. Buddy Ratner at the University of Washington, is to promote integration or “healing” of the implant into the host’s body [8, 9, 14, 15]. Either goal requires a detailed understanding of the physiological reaction to an implant’s surface. It is already clear that the signaling pathway in the foreign body response is triggered by the nanometerscale characteristics of the proteinaceous layer which coats an implant surface [8]. Thus attempts to control the foreign body reaction, like studies of the structure and mechanisms operating in cell membranes and studies of integral membrane proteins, increasingly focus on the nanometer scale. Another niche has recently opened for thin, biocompatible films in the field of coatings for microfluidic devices. Fouling of flow systems has long been a major problem in industry and medicine, impacting water filtration, heat transfer systems, and surgical implants such as shunts [10]. Passivation of the plastic, ceramic, and metal surfaces involved in these systems has often been satisfactorily achieved; however, advances in microfabrication techniques should permit us to use much more compact, efficient devices to accomplish the same ends. Many proposed devices approach cellular dimensions; thus even intermittent adhesion of just a few cells could block flow in one or more device “channels”, seriously compromising function. To avoid catastrophic device failure, passivation coatings will need to approach molecular dimensions and increase coating uniformity while maintaining the strength, durability, and uniformity of their macro-scale predecessors. Surfactant monolayers and bilayers are natural candidates in this area [10]. The nanoscale properties of the lung surfactant monolayer, which has an explicitly dynamic physiological role, are another growing area of research. At the end of this chapter we summarize our own and others efforts to create robust structure–function relationships for these films, which in monolayer form coat the mammalian lung with a complex mixture of lipid, fatty acid, and peptide, lowering surface tension to permit easy breathing [16, 17]. Recent work has given new insight into the roles played by different components in native lung surfactant [18–23], the importance of micrometer and nanometer scale domain organization within the monolayer [24–35], and novel mechanisms by which to control monolayer viscosity [36, 37] and dynamic respreading [21, 29, 31, 34, 38, 39], two key parameters in lung surfactant functionality.

2. METHODS 2.1. Isotherms History Langmuir troughs have been used for almost a century to study films at the air–water interface. The first “Langmuir” trough was invented by a German housewife, Agnes Pockels, who used it to measure the minimum interfacial area required for an oil film on a water surface. She communicated her results to Lord Rayleigh, who facilitated publication of her work [2]. Some years later, Dr. Irving Langmuir used an improved version of the device to study monolayers of fatty acid on a water surface [3]. His work achieved much wider recognition than Pockels’, with the result that the instrument she invented became known as the Langmuir trough.

Mechanics There are currently several commercially available Langmuir troughs (e.g., Nima Technologies, Kibron, KSV, etc). The Nima 301S in Figure 4 demonstrates the essential features: (1) a well made of an inert material to hold the surfactant film and water or other subphase;


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Figure 4. NIMA 301S with confined area for surfactant (well), barriers, and surface tensiometer labeled 1, 2, and 3, respectively. The copper inlets on the left permit water to flow through an aluminum base beneath the Teflon trough, an inexpensive method of temperature control. A surfactant monolayer atop a water surface can be symmetrically compressed or expanded. Reprinted with permission from NIMA Technology Ltd.

(2) a motorized, movable barrier to compress the surface film while permitting the subphase to remain evenly distributed in the well; (3) a surface tension measuring device (tensiometer). Not shown is the computer with software and hardware required to control the barrier motors, supply a control signal to the heating/cooling devices used to maintain a fixed trough temperature, and convert the electronic signals of the barrier motors and surface tensiometer into the surface pressure versus area plot or isotherm exemplified in Figure 5. For a single species of surfactant of known molecular weight, or for a surfactant mixture with a calculated number-average molecular weight, an isotherm indicates the average area occupied per molecule at each surface pressure. The gradient of the isotherm also provides an indication of the surfactant’s two-dimensional phase (gradient in the gas phase ∼ 0 < gradient in the liquid phase < gradient in a solid composed of tilted molecules < gradient in an untilted

Surface Pressure (dyn/cm)

25

20

15

10

5

0 19

20

21

22

23

Area/Molecule

24

25

26

(A2)

Figure 5. Isotherm of palmitic acid on 25 C water. The arrow points to a characteristic kink in the isotherm which occurs at the transition from a tilted solid at lower pressures to an untilted solid at pressures above 22.55 dyn/cm. Reprinted with permission from [259], I. R. Peterson et al., Langmuir 8, 2995 (1992). © 1992, American Chemical Society.

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solid phase). Regions in the isotherm where the gradient goes to zero or changes abruptly from one finite value to another identify the surface density and pressure at which phase transitions occur.

Uses Isotherms are a standard method of characterizing the phase behavior of a new surfactant. Isotherms are also used to evaluate molecular interactions; for instance, the surface pressure at which a dissolved protein can insert into an established film is routinely used as an indication of the strength of attraction between a particular surfactant and protein [40–45]. The viscous or elastic response of a monolayer to area dilation can also be measured simply by changing the area available to a monolayer and measuring the time-dependent response of the surface pressure [46–48]. Various methods have been developed in conjunction with the trough to measure monolayer shear viscosity as well [36, 49–53]; we return to these later in the section devoted to shear viscosity of monolayers. However, a Langmuir trough alone cannot identify surfactant structure; additional measurements must be performed. Spectroscopic [11, 54], scattering [55], imaging [32, 56, 57], and surface potential [58, 59] techniques have thus been adapted for use with the Langmuir trough. For example, the isotherm of pure palmitic acid in 25 C purified water in Figure 5 shows a kink at a surface pressure of approximately 22.5 dyn/cm. This kink indicates that a phase transition is taking place, but it is not possible to determine from the isotherm alone what the structures in either the beginning or ending phase are. Various complementary techniques have been developed to provide this structural information.

Complementary Techniques Grazing incidence X-ray diffraction data of palmitic acid under these conditions show diffraction peaks for surface pressures above 2–3 dyn/cm, indicating that the prekink phase is crystalline. Further, the orientation of the diffraction peaks indicate that the solid phase is composed of a centered rectangular lattice crystal in which the long axis of the palmitic acid molecules is on average tilted with respect to the surface normal; this tilt gradually decreases as surface pressure is increased, disappearing at the kink in the isotherm [32, 55]. Similar conclusions could be drawn using Brewster angle microscopy images; before the kink in the isotherm, image contrast varies strongly within the plane of the monolayer or as the trough is rotated (Fig. 6A), indicating a plane composed of randomly oriented crystals each made up of tilted molecules. After the kink, image contrast and dependence on trough orientation disappears, indicating molecular orientation is parallel to the surface normal. As surface

A

B

100 µ

Figure 6. Low-resolution Brewster angle microscopy images of a palmitic acid monolayer on 22 C water. (A) Before the kink in the isotherm, contrast varies strongly throughout the image due to the random orientation of crystallites of tilted molecules with respect to the illumination light. (B) After collapse, the image is uniform except where extremely bright three-dimensional collapsed surfactant appears, indicating that the monolayer now comprises crystallites of untilted molecules.


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pressure increases further, the monolayer collapses into coexistence with surface-associated bulk surfactant, which appears as extremely bright clusters hundreds of micrometers long in the BAM image (Fig. 6B). In the above example, both BAM and GIXD provide the same qualitative information about the crystalline phase of the surfactant, but only GIXD data can be quantitatively analyzed to determine precise dimensions and orientation of the molecular crystal formed. On the other hand, a basic BAM microscope is relatively inexpensive and simple to construct, and BAM images provide data at every point in the isotherm, not just the crystalline regions. This is generally true of microscopy, and we thus rely on imaging as our primary characterization technique. The following three sections provide a detailed explanation of the imaging techniques: in-situ epi-fluorescence and Brewster angle microscopy of monolayers at the air–water interface and atomic force microscopy of monolayers and bilayers transferred to a solid substrate. In a fourth section, we review modern methods of interfacial rheometry.

2.2. Epi-fluorescence Microscopy Fluorescence microscopy (FM) is the imaging technique most often employed to observe surfactant films. FM has been used to characterize lipid organization and peptide–lipid interactions in monolayers at the air–water interface and on solid supports [31, 34, 60–66], to monitor formation of solid-supported bilayers [67, 68], and to study in detail free air bubbles and vesicles [69, 70]. FM also allows assessment of dynamics through more quantitative techniques such as fluorescence recovery after photobleaching (FRAP) (a measurement of molecular diffusion made by observing the time required for fluorophores to diffuse into a deliberately photobleached area of membrane) [71–75]. FM can also be used to measure molecular association through colocalization of differently labeled molecules of interest [76] or through the more quantitative measurement of fluorescent resonant energy transfer (FRET) between appropriate dye pairs [77–80]. The last few decades have seen considerable instrumental development in this essential tool of modern biology. Although most biologists prefer to use inverted microscopes with high numerical aperture oil- or water-immersion objectives, monolayers on a Langmuir trough are most easily observed with an upright epi-fluorescence microscope using dry, long working distance objectives with moderate numerical apertures. Observation of a fluid interface also faces demanding vibration isolation requirements and requires faster exposure times than work on most other specimens. In this section we examine solutions to these challenges.

Physics of Fluorescence and the Fluorescence Spectrum We begin with a brief review of the characteristic features of a fluorescence spectrum. Fluorescence emission results from absorption of light of sufficient energy to move a molecule into an electronically and vibrationally excited state. Excitation is swiftly followed by relaxation to the lowest vibrational state available within the excited electronic state. Approximately one nanosecond after its absorption of light energy, the molecule will either emit a photon, returning to the ground electronic state, or enter a “forbidden” excited state (triplet state) in which the molecular bonds may be broken, destroying the ability to fluoresce; the latter process is called photobleaching. Due to the loss of energy through vibration, the emitted photon is always of lower energy (longer wavelength); the difference in wavelength between peak absorption and emission is called the Stokes shift (Fig. 7). The Stokes shift gives fluorescence a competitive advantage over other imaging techniques, since a well-chosen dye can move the molecular signal into an uncrowded spectral region, increasing signal to noise. Besides an acceptably large Stokes shift, at least five other parameters should influence dye choice: (1) the presence of a covalent or other highly selective binding between the dye and the molecule of interest; (2) , the absorption coefficient at excitation wavelengths; (3) the ratio of photons absorbed to photons emitted or dye quantum efficiency (QE); (4) , the average number of cycles before irreversible photobleaching occurs; (5) the degree of spectral overlap between dye molecules in multicolor experiments. Most of this information is readily available from vendors (e.g., www.probes.com). Although

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Relative Intensity

Stokes Shift

Absorption (Excitation) Fluorescence Emission

300

400

500

600

700

Wavelength (Nanometers) Figure 7. Excitation and emission spectra of a model fluorophore with moderate Stokes shift. Figure adapted from the Molecular Expressions website with the kind permission of Dr. Michael Davidson (http://microscopy.fsu.edu).

directed research has resulted in fluorophores with significantly improved binding, , QE, and , spectral overlap between dyes remains a significant problem. Both absorption and emission peaks are intrinsically broad (cf. Fig. 7) because a fluorescent molecule can absorb/emit any photons which have the energy to move the molecule from any vibrational state in the ground/excited electronic state to any other vibrational state in the first excited/ground electronic state. In multicolor experiments therefore, the spectral characteristics of the imaging system are crucial.

The Fluorescence Microscope The purpose of a fluorescence imaging system is to gather as much of the light emitted by the specimen as possible and project an image of that light onto an appropriate detector. Thus the microscope must deliver satisfactory spectral and spatial resolution. The architecture of an epi-fluorescence microscope, shown in Figure 8A, is better for this purpose than a transmitted light microscope configuration (Fig. 8B). Two essential pieces of a modern epi-fluorescence microscope are not shown: a computer with the appropriate software and hardware to control mechanized components and an electronically controlled shutter after the lamphouse. In quantitative FM, it is absolutely essential to be able to control exposure time with millisecond accuracy; intensity comparisons between colors or as a function of time are impossible without this capability. Most modern microscope control software/hardware packages also offer at least basic image arithmetic capability. Two packages which we have been satisfied with are Metamorph by Universal Imaging Corporation and Image Pro Plus by Media Cybernetics. For image manipulation alone, the shareware NIH Image works well.

Light Sources Spectral response is primarily determined by a convolution of the spectral characteristics of the light source, dye molecule, optical filters housed in the type of turret shown in Figure 8A, and the detector. As costs have decreased and reliability improved, more researchers are turning to lasers to obtain truly monochromatic excitation light; however, the most economical and commonly used sources are still high-intensity mercury or Xenon arc lamps. For quantitative multicolor work, many researchers prefer the more uniform spectral output of a Xenon source; however, images collected using many common dyes will be significantly brighter with a mercury source due to the strong overlap of mercury emission and dye absorption peaks (Fig. 9).

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Emission Light Barrier Filter

CCD Camera

Arc-discharge Lamphouse

Eyepieces

Mercury (HBO) Lamp

Filter Optical Block Turret Breathshield (UV Shield) Objective Stage

Objective

Excitation and Emission Light (Mixture) Specimen

Microscope Frame

Condenser

Condenser Turret Field Lens

Excitation Light

Base

Excitation Filter

Figure 8. (A) Epifluorescence microscope with cutaway showing incident and emitted lightpath. (B) Transmitted light fluorescence showing intermingling of excitation with emitted light. Adapted from the Molecular Expressions website with the kind permission of Dr. Michael Davidson (http://microscopy.fsu.edu).

100

365 436

Mercury 546

405 % 337

0

300

577 and 579

900

600

100

Xenon

828

%

0 300

600

900

Figure 9. Relative intensity of mercury and xenon spectra. The left and right shaded columns denote wavelengths of significant absorption probability for two common fluorophores, cascade blue and Texas red, respectively. Note that a relatively narrow filter used with the mercury source, appropriately centered on an emission peak, would deliver more excitation energy than a similar filter used with a xenon source. Adapted from J. Reichman, “Handbook of Optical Filter for Fluorescence Microscopy (HB1.1),” Chroma Technology Corp (2000) with permission from Chroma Technology Corp.

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Optical Filters Fluorescence Cubes Figure 9 illustrates that both lamp and dye spectral characteristics are quite broad; however, fluorescence derives its advantage as an imaging technique from separating the light originating from the molecule of interest from that produced by other dyes, sample autofluorescence, or ambient light. This separation is accomplished primarily by the three filters contained in a filter block or cube housed in the microscope filter turret (Fig. 8A): the excitation filter, the dichroic mirror, and the emission filter. The excitation filter should be chosen to transmit only those wavelengths which will most efficiently excite the dye molecule; the dichroic mirror, at an angle of 45 degrees from the incoming light direction, must both reflect the excitation light down onto the sample and transmit the light emitted by the fluorescing dye; the emission filter blocks any residual excitation light transmitted by the dichroic, while passing most of the light emitted by the dye. Figure 8A shows the filter turret position in an Olympus epi-fluorescence microscope, while Figure 10A sketches the cube geometry and spectral effect; Figure 10B shows the spectral transmission curves for the filters in a commonly used Olympus cube, the U-MWIBBP. The U-MWIBBP is called a “narrow-pass” or “band-pass” cube because the emission filter only passes wavelengths in the narrow range 515–550 nm to the detector. Narrow-pass cubes are the appropriate cube choice for quantitative FM in which the dye signal must be rigorously separated from all other light sources; however, for qualitative work or when the eye will be the detector, a long-pass filter will create a brighter, more pleasing image.

Objectives Both spatial resolution and image brightness are dramatically affected by the numerical aperture (NA) of the microscope objective used, as shown in Table 2, where NA is defined as the product of the index of refraction between objective and sample multiplied by the sine of the half-angle of light collected by the objective (Fig. 11) and Mag is the specified magnification of the objective (e.g., 10 for a 10× objective). Image collection is optimized by using the lowest magnification, highest NA objective appropriate for each experiment. The highest numerical apertures are achieved with oil immersion objectives, i.e., objectives used with a small drop of oil bridging the submillimeter working distance separating the outermost objective lens from a fluid-immersed sample resting just beneath a coverslip. However, this is not a practical geometry for observing a surfactant film floating freely on a (B) 100

To Detector

Emission Filter Dichroic Mirror Excitation Filter

Lamp Light

Transmittance (%)

(A)

BA 515–550 BP 460–490 DM 505

50

0 To sample

350

400

450

500

550

600

650 nm

Wavelength U-MWIBBP Cube Figure 10. (A) Geometry of a fluorescence cube, with lamp light incident from the right encountering first an excitation filter, then a dichroic mirror tilted at 45 to reflect the excitation light down onto the sample; light emitted by the sample passes through the dichroic and emission filter to the detector. (B) Olympus U-MWIBBP cube, suitable for use with FITC or analogous dyes. Both the excitation filter (BP) and the emission filter (BA) are narrow-pass filters. The dichroic mirror, DM505, is specified to block 50% of photons with a wavelength of 505 nm but transmits 80% of light with wavelength greater than about 530 nm. Adapted from the Molecular Expressions website with the kind permission of Dr. Michael Davidson (http://microscopy.fsu.edu).


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Table 2. Relationship between numerical aperture, spatial resolution, and image brightness of a microscope objective. Spatial resolution Resolution =

Image intensity

122 NA

I∝

NA 4 Mag2

water surface. Although some researchers have chosen to equip the bottom of their troughs with a transparent window, the objective must still focus through a fairly thick water layer to image the surfactant film. Thus a long-working distance objective is required, whether using an upright or inverted microscope. However, affixing a window to the trough body introduces the possibility of contamination from the glues and sealants used to attach the window and leakage if and when these sealants fail or the window breaks; we thus argue that monolayer imaging is best accomplished by using an upright microscope equipped with relatively longworking distance, dry objectives. Since objective diameter (A in Fig. 11) must be limited in order to permit interchangeability, long working distance objectives typically have smaller NAs and thus poorer resolving and collecting power. In our work, we rely on either a 10× objective with an NA of 0.4, or a 40× objective with an NA of 0.6. Our resolution using the 40× objective is therefore about half a micrometer, three times worse than that achievable with a good oil immersion objective (NA of 1.4); however, the real disadvantage arises in signal to noise: images are almost 30 times dimmer.

Cameras From the preceding analysis, monolayer imaging might be expected to require a much more sensitive and expensive camera than most other biological research. However, in practice, most users of fluorescence microscopy want very sensitive detectors. For example, phototoxicity in live cell work (focused excitation light can be more than 1000× a typical solar flux [81]) motivates cell biologists to drastically limit exposure time during image collection. Single-molecule FM similarly places extreme demands on detector sensitivity and speed. Thus surfactant monolayer work has benefited from the general drive to develop highly sensitive, fast detectors. Modern, moderately priced cameras (∼$10–20 000) typically have detector quantum efficiencies in the visible of 40% or greater; more expensive, back-thinned cameras have efficiencies close to 1. However, camera speed varies widely due to differences in image read-out time. In monolayer work, experimental conditions change as quickly as one can compress or expand trough surface area, so image collection needs to occur at video rate or faster to minimize blurring. Angular Apertture

A Light Cone

A µ

Working Distance

NA=n*sin(µ) Figure 11. Graphical illustration of numerical aperture or NA and working distance for a microscope objective. Adapted from the Molecular Expressions website with the kind permission of Dr. Michael Davidson (http://microscopy.fsu.edu).

14


Most cameras use the relatively slow line transfer; faster cameras use the interline transfer geometry, in which every other line of pixels is masked. In an interline transfer camera, the first step in reading out an image for processing moves the electrons in each exposed pixel well to its corresponding pixel in the masked line of pixels just beneath it. This transfer is very quick, but the next step of reading out the electrons from the masked line can take many tens of milliseconds, seriously limiting frame rate. A much faster geometry is the frame transfer geometry, which can achieve frame rates many times that of video cameras; this type of camera is very good for experiments requiring a series of fast, timed images such as FRAP.

2.3. Brewster Angle Microscopy Introduction Brewster angle microscopy (BAM) is a noninvasive imaging technique based on reflectivity of light at the Brewster angle. It can provide in-situ, instantaneous information about the domain morphology of macroscopic structures associated with phase transitions in Langmuir monolayers at the air–water interface [82–87] and Langmuir–Blodgett films deposited on solid substrates [88]. Film thickness or molecular tilt angle can also be derived from the relative intensities in the BAM images [89–92]. This section outlines some of BAMs theoretical underpinnings, various designs, and recent uses.

Components of the BAM BAM was invented as an alternative to fluorescence microscopy, which requires the addition of a fluorescent probe that may change the phase behavior of the monolayer under study [93, 94]. BAM can also image structures formed within the solid phase of a monolayer [83, 95], where most fluorescent probes would normally be excluded [93]. The basic principle underlying BAM is the strong dependence of reflectivity of p-polarized light from an interface on both the incidence angle and index of refraction. At the Brewster’s angle for a homogeneous interface, reflectivity vanishes; any material added to that interface will then be the only source of reflection. A home-built, combination BAM–Langmuir trough setup is shown in Figure 12. The incident light is produced by a 30-mW 690-nm diode laser (a), whose polarization axis is oriented in the plane of incidence. A Glan-Thompson or Glan-laser polarizer (b), mounted on a rotation stage for fine-tuning, further defines its polarization. To aid in beam positioning and

Figure 12. Home-built BAM–Langmuir trough instrument: (A) 690 nm; 30 mW diode laser; (B) Glan-Thompson polarizer; (C) iris; (D) lens tube; (E) absorber; (F) analyzer; (G) zoom lens; (H) iris; (I) near-IR CCD camera; (J,K) rotation stages; (M) motor.

15


reduce scattered light, an iris (c) may be placed in the beam path, and the lens tube (d) may house a beam expander to provide uniform illumination at low magnification. Omitting (c) and (d) is, however, often necessary in order to provide adequate incident light intensity at high magnification. A small piece of dark glass, placed at the bottom of the trough, functions as an absorber to reduce scattered light from the trough. It is also tilted to direct specular reflection away from the detector. The reflected light path contains an analyzer, i.e., another Glan-Thompson polarizer, an iris, a zoom lens (f, g), and a near-IR sensitive CCD camera (i). The zoom lens has a large depth of field, which is the interval around the working distance that appears in focus. This is to maximize the in-focus area of the field of view (FOV), because the oblique angle of incidence puts most of the FOV outside the working distance of the lens. Goniometers (j, k), mounted on vertical translation stages, allow adjustment of detector and incident height and angle. Finally, a motorized micrometer translation stage (l) scans the zoom lens’ focus to obtain in-focus images of the whole area.

Reflection and Transmission of Polarized Light at an Interface Vector Illustration Light is an electromagnetic wave, composed of orthogonal electric and magnetic field vectors whose magnitudes oscillate sinusoidally. The light wave propagates in a direction perpendicular to these vectors. Either the electric or magnetic field vector may be used to specify the polarization angle; typically, one refers to the direction of the electric field vector. Light whose electric field is parallel to the plane of incidence is called p-polarized, where light whose electric field is perpendicular to the plane of incidence is called s-polarized. In this text, vectors are indicated by arrow superscripts (E . When light strikes an interface, the plane of incidence is defined as the plane formed by the incident, reflected, and transmitted beams. These are shown as Ei , Er , and Et in Figure 13. In the general case of reflection from an interface, the incident polarization is at an arbitrary angle () to the plane of incidence. This polarization vector is a linear

p Er

Ei

s

θi

Air (n1) Substrate (n2)

Et

Ei Et

Ep α

α α

Er

Es

Figure 13. Reflection at arbitrary incidence and polarization angles. The polarization of the reflected light is enriched in s-component, while that of the transmitted light is enriched in p-component. This causes a change in polarization angle ().

16


combination of two components: Ep , parallel to the plane of incidence, and Es , perpendicular to the plane of incidence. The relative magnitudes of Ep and Es differ in the reflected and transmitted beams, resulting in a change of the overall polarization. p-polarized light incident at Brewster’s angle on an ideal substrate produces no reflected beam. For a real substrate, the interface is not ideally smooth or sharp; here, Brewster’s angle of incidence results in a nonzero minimum of reflectivity. The simpler case of the ideal interface is presented first. Dipole Illustration The reflected and transmitted beams arise from oscillations that are stimulated in the medium by the incident light. The direction of these beams can be calculated from the law of reflection (Eq. (1)) and Snell’s law (Eq. (2)) where n1 is the refractive index of the incidence medium (usually air), n2 is the refractive index of the medium through which the light is transmitted, and the angles between the incident, reflected, and transmitted beams with respect to the surface normal are given by i , r and t . i = r

(1)

n1 sin i = n2 sin t

(2)

The electric field vector’s oscillation in amplitude stimulates dipole oscillations in the medium. For p-polarized light at Brewster’s angle of incidence, these oscillations have their axes aligned in the reflected beam direction (Fig. 14). They therefore contribute only to the transmitted beam, since the electric fields produced by oscillating dipoles do not propagate along the dipole axes. Derivation from Maxwell Equations Maxwell’s equations describe the behavior of electric and magnetic fields within a medium [96, 97]. a • E =

1 0

(Gauss’s law; here =charge density)

b • B = 0 c × E = −

B t

(3)

(Faraday’s law)

E d × B = 0 J + 0 0 t

(Ampère’s law with Maxwell correction)

Ei Er

θB n1 n2

Et Figure 14. p-Polarized light at Brewster’s angle of incidence. p-Polarized light incident at B stimulates dipole oscillations whose axes are aligned in the reflected beam direction. These produce electric fields that contribute to the transmitted beam but not the reflected beam.


17

Boundary conditions derived from these equations require that the total electric and magnetic fields (both parallel and perpendicular components) are equal across the interface. They are given below for the absence of free surface charge or current, as is the case for the air–water interface. The subscript 1 represents one side of the interface (air), while the subscript 2 represents the other side. a 1 E1⊥ − 2 E2⊥ = 0

c E1 − E2 = 0

b B1⊥ − B2⊥ = 0

d

1 1 B1 − B =0 1 2 2

(4)

Both incident and reflected waves contribute to the total fields E1 and B1 in the incidence medium, while the total fields E2 and B2 in the transmittance medium arise from the transmitted wave alone. For the p-polarized wave, these relationships are given by Eqs. (5) and (6): Bi + Br = Bt

(5)

Ei cos i − Er cos r = Et cos t

(6)

The magnetic fields can be rewritten in terms of the electric fields according to Eq. (7): c E = vB = B n (7) n B= E c Equations (5) and (6) can then be expressed as follows, where furthermore both i and r , being equal, are written as the same angle : n1 Ei + n1 Er = n2 Et

(8)

−Ei cos + Er cos = −Et cos t

(9)

The transmitted electric field Et can be eliminated from Eq. (9) by rearranging Eq. (8), which then gives rise to the reflection coefficient rp = Er / Ei (Eq. (10)). n1 E + Er = Et n2 i n − Ei cos + Er cos = − 1 Ei + Er cos t n2 n1 n − Ei cos + Er cos = − Ei cos t − 1 Er cos t n2 n2 n n − Ei cos + 1 Ei cos t = −Er cos − 1 Er cos t n2 n2 n1 n1 − Ei cos − cos t = −Er cos + cos t n2 n2 cos − E rp = r = Ei cos + rp =

n1 n2 n1 n2

cos t cos t

n cos − cos t n cos + cos t

(10)

where n = n2 / n1 . The reflectance of the s-polarized light can be derived in a similar fashion, by rearranging Eq. (11). The transmission coefficients are derived by eliminating Er rather than Et . The reflectivity rp of p-polarized light is given above in terms of the angles of incidence and

18


transmittance. Brewster’s angle of incidence, B , at which rp is zero, is obtained by multiplying the numerator and denominator by n writing rp in terms of the incidence angle, and using Snell’s law to eliminate the transmittance angle. n1 sin i = n2 sin t (Snell’s law) n sin i = 2 sin t = n sin t n1

n2 cos i − n cos t rp = 2 n cos i + n cos t cos2 t = 1 − sin2 t

(trigonometric relationship) cos t = 1 − sin2 t n cos t = n2 − n2 sin2 t 2 n cos t = n2 − n sin t n cos t = n2 − sin2 i

Thus replacing n cos t , 2

rp =

n cos i − n2 cos i +

n2 − sin2 i n2 − sin2 i

(11)

rp is then set to zero, and this equation solved for sin2 B . n2 cos B − n2 − sin2 B n4 cos2 B = n2 − sin2 B 0= 2 2 4 2 2 n cos B + n2 − sin2 B n 1 − sin B = n − sin B n4 − n2 = n4 − 1 sin2 B n2 n2 − 1 = n2 − 1 n2 + 1 sin2 B n2 + 1

1 = 2 n sin2 B 1 2

sin B

−1=

1 n2

(12)

The Brewster angle condition (Eq. (14)) arises by applying to Eq. (12) the trigonometric relationship in Eq. (13). csc2 = 1 + cot2 1

1 tan2 sin tan B = n 2

(13)

−1=

(14)

The Fresnel equations, or coefficients for reflection and transmission, are rewritten below for ready reference. They give the amplitudes of the p- and s-polarized components of both the reflected and transmitted waves. n2 cos i − n2 − sin2 i cos i − n2 − sin2 i rs = (15) rp = n2 cos i + n2 − sin2 i cos i + n2 − sin2 i tp =

2n cos i n2 cos i + n2 − sin2 i

ts =

2 cos i n2 cos i + n2 − sin2 i


19

These equations apply at an ideal or Fresnel interface, where the surfaces are smooth and the transition from n1 to n2 is abrupt. The air–water interface approximates such a system.

Factors Contributing to Reflected Intensity Figure 15 shows the effects of variation in surface layer thickness and molecular tilt angle, two factors that produce contrast in the BAM image. As the optical axis of the detector is rotated with respect to that of the molecular layer, tilt-angle-dependent intensities should change from light to dark while thickness-dependent intensities will remain constant [88, 98]. This rotation may be achieved either by rotating the sample around the surface normal or rotating the analyzer that is placed in front of the detector. The following subsections present the mathematics behind the intensity dependence on these two surface characteristics. Layer Thickness The general form of the harmonic wave equation may be written as follows (Eq. (16)): x t = Aeikx−

t

(16)

where A represents the amplitude, k (the propagation constant) is equal to 2!/, and kx − t is the phase of the wave, expressing its dependence on space and time. This wave has a real component, A coskx − t , and an imaginary component A sinkx − t . The p- and s-components of a reflected wave, which are themselves waves, may be expressed in this form (Eqs. (17) and (18)). Rpp = rp ei#p Rss = rs e

(17)

i#s

(18)

Here, #p and #s represent p t and s t, or the phase shift of each component with respect to the incident wave. The ratio Rpp /Rss (Eq. (19)) provides all information about the reflected wave, including its direction of polarization and whether its polarization is linear or elliptical. Rpp rp = ei#p −#s = cos % + i sin % Rss rs

(19)

For linearly polarized light, the relative phase shift % = #p − #s is zero, and the ratio Rpp /Rss is equal to the ratio of the reflection coefficients = rp /rs . Elliptically polarized light will exhibit a nonzero relative phase shift.

T1

n(z)

T2

n1

n1

n2

n2

n(z)

b a

Figure 15. Effect of monolayer characteristics on reflected intensity. (a) Multilayer domains (bright) in a continuous trilayer (dark) of 4 ,8-dialkyl[1, 1 -biphenyl]-4-carbonitrile. Reprinted with permission from [89], M. N. G. de Mul and J. A. J. Mann, Langmuir 14, 2455 (1998). © 1998, American Chemical Society. (b) Condensed phase domains of dipalmitoylphosphatidylglycerol (DPPG) at analyzer position of 60 . The variations in intensity result from differences in the azimuthal tilt angle of the DPPG molecules. Reprinted with permission from [86], D. Vollhardt et al., J. Phys. Chem. B 104, 4115 (2000). © 2000, American Chemical Society.

20


Upon reflection at the Brewster angle, a phase shift of !/2 is produced between Rpp and Rss . The p-polarized reflected wave therefore becomes purely imaginary. This is shown in Eq. (20), where the bar superscript over ¯ B indicates that the polarization has changed from linear to elliptical, and the B subscript indicates Brewster’s angle of incidence [99, 100]. Rpp B = i¯ B Rss B

(20)

The existence of p-polarized reflection depends on whether this imaginary component also vanishes. Drude first defined the ellipticity ¯ as the ratio of p and s amplitudes in elliptically polarized light, rewriting it as in Eq. (21) [100, 101]. This quantity differs from the amplitude ratio , which does not specify polarization. 2 2 ! n1 + n2 + nz 2 − n21 nz 2 − n22 ¯ = dz (21) n21 − n22 − nz 2 For an ideal interface, the refractive index nz is equal to either n1 or n2 at any value of z. The numerator within the integral in Eq. (21) then goes to zero, resulting in zero reflectivity. Monolayer-free areas on a substrate are considered a Fresnel interface. Thus, the use of p-polarized light and Brewster angle incidence in BAM eliminates background reflection from these areas. A monolayer with thickness h introduces a region of differing refractive index nz , which produces a real interface where the transition between n1 and n2 is gradual or interrupted over the region h. The integral is therefore nonzero, resulting in nonzero reflectivity. If the monolayer’s refractive index is a constant n0 , the ellipticity (and therefore the reflectivity) also exhibits thickness dependence [89, 102]: 2 2 !h n1 + n2 n20 − n21 n20 − n22 ¯ B = (22) n21 − n22 n20 2 and rpp as given in Eq. (20) is proportional Then, since the reflectivity is proportional to rpp to the ellipticity, the reflectivity scales as the square of the monolayer thickness [89, 102]

2 r = rpp ∝ h2

(23)

Thicker domains therefore produce more reflection, as seen in Figure 15A, where the bright domains in the image are multilayers atop a dark (continuous) trilayer [89]. Tilt Angle Reflectivity also provides information about the optical properties of the monolayer and the tilt angle of its constituent molecules. A tilted surfactant molecule requires two angles in order to define its position: the polar tilt angle L and azimuthal tilt angle *L . The reflected light intensity depends on these two angles. Figure 15B shows a surfactant monolayer with a multitude of domains, whose intensities vary with azimuthal tilt angle [86]. The dependence of intensity on tilt angle can be illustrated by a vector diagram, as in Figure 16, where the xz plane is the plane of incidence. The monolayer rotates the incoming electric field according to the orientation of the surfactant hydrocarbon tails. The molecules may be tilted out of the plane of incidence, and therefore their orientation (Eq. (24)) has a y-component. s = cos *L sin L xˆ + sin *L sin L yˆ + cos L zˆ

(24)

However, the orientation of the analyzer is such that only p-polarized reflected light is detected. The reflected electric field as seen by the detector is given in Eq. (25). E = cos *L sin L xˆ + cos L zˆ

(25)

Modifying either *L or L changes the reflected electric field’s amplitude, resulting in an intensity shift.


21

E = incident electric field vector E′ = reflected electric field vector O = optical axis of detector

z 90-θB-θL

De

tec

tor

e

z

E′ θL x

x

90-θB

O

urc

So

E θL

φL

θB φL

θL = molecular tilt angle. Constant for each surface pressure. φL = azimuthal tilt angle. Varies among sample domains at each surface pressure. Figure 16. Rotation of the electric field due to interaction with tilted surfactant molecules. The reflected electric field vector E has components along the x and z axes; the detector only “sees” the component of E that lies along O, the optical axis of the analyzer.

Hosoi et al. [98] and Lautz and Fischer [103] extracted information about the molecular tilt angle L from variations in reflected intensity corresponding to different regions in the BAM image. Overbeck et al. [90, 91] and Tsao et al. [92] used modifications of Berreman’s 4 × 4 matrix method [104] to calculate intensity for different values of azimuthal tilt angle *L , using their results to propose model azimuthal tilt angle distributions for observed experimental images.

Image Quality and Information Content in Brewster Angle Microscopy Representative images taken with the BAM, of a line grating and DMPE monolayer, are shown in Figure 17. Since different areas of the object are at different distances from the lens in a BAM microscope (Fig. 18), the upper and lower regions of the image appear blurred. This is most evident in the perpendicular images of the 40 line pairs/mm grating (Fig. 17B), where the ends of the lines appear thicker even though they are a uniform thickness in the actual object. Digital correction of the images (i.e., shrinking or stretching of certain areas in the image) can be applied to remove some of this distortion [85, 88, 89]. Choosing a detector lens with a large depth of field (DOF) also focuses regions at a wider range of distances from the lens. However, DOF is inversely related to resolution, according to Eqs. (26) and (27). The resolution is given by xmin the minimum separation between two just-resolved objects near the focal plane of the lens. A is the numerical aperture, so is the object distance of the lens, and d is the image diameter within which the image is considered in focus. 122 A= (26) xmin DOF =

2Adso so − f f 2 f 4 − A2 d 2 so2

(27)

A long-wavelength laser, requiring a near-infrared-sensitive detector, provides the largest depth of field. Alternatively, scanning the detector’s focus over a range of distances obtains

22


distortion

(a)

(b)

Figure 17. BAM images at high magnification, from the instrument in Figure 1. (a) 40 line pairs/mm grating oriented perpendicular to the plane of incidence. A 2× beam expander (not normally used with the BAM) is included in the incident light path. 128× attenuation filters placed after the incident light iris. Laser power ∼7 mW; FOV 375 × 420 m. (b) DMPE on 20 C water, no beam expander, surface pressure = 4 dyn. Laser power ∼30 mW (full power); FOV 1300 × 1425 m.

lens object

image

w f

O d

f

f = focal length w = working distance d = Depth of field O = optical axis

image distance

image

lens

f

im

ag

ed

im

ist a

ed

e1

ist

an

ce

2

f

nc

ag

object

air water

d w

O Figure 18. Why BAM requires a lens with a large depth of field. (a) Object perpendicular to optical axis. Within the range d, the entire image appears focused at a certain distance from the lens. (b) Object not perpendicular to optical axis. Each point on the object produces a focused image at a different distance from the lens. A distorted image therefore appears in the image plane (see Fig. 17). A large d allows a greater amount of the field of view to appear focused.


23

in-focus images of different regions. These images can then be digitally assembled (Fig. 19) to produce a focused picture of the entire monolayer. This strategy was applied in the earliest BAM [82] further developed by a number of researchers [105, 106] and is commercially available through the German companies Optrel GbR and Nanofilm Technologies. Data acquisition is slower with this method, since the acquisition of each image requires several seconds. Dynamic images of a moving monolayer therefore cannot be obtained by scanning the detector focus.

Modern Versions and Applications of the BAM The Brewster angle microscope has evolved considerably since its initial development (Fig. 20) in the early 1990s. More recent designs seek to improve upon the existing characteristics of the BAM or to modify it for a specific purpose. For example, another creative way to eliminate the distortion due to the limited depth of field of the detector optics involves custom lenses that place the entire field of view at the working distance of the objective (Fig. 21) [107]. Marshall, Dennin, and Knobler employ mirrors to establish the Brewster angle, allowing the laser and detector to be placed parallel to each other [108]; this BAM is useful in applications such as Langmuir–Blodgett deposition, where it may be necessary to conserve space (Fig 22). Harke, Teppner, Motschmann, and Orendi, capitalizing on the similarity between several methods of surface characterization, built a device in which the incidence plane and angles of incidence and detection can be easily changed to perform multiple techniques, including Brewster angle microscopy and ellipsometric imaging [105]. Optrel GbR now markets this device as the Multiskop (Fig. 23) and has recently added an attachment for contact angle measurement. Its use as an ellipsometer has especially benefited researchers in fields as diverse as food science [105, 109, 110] and polymer science [111] and the design of functional materials [112]. Both home-built [113] and commercial BAMs [86, 87, 112, 114, 115] are increasingly employed as experimental tools for surface characterization. This is particularly evident in the development of the MiniBAMplus (Fig. 24) (Nanofilm Technologies), which utilizes the minimum degrees of freedom necessary for obtaining an image and is intended as a standardized method for checking the quality of Langmuir–Blodgett monolayers (www.nanofilm.de). Table 3 shows the 2003 materials only, US dollar cost of assembling a home-built BAM with motorized detector focus, video monitor, and computer (analytical software not included). Commercial BAMs, while often several times the monetary cost, include mechanical features and software that may not be easily or quickly replicated. An investigator planning on

Computer

Framegrabber software

VCR Annotation text software

Camera

Sample Thermistors, pressure sensor in Langmuir trough Figure 19. Example schematic for digital analysis of data.

24


computer Argon Laser

video camera

Ob1

C L5

monomode fiber

L4 L2

θB

Ob2

L1

or

L3

,

cu O

ot

m

P Q

Ob3

A

Figure 20. Early BAM. Reprinted with permission from [82], S. Henon and J. Meunier, Rev. Sci. Instrum. 62, 963 (1991). © 1991, American Institute of Physics.

utilizing Brewster angle microscopy should estimate the value of the time spent in design and programming, as well as administrative and overhead costs associated with obtaining individual components, in order to determine the true economic feasibility of assembling a home-built BAM versus purchasing a commercial one.

2.4. Atomic Force Microscopy Overview The general class of microscopes known as scanning probe microscopes (SPM) are all descendants of the scanning tunneling microscope (STM), which was first described in 1982 [116]. The significance of this development was demonstrated by the Nobel Prize in Physics in 1986. As in all SPMs, the STM operates by bringing a probe into close proximity to the surface to be imaged and then uses some aspect of the probe–surface interaction to develop a feedback loop that also eventually determines the image. For the STM, the probe is a metal tip brought sufficiently close to a conducting or semiconducting surface so that sufficient electrons can tunnel between the surface and the tip. As the tip is rastered across the surface, the most common imaging mode is to adjust the distance between tip and sample surface to maintain a constant tunneling current. This adjustment of the tip-sample surface

M1 L1

L2 D

M2

L3 100 mm

Figure 21. Custom-designed BAM objective, which places the entire field of view at the working distance. Reprinted with permission from [107], C. Lheveder and S. Henon, Rev. Sci. Instrum. 69, 1446 (1998). © 1998, American Institute of Physics.


25

Laser

Camera

F L2

Z Y X

A P L1 M1

M2

10 cm Figure 22. Compact BAM for Langmuir–Blodgett deposition. The detector and laser are parallel; placement of mirrors M1 and M2 produces the Brewster angle condition. Reprinted with permission from [108], G. Marshall et al., Rev. Sci. Instrum. 69, 3699 (1998). © 1998, American Institute of Physics.

becomes the three-dimensional image of the scanned region, with atomic resolution possible for certain surfaces [117–119]. The STM has been an important tool in elucidating the structures of conducting surfaces; however, the use of the STM for biological and organic surfaces has been limited by the inherent lack of conductivity of organic materials, which makes both imaging and interpreting images difficult. The atomic force microscope (AFM), unlike its predecessor the STM, uses the force between the sample and a spring-loaded cantilever as the source for creating the feedback loop necessary to create an image [120, 121]. Since force (and not tunneling) is the imaging mechanism, the conductivity of the sample is unimportant, making the AFM a more general instrument for surface characterization. Over the past decade, the AFM has eclipsed the STM in general utility and has become the most important and one of the most common instruments for studying surface structure and organization at high resolution [122–131]. The inspiration behind the AFM was the realization that it is easy to make a cantilever with a spring constant weaker than the equivalent spring between atoms [120]. While an interatomic spring constant is on the order of 10 N/m, a piece of aluminum foil 4 mm long by 1 mm wide has a spring constant of only 1 N/m. Thus by sensing the angstrom-size displacements of such a soft cantilever spring, one could image atomic-scale topography. Furthermore, the applied force would not be large enough to push atoms out of their atomic sites [132]. As the tip, which is in contact with the surface, rasters across the area of interest, the cantilever is deflected by the variations in the surface contours. In most commercial instruments,

26


The Multiskop a

d

b

c

Figure 23. Commercially available BAM. Optrel GbR’s Multiskop (a) is adapted from Harke et al. The detector and laser are mounted on a two-circle goniometer that allows imaging of substrates at a variety of angles: horizontal (a), vertical (c), and in between (b). Several surface characterization techniques, including Brewster angle microscopy and ellipsometry, can be performed with the Multiskop; it may be used in conjunction with a Langmuir trough (c) and has an attachment for contact angle measurement (d). (a–d) Reprinted from www.optrel.de with permission from Optrel GbR (Dr. Hubert Motschmann). (b) Reprinted with permission from [105], M. Harke et al., Rev. Sci. Instrum. 68, 3130 (1997). © 1997, American Institute of Physics.

Figure 24. Another commercially available BAM. Nanofilm Technologies’ MiniBAMplus (pictured above) is an intermediate-resolution tool for rapid characterization and monitoring of ultrathin organic films. The detector and laser are fixed on the same mount and can be placed above the monolayer for visualization with minimum adjustment of controls. Their BAM2plus (not shown) is a high-end research device that offers greater variability. Reprinted from www.nanofilm.de with the permission of Nanofilm Technologies (Andreas Hadrich).

27

Nanostructure and Dynamics of Biocompatible Surfactant Monolayers and Bilayers Table 3. Components of the home-built BAM in Figure 12a . Component

Company

Product no.

Price

Qty

Light path Diode laser Glan-laser polarizer Iris Lens tube Zoom lens Near-IR CCD CCD power supply

LaserMax Inc. Newport Thorlabs Thorlabs Optem Sony Sony

LAS-200-685-30 10GL08AR1.6 SM1D12 SM1L20 multicomponent XC-E150 DC 700

$628.00 $795.00 $49.00 $18.00

1 1 1 1

$750 $325

1 1

Incident mount Rotation stage Rotation stage Cage to hold laser Horizontal rail Carrier Large angle bracket m translation stage Mounting plates

Newport Melles Griot Thorlabs Newport Newport Melles Griot Newport Newport

RSP-1T 07-TRS-001 multicomponent PRL-24 PRC-3 07 TSH 004 UMR5.25 338284

$130.00 $589.00

1 1

$305.00 $83.00 $81.00 $248.00 $44.00

1 1 1 1 4

$130.00 $589.00 $98.60 $305.00 $83.00 $81.00 $248.00 $176.00

Detector mount Rotation stage m translation stage Motorizer Motion controller Vertical stage Rod assembly Rod clamp Mounting plate Angle bracket

Newport Newport Newport Newport Newport Newport Newport Newport Melles Griot

RSP-2 UMR 8.25 CMA 25CC 861 MVN-80 410-RC 340-RC 410-RCP 07-TSH-004

$203.00 $221.00 $500.00 $209.00 $800.00 $489.00 $84.00 $95.00 $81.00

1 1 1 1 1 1 1 1 1

$203.00 $221.00 $500.00 $209.00 $800.00 $489.00 $84.00 $95.00 $81.00

Image processing Video monitor (b/w) Computer PC monitor

Sony Dell Dell

SSM125 Dimension 2400 M992

$295.00 $499.00 $249.00

1 1 1

$295.00 $499.00 $249.00

TOTAL

Total $628.00 $795.00 $49.00 $18.00 $1, 680.00 $750.00 $325.00

$9, 680.60

a

With a motorized zoom lens replacing the motorized objective scanning feature in Figure 12. List prices (2003) for components are shown in U.S. dollars as a guideline. Additional costs are involved for software, shipping, administration, and overhead.

the cantilever deflection is measured by optically amplifying the motion of the cantilever. This is achieved by bouncing a laser beam off the reflective end of the cantilever to a split photodiode detector [121]. Figure 25 shows a schematic of the “optical-lever” sensing system. A feedback loop then controls the voltage to the vertical (z-direction) piezo element on which the cantilever is mounted so that the force (or deflection) is held constant as the tip is scanned across the surface of the sample with the horizontal (x-direction) piezo element [118]. An image is built up of many such x-scans, each displaced by a fixed distance in the y-direction. The forces that act upon the tip and the sample are responsible for the cantilever deflection and ultimately the image that is produced. However, these forces are not wellunderstood. The dominant attractive force between the tip and the surface is the van der Waals force. Lateral shearing forces caused by dragging the tip can be substantial in some modes of operation, as can be the capillary force resulting from condensation of water or other liquids when imaging in air [133, 134]. These last two forces can decrease image resolution and cause severe damage to the sample or the tip. Imaging under liquids can minimize these capillary forces [121]. To isolate the AFM from external vibrations, the AFM is placed on a concrete or granite block suspended with elastic cords. This setup removes all but the very lowest frequency vibrations from being transferred to the AFM [119]. Most commercial instruments fix the sample to the AFM piezo drives via a magnet; the substrate must be rigidly fixed (glued or taped) onto a magnetic steel disk. In most cases, the substrate is freshly cleaved mica

28


Laser Diode

Quad Photodiode

Beam Path Mirror

Cantilever Substrate Flexible Cantilever

XYZ Piezoelectric Scanner

Sample

Figure 25. Optical sensing system. The laser light bounces off the cantilever, onto a mirror, and then onto the photodiode. This optical lever magnifies the motion of the cantilever, making it possible to sense 0.1 nm motions of the cantilever tip. The split photodiode measures the difference signal between different quadrants of the photodiode, increasing the sensitivity of the system to both height and lateral force variations.

or silicon, but other surfaces such as quartz or graphite can be used as long as they are smooth. AFM tips were originally made out of crushed diamond and glued to the cantilever. Modern AFM tips and cantilevers are fabricated together from silicon, silicon oxide, or silicon nitride using microlithography techniques. Typical lateral dimensions of the cantilever are 100–200 m long, with a thickness on the order of 1 m. Spring constants are in the range 0.1–1 N/m [135]. A schematic of the tips and cantilever is shown in Figure 26. Among the first nonconductors imaged with the AFM were organic thin films [136] and biological crystals [137]; this work was done prior to the development of the optical lever AFM in 1989 [121]. These early AFMs used a tunneling sensor to detect the motion of the cantilever tip and were difficult to align and operate [120]. From the early optical lever instruments came the first commercial AFM instruments in the early 1990s. Further developments lead to AFMs compatible with a variety of environments, including liquids or under vacuum. It is even possible to image surfaces held at low temperatures [138]. Lipid and fatty acid monolayers and multilayers supported on rigid mica, glass or silica substrates have been resolved both in air and in fluid since the AFM was first developed [139–147] and are probably among the best subjects for AFM investigation [25, 124–126, 148–152]. Supported monolayers and bilayers are quite easy to make, are generally flat compared to AFM tip curvatures, and are generally tough enough to image with contact mode AFM. In recent work, there has even been some success in imaging monolayer structures on at a water–air interface [153]. In addition to imaging, AFM has been expanded in recent years as a singlemolecule force sensor. Several excellent reviews have been published that provide in-depth tip

cantilever

Figure 26. Cantilever with tips. The cantilevers and tips are fabricated by using microlithography and etching techniques. The width and lengths of the cantilever legs determines the spring constant of the cantilever. The glass substrates are about 2 mm × 4 mm, while the cantilever legs are 100–200 m.


29

analyses of both the problems and insights of single molecule force spectroscopy [154, 155], which will not be the focus here.

Imaging Modes Significant advances have been made in AFM technology in the last few years. New scanning modes have allowed for imaging of electrochemical, magnetic, and conducting surfaces, as well as blends, composites, and extremely soft surfaces. The following is a survey of some of the available AFM modes. Contact In the conventional contact mode, the tip is in “direct contact” with the surface and then rastered to form an image. The resulting image is a topographical map of the surface of the sample. Of course, exactly what is meant by direct contact on the molecular scale is still rather ambiguous, although there have been both experimental [147, 156] and theoretical studies [157] of what really makes up the molecular contacts that result in AFM imaging. Under ambient conditions, most hydrophilic and high-energy surfaces [133] (such as metals, oxide covered silicon, mica, to name a few typical surfaces used in AFM) are covered by a layer of adsorbed gases (condensed water vapor and other contaminants). When the scanning tip touches this layer, capillary action can cause a liquid meniscus to form and surface tension pulls the cantilever down into the layer. This large attractive force can cause substantial damage to both sample and probe and can create artifacts in image data (Fig. 27) [133]. To overcome this force, the AFM is often operated with the sample and tip immersed in liquid. While this can help eliminate the capillary force, hydrated samples are often substantially softer, and tracking forces can still reduce image quality and cause sample damage [158]. In addition, many samples, such as semiconductor wafers, cannot be immersed in fluid. However, contact mode imaging gives the highest resolution and the most accurate height information and is the only imaging modality that routinely can achieve molecular resolution [124]. Noncontact In noncontact mode, the scanning probe is held a small distance away from the sample (see Fig. 27) [159–161]. This avoids some of the problems associated with contact mode, but creates others of its own. The long-range attractive van der Waals forces and double-layer electrostatic forces acting between the tip and the sample are substantially weaker than the forces accessed in contact mode. These forces are so weak that the tip must be given a small oscillation so that ac detection methods can be used. Hence, the resolution obtained with noncontact mode is substantially lower than can be achieved in conventional contact mode. However, this mode has been useful in imaging soft biological samples and weakly adsorbed surfactant molecules [160] without inducing sample distortions due to the tip.

Figure 27. Comparison of contact mode, noncontact mode, and tapping mode scanning techniques. Strong adhesion forces in contact mode can cause sample damage and surface distortion. Noncontact mode, in which the tip traces contours of constant short-range forces, such as the double-layer electrostatic force, generally gives lower resolution [159, 161]. Tapping mode reduces forces on the surface with a small decrease in resolution [158].

30


Tapping Mode Tapping mode imaging overcomes the limitations of traditional scanning modes by oscillating the cantilever assembly at or near the cantilever’s resonant frequency using a piezoelectric crystal, generally at a frequency of 50 000–500 000 cycles/s [158, 162]. The oscillating tip is then moved toward the surface until it begins to lightly touch the surface. As the oscillating cantilever begins to intermittently contact the surface, the cantilever oscillation is reduced (damped) due to energy losses caused by the surface contact. The reduction in oscillation amplitude is used to identify and measure surface features. This technique prevents the tip from sticking to the surface and causing damage during scanning. Unlike contact and noncontact modes, when the tapping tip contacts the surface, it has sufficient oscillation amplitude to overcome the tip–sample adhesion forces (see Fig. 27). Also, the surface material is not pulled sideways by shear forces, as the applied force is always vertical. In fluids, similar advantages are found [162]. When doing tapping in fluid, the fluid tends to dampen the cantilever’s natural resonant frequency, so instead the entire fluid cell is oscillated to drive the cantilever into oscillation. The other aspects of tapping in fluid are similar to tapping in air. The key advantage of tapping mode imaging over conventional contact AFM is the low forces generated during scanning. Tapping mode has been used successfully to reproducibly image numerous fragile samples [163] and is probably the most common mode of imaging used in biological samples today. Lateral and Chemical Force Microscopy This SPM technique identifies and maps relative differences in surface frictional characteristics. Lateral force microscopy (LFM) is particularly useful for differentiating among materials on surfaces, such as different components in a polymer blend, composite, coating, or other surface layers. In the LFM technique, the probe is scanned perpendicular to its length (see Fig. 28). The torsion, or twisting, of the cantilever supporting the probe will increase or decrease depending on the frictional characteristics of the surface [164]. The AFM can simultaneously measure and record topographic data and the lateral force data. LFM can be extremely useful for identifying surface compositional differences, where the materials have differing frictional characteristics, but the topography is relatively undifferentiated [165]. In another use, the LFM can be used as a chemical force microscope (CFM). Here, the tip is functionalized with one chemical species and scanned over a surface to detect adhesion differences between the species on the tip and those on the surface of the sample [166–168]. This has evolved into the force spectroscopy measurements in use today using various ligands or receptors bound to the AFM tip or substrate [169].

B

A

D

C

Scan Direction

Figure 28. Movement of the cantilever in lateral force microscopy.


31

Sample Preparation Samples for the AFM must be firmly mounted to a solid substrate, regardless of the imaging mode, except in unusual circumstances [153]. In our lab, this generally requires Langmuir– Blodgett or Langmuir–Schaeffer deposition of monolayers from the air–water interface [170]. Transfer to a substrate is used when the scientific questions regard the detailed structure of the monolayer. If a monolayer or bilayer of lipid is needed as a substrate, vesicle deposition techniques are often used [171–173]. More elaborate configurations can be used, such as substrates with adsorbed polymer layers onto which a lipid or lipid/protein monolayer or bilayer is draped [174–176]. Deposition of monolayers or bilayers from an interface generally requires that a minimal reorganization of the films occur on transfer to the substrate. While changes at the molecular level must be dealt with in different ways [150], it is possible to make sure that minimal changes in the films occur at optical resolution [170] by constantly monitoring the monolayer during the deposition process with fluorescence optical microscopy. An alternative is to correlate AFM structural information with electron microscopy [177] or X-ray or electron diffraction [178]. Fatty acid [124, 126, 150], lipid [34, 170], or lipid–protein [27, 30, 76, 149, 150, 179] monolayers are typically examined by AFM after transfer to hydrophilic mica or oxidized silicon wafer substrates. Silicon wafers must be cleaned in a hot “piranha” solution (H2 O2 :H2 SO4 , 3:7 v/v) to remove organic contaminants while leaving the native oxide intact and then stored in clean water until use. Mica substrates are generally cleaved with ordinary adhesive tape immediately before use. As an alternative, the monolayer can be deposited onto hydrophobic lipid [144], self-assembled monolayers [146], or hydrogen-passivated silicon surfaces [Takamoto 1998, #119; Schwartz 1992, #177]. The choice of substrate dictates whether the hydrophilic or hydrophobic side of the monolayer is imaged and whether the AFM imaging is done in air or under liquids. To prepare the monolayers for AFM imaging, monolayers are spread onto a conventional, temperature-controlled Langmuir trough [35]. A Wilhelmy plate-type transducer for surface pressure measurements controls a feedback loop for constant pressure depositions. Fluorescence microscopy (FM) is often used to visualize the surface of the spread film by incorporating a small percentage (∼1 mol%) of fluorescent probe into the monolayer lipids [35, 180, 181]. As the fluorescent molecules preferentially partition into the less ordered phase, contrast is generated, with the less ordered phase bright and the more highly ordered phase dark. Resolution is typically on the order of 0.5–1 m. Langmuir–Blodgett Deposition Langmuir–Blodgett deposition can be used with either hydrophilic or hydrophobic substrates and should result in a complete monolayer coating on both sides of the substrate. For the hydrophilic substrate, the bare, clean substrate is held beneath the monolayer film perpendicular to the air–water interface, while the monolayer is compressed to the desired surface pressure. At this point a feedback loop is engaged to maintain the surface pressure at the desired value by either compressing or expanding the available surface area of the trough. After a few minutes, to permit the monolayer structures to stabilize, the substrate is slowly withdrawn from the subphase (rates of a few millimeters/minute have worked well in our experiments) until it is fully removed from contact with the subphase (Fig. 29A). The change in area of the trough during deposition divided by the surface area of the substrate, or transfer ratio, is a quantitative measure of the fraction of substrate surface successfully coated with a monolayer film. Values equal to or slightly greater than one imply that the deposition was successful (transfer ratios greater than one are possible due to other mechanisms of removing surfactant from the interface, e.g., by slow solubilization or by leakage past the trough barriers). The same procedure is followed for deposition onto a hydrophobic substrate, except that the clean substrate starts in air and is slowly pushed through the interface to acquire its surfactant monolayer coating (Fig. 29B). Langmuir–Schaeffer Deposition Langmuir–Schaeffer deposition can only be used with hydrophobic substrates and does not furnish a transfer ratio by which to evaluate the coating process. As in Langmuir–Blodgett deposition, the monolayer is slowly compressed to the

32


Figure 29. Left: Langmuir–Blodgett deposition of a surfactant monolayer onto a hydrophilic substrate. Right: LB deposition onto a hydrophobic substrate. Note the difference in contact angle for the two cases.

desired surface pressure, and the feedback loop to maintain surface pressure is engaged. Once the monolayer structures are judged to have reached equilibrium, the substrate is oriented parallel to the air–water interface and gently lowered into contact with the monolayer. Depending on its weight, it may be possible let the substrate float on the monolayer in order to promote good adhesion between surfactant tails and the hydrophobic surface; if the substrate is too heavy, it should be held in contact with the monolayer for this period. Then, the substrate is smoothly pushed or dropped through the interface into the subphase (Fig. 30). In a successful deposition, only the downward facing side of the substrate will attain a complete surfactant monolayer coating. Reverse Langmuir–Schaeffer Deposition Reverse Langmuir–Schaeffer deposition can only be used with a hydrophilic substrate and is the only technique we are aware of which permits continuous observation of the surfactant monolayer throughout deposition. Figure 31 shows the schematic and photo of the monolayer deposition device. A mica or silicon wafer is cut into a 1-cm diameter circular disc (the typical size for a Nanoscope FM system), which is held in place by four small posts to keep the substrate from moving. A 15 knife-edge was machined to cut the monolayer at a prescribed area per molecule when the subphase level was lowered. The distance from the knife-edge to the substrate top surface is kept small to prevent the monolayer from deforming during deposition. The four sides of the base are partially shaved off, leaving it in a cross shape and allowing water to drain under the substrate. The substrate can be placed directly onto the piezoelectric scanner of the NanoScope AFM after mounting to a stainless steel disc as described previously (Digital Instruments, Santa Barbara, CA) for AFM imaging.

Figure 30. Langmuir–Schaeffer deposition method with a hydrophobic substrate.


15°

A

33

B

Φ.750

Φ.645

Figure 31. (A) Schematic diagrams and (B) photo of deposition stage. The stage is constructed of stainless steel. Four small prongs hold and center the substrate (1-cm diameter mica or silicon wafer circles, white arrow in the photo). Holes in the bottom and sides of the stage (black arrow) allow the subphase to slowly drain away as the subphase level is lowered in the trough, thereby depositing the monolayer onto the substrate (black arrow in the photo). A rim with a 15 knife edge cuts and holds the monolayer at a constant area/molecule, hence surface pressure, when the subphase level is lowered. The rule is in inches. Reprinted with permission from [30], J. Q. Ding et al., Langmuir 19, 1539 (2003). © 2003, American Chemical Society.

The transfer unit is submerged in the subphase before the monolayer is spread. For the deposition to work, the knife-edge has to lie just below the subphase surface (90 wt %) of dipalmitoylphosphatidylcholine (DPPC), unsaturated phosphatidylcholines and phosphatidylglycerols, fatty acids, and cholesterol. Bulk phase endogenous surfactant contains about 10 wt % of the lung surfactant specific peptides, SP-A, SP-B, SP-C, and SP-D. Only SP-B and SP-C participate in the biophysically active monolayer form of lung surfactant; both SP-B and SP-C are relatively small, hydrophobic, and cationic (78–79 amino acids and 33–35 amino Table 4. Biophysical properties of lung surfactant and physiological actions supported. Biophysical requirement

Physiological action

Low minimum surface tension under compression Variable surface tension on compression–expansion Rapid respreading of surfactant after compression past monolayer collapse Rapid adsorption to the air–water interface

Decreased work of breathing Enhanced uniformity of alveolar recruitment Increased alveolar stability; continuous surface tension control Continuous surface tension control throughout breathing cycle


43

acids, respectively) [209]. Biophysically, SP-B and SP-C are thought to play crucial roles in promoting rapid adsorption and spreading of lung surfactant [195, 210, 211]; however, because SP-B and SP-C are difficult to isolate from natural sources, all commercial RLS formulations contain significantly lower amounts of one or both peptides than do native surfactants [207, 212]. Extrapolating from NMR data for a related peptide, the most likely structure for SP-B is five amphipathic helices in which the polar faces of the helices interact with phospholipid headgroups; SP-B often forms homodimers which may provide a link between adjacent surfactant membranes [213, 214]. The NMR structure of porcine SP-C is consistent with a membrane-spanning -helix in which positively charged residues near the N-terminus of the peptide interact with phospholipid headgroups. For both peptides, amino acid sequences are highly conserved between species [213]. SP-A and SP-D, which have been shown to be excluded from the monolayer form of lung surfactant, comprise about 8% of native surfactant but are absent from all clinically used replacement surfactants [209]. SP-A has been shown to strongly influence surface activity and bulk phase packing of lung surfactant in a calcium- and pH-dependent manner; moreover, in vitro and animal model experiments generally show a strong correlation between SP-A concentration and surfactant tolerance of ARDS-like conditions [20, 212, 215–217]. SP-D on the other hand has no known role in lung surfactant biophysics [209]. Both SP-A and SP-D belong structurally to the collectin family of host defense proteins and so may play a role in protecting the airways from bacterial or viral infection [217–219]. Recent experiments with mice genetically modified to lack one of the four surfactant peptides demonstrated that only SP-B deficiency was a lethal mutation; however, the implications for replacement lung surfactant design are complicated since, along with markedly changing surfactant structure, the SP-B mutation arrested normal development of some lung tissues [213, 217]. Although not lethal, SP-A and SP-D mutations conferred increased susceptibility to pulmonary infection, supporting the putative host defense role for these proteins [220–222]. Although a consensus has recently emerged that viable replacement surfactants should contain some amount of a peptide analogous to SP-B, the current wide variations in replacement surfactant composition (Table 5) are likely to persist until more complete data establishing an optimal lung surfactant composition emerges [212]. The startling heterogeneity of replacement lung surfactant composition and confusion surrounding the “optimum formulation” reflects the general situation of biomaterials science: currently used biomaterials are materials which were readily available when a critical need was identified and were thus drafted into service. In general, these materials perform adequately but have significant, long-standing drawbacks [8, 14]. As in other areas of biotechnology, the emphasis in lung surfactant research is slowly shifting from adaptation of existing materials toward nanoscale structure–function studies to guide rational design [15]. One exciting area of materials development is the construction of synthetic analogues for SP-B and SP-C which mimic the activity of the native peptides but are easier to manufacture and process. For a recent review of these efforts, please see references 214, 223, and 224. Current research on the physical properties of endogenous, commercial replacement lung surfactants and model lung surfactant mixtures strives to link the biophysical requirements listed in Table 4 with specific lipid/peptide mixtures, micro- and nano-scale structures, and rheological characteristics. As well as the basic scientific value in developing structure– function relationships for a dynamically active film, there is a strong societal interest in establishing more effective formulations for clinical use in NRDS and potentially in ARDS. Although considerable progress has been made, large areas of confusion persist.

3.4. Composition, Phase Behavior and Monolayer Structure Chain length and the ratio of unsaturated to saturated chains in a surfactant mixture strongly influence monolayer phase behavior in vitro [55, 180, 225]. Experimentally, phase behavior of surfactant monolayers is studied either in a Langmuir trough [226, 227] or by expanding and compressing a captive bubble [228]. At large areas per molecule, the molecules are in a two-dimensional “gas” phase and exert little influence on each other. As the monolayer is

44


Table 5. Replacement surfactants in current use worldwide. Name

Source (animal or synthetic), Manufacturer

Infasurf

Bovine extract (organic solvent extract of alveolar surfactant washed from intact calf lungs), ONY labs, USA

Survanta

Supplemented bovine extract (organic solvent extract of finely ground lung tissue with added synthetic DPPC, palmitic acid, and tripalmitin), Abbot Laboratories, USA Porcine extract (organic extract of minced pork lung), Chiesi Farmaceutici, Italy

Curosurf

Exosurf

Synthetic (dry powder reconstituted in saline), GlaxoWellcome, USA

ALEC

Synthetic (dry powder reconstituted in saline), Britannia Pharma., UK

Composition, wt %

Ref.

Phospholipid, cholesterol, protein 93% phospholipids 5% cholesterol 1.5% protein phospholipid breakdown: 83% PC, 6% PG, 4% PI and PS, 3% PE, 2% SPH Phospholipid, triglyceride, fatty acid, protein 75–92% phospholipids 2–7% triglycerides 6–14% free fatty acids 0–1% surfactant protein Phospholipid, protein 99% lipids 1% surfactant protein phospholipid breakdown: 65–75% PC, PE + SPH 12–22% Phospholipid, alcohol, polymer detergent 84% DPPC 9% hexadecanol 7% tyloxapol (surfactant polymer of the nonionic detergent Triton X-100) Phospholipid 70% DPPC 30% POPG

http://www.fda.gov/cder/ consumerinfo/druginfo/ infasurf.htm [209]

http://www.survanta.com/ _html/generalinformation/ hcp_fullprescrib.cfm [209]

http://www.fda.gov/cder/ consumerinfo/druginfo/ curosurf.htm [209] http://www.drugbase.co.za/ data/pi/exosurf.htm [209]

[209]

compressed, molecules interact to create a disordered fluid or LE phase; further compression leads to a first-order transition to a liquid-condensed or LC phase which has long-range order and is less fluid and less compressible than the LE phase. Most trace impurities, such as fluorescent dyes, preferentially locate in the more disordered LE phase. Above a critical temperature that varies with lipid species and subphase composition, only the LE phase is present [56]. The unsaturated lipids in native and replacement lung surfactants generally have critical temperatures below 20 C and thus always form LE phases at physiological temperature [229]; the disaturated lipids, however, have critical temperatures well above physiological temperature and so form LC phases at sufficiently elevated surface pressure [230]. Fluorescence and BAM images of native and replacement lung surfactant monolayers generally display coexisting LC and LE phases over a wide range of surface pressures and clinically relevant temperatures (20–37 C) [34, 76, 231, 232]. The LC phases have been shown to contain most of the DPPC in the mixture, along with any fatty acids and other saturated lipids [32, 233]; the LE phases contain the unsaturated lipids and any lung surfactant proteins in the mixture [234]. The partitioning of interfacial area between LC and LE phases thus depends on surfactant composition, surface pressure, temperature, and ionic species present in the subphase. Certain minor lipid species such as the cholesterol found in native surfactants or the palmitic acid and hexadecanol added to Survanta [19, 212] and Exosurf [235], respectively, have a particularly strong effect on LE/LC coexistence. Adding either palmitic acid or hexadecanol to mixtures of DPPC and POPG increases the solid phase fraction in the monolayer at fixed temperature and surface pressure; either molecule can combine specifically with DPPC to create a new crystal of untilted, highly uncompressible molecules [32, 33]. Pure monolayers of DPPC require much higher surface pressures or lower temperatures to achieve a zero molecular tilt; thus increasing palmitic acid or hexadecanol content is equivalent to increasing surface pressure or decreasing temperature for DPPC-containing


45

monolayers. However, cholesterol appears to have a more complicated effect, increasing order at low surface pressures and disordering DPPC monolayers at high surface pressure [227]. Interestingly, whereas palmitic acid and hexadecanol are added to improve in vivo performance of clinical surfactants, several studies have documented a correlation between the presence of cholesterol and degraded lung surfactant performance [21, 236]. This correlation between crystallinity at higher surface pressures and enhanced in vivo performance suggests that there may be important functional reasons to the different degrees of order attained in the monolayer at the different stage of the breathing cycle. As the monolayer is compressed, the surface pressure reaches a limiting value beyond which the monolayer becomes unstable and the monolayer “collapses” into the bulk. The collapse surface pressure is dependent on the speed with which the monolayer is compressed [209]; however, in the range of timescales relevant for breathing, the value is consistent and reproducible. Under dynamic compression in a Langmuir trough at physiological temperature, clinical lung surfactants will collapse at surface pressures in excess of 60 dyn/cm (surface tension below 10 dyn/cm) [237, 238]. Whether the collapsed surfactant is lost to the subphase or is drawn back onto the interface during expansion (respreads) is crucial to surfactant performance, as lung surfactant monolayers must typically reside at the alveolar interface for hundreds of breathing cycles before replacement [239]. Surfactant respreading from the collapse structures to the useful monolayer may be estimated by comparing the onset area of collapse in successive isotherms [209]. A “good” lung surfactant will show an onset area ratio for successive cycles close to 1, whereas a more typical lipid or fatty acid will lose most of the overcompressed material irretrievably to the bulk [31]. Surfactant peptides SP-B and SP-C are strongly correlated with better adsorption, respreading, and surface tension reduction by lung surfactant mixtures [209]. Although various models for the action of these peptides exist, the precise mechanisms by which SP-B and SP-C improve surfactant performance are still unclear. Until the early 1990s a dynamic model of peptide action called the “squeeze-out process” dominated lung surfactant theory. In the squeeze-out model, the surfactant peptides facilitate removal of unsaturated lipids from the lung surfactant monolayer during the compression limb of the breathing cycle [22, 240–243]. Squeeze-out thus rationalizes the presence in lung surfactant of large fractions of unsaturated lipids (cf. Table 5), which are known to have good adsorption rates but poor dynamic surface tension-lowering properties. After adsorption, squeeze-out would gradually remove the unsaturated lipids and surfactant proteins, enriching the monolayer in saturated phosphatidylcholines, which have unusually good dynamic surface tension reduction properties. Squeeze-out induced compositional changes in the lung surfactant monolayer should be reflected in monolayer phase behavior, particularly in the partitioning of interfacial area between condensed-phase saturated lipids and fluid-phase unsaturated lipids. Several groups have now tested squeeze-out using images and isotherms collected during dynamic compression–expansion cycles of monolayers confined in a Langmuir trough. In both model surfactant mixtures [34, 76, 231] and extracted lung surfactants [232], the reproducibility of isotherms for peptide-containing mixtures under repeated cycling demonstrates that surfactant peptides actually enhance lipid retention. Interestingly, the peptides have been shown to maintain discrete fluid and crystalline domains in the monolayer at all surface pressures both on the trough and, most recently, on the surface of lung surfactant coated air bubbles (Fig. 38) [153, 244]. Fluorescently labeled SP-B analogues have been shown to colocalize with unsaturated lipids [34, 76, 231] and can significantly enhance the collapse pressure (maximum dynamic surface pressure) attainable by unsaturated lipids alone. Thus the surfactant peptides actually retain unsaturated lipid at high surface pressures. Fluorescence images also demonstrate that either surfactant peptide fosters the appearance of three-dimensional surfactant aggregates at collapse similar to those observed in TEM studies of the alveolar surface of excised lungs [17] (Fig. 39); these aggregates reincorporate quickly when the film re-expands [29, 76, 245]. However, in the presence of the high levels of serum proteins associated with ARDS, no respreading occurs and surface tension gradually climbs as the interface is denuded of effective lung surfactant [31].

46


50 µ Figure 39. Fluorescence micrograph of a model lung surfactant monolayer on 25 C buffer at a surface pressure of 20 dyn/cm. The mixture is 68/20/10/2 (w/w/w/w) DPPC/POPG/PA/Texas Red DHPE with 2 wt % of a 25 amino acid SP-B analogue peptide. The bright areas contain most of the POPG, SP-B analogue, and dye lipid, while the dark, flowerlike structures are crystalline domains containing DPPC and PA.

Nanoscale morphological studies using monolayers transferred to a solid substrate reveal structural details inaccessible to optical microscopy. In particular, several groups have shown that the heterogeneous lung surfactant monolayer has regions of significantly different height or thickness [25, 26, 76, 150]; comparison of AFM and FM images has demonstrated that these regions correspond to fluid and solid domains. These studies have also generally confirmed the extensive overlap in SP-B and SP-C function seen in the optical microscopy studies. Examination with AFM of the structures formed by model surfactant mixtures of DPPC and DPPG or DPPC and POPC with SP-C demonstrated that compression beyond the equilibrium spreading pressure of the mixture resulted in the formation of stepped multilamellar structures at the edges of monolayer domains with a step height approximately that of a DPPC bilayer [26, 246]. Similar studies using mixtures of DPPC, POPG and palmitic acid with SP-B, DPPG and POPG with SP-B, and DPPG with SP-B all found that SP-B also promotes the formation of multilamellar structures at surface pressures exceeding the equilibrium spreading pressure of the most soluble component of the lipid mixture [29, 76, 150]; however, these structures are not confined to the edges of domains. A later study using DPPC, POPG, and palmitic acid aimed at elucidating which surfactants participate in multilamellae formation: interestingly, only SP-B and POPG were required, dashing expectations that the cationic peptide would also preferentially associate with the anionic fatty acid in the mixture [30]. A striking difference between the action of the two peptides appears to be that SP-B promotes formation of more extended collapse structures whereas SP-C associated structures are relatively compact [29]. This difference would be a logical result of differences in peptide structure: SP-C, as a smaller, membrane-spanning peptide, should be less able to bridge and aggregate-adjacent surfactant bilayers than either monomeric or dimeric SP-B. Redundancy in peptide action is also consistent with the clinical result that mixtures lacking just one of the peptides can still perform adequately as replacement surfactants [196, 197, 247]. The positive correlation between multilamellar formation and increased retention of surfactant at the interface has led to a new model of surfactant peptide action and biphasic monolayer collapse called the buckling model, illustrated in Figure 40. In buckling, the phase separation maintained by SP-B and/or SP-C within the lung surfactant monolayer is the key to retention of surfactant during the dynamic compression–expansion of breathing. When overcompressed, adjoining areas of incommensurate spontaneous curvature buckle or fold into the subphase, like crumpling a precreased sheet of paper. Because surfactant folds remain connected to the monolayer, they are more easily drawn back into the monolayer during expansion, increasing retention [76, 248]. This theory is consistent with the evidence summarized above, but not with recent results demonstrating reversible collapse in a monophasic fatty acid film without surfactant peptide [57, 249, 250]. Another new study has shown that the buckling model is inconsistent with the behavior of single-component lipid monolayers as well; retention of a DPPG monolayer during dynamic cycling can be achieved simply by increasing the salinity of the subphase. Examination by AFM of transferred DPPG films


47

50 µ Figure 40. Fluorescence micrograph of a model lung surfactant monolayer on 25 C buffer after collapse. The mixture is the same as in Figure 39. The arrow points to collapsed surfactant aggregated beneath the monolayer.

shows a smooth, homogeneous monolayer just prior to collapse; moreover, the structures formed during reversible collapse in this system are primarily vesicles, not the stacked sheets predicted in buckling [251]. Thus, the available evidence does not identify specific surfactant mixture properties required for reversible collapse to multilamellae or support that collapse to a monolayer-connected, multilamellar structure is actually necessary for surfactant retention. More detailed morphological work using a wider variety of surfactant mixtures will be required to answer the role of monolayer and collapsed aggregate structure in promoting surfactant retention at the interface. These studies are particularly urgent, given that failure of surfactant adsorption and retention have been implicated in biophysical studies of the ARDS condition [31, 206].

3.5. Structure and Transport The last two biophysical requirements listed for functional lung surfactants in Table 4 deal with transport. The bulk and surface viscosity of clinical surfactants potentially influences their delivery and distribution in the lungs and varies significantly with composition, concentration, temperature, ionic environment, and physical formulation [252]. Adsorption and flow are required to establish a complete surfactant monolayer within the lung and thereafter to correct surface concentration differences arising during normal breathing. Surfactant flow has other functional implications as well: surface shear viscosity is a determining factor in the surface tension gradient-induced flow of contaminants from the deep lung [195]. However, rheology is by far the least explored of lung surfactant properties; we hope to see significant advances in this area in the future. In this section we focus on the relatively new field of surface shear viscosity measurements. Saturated dipalmitoylphosphatidylcholine (DPPC) is the majority component of human and mammalian lung surfactant (LS), in addition to being ubiquitous in biological membranes. Understanding the shear response of DPPC under various conditions is thus a first step in understanding shear response of replacement lung surfactants used in treatments of RDS. Qualitative measures of surface shear for DPPC have been reported by several investigators; however, quantitative values vary widely [52, 185, 188]. As mentioned in the viscometry methods section, these differences may be attributable to the strong temperature and surface pressure dependence of morphology in DPPC [52, 180], which would lead to systematic errors in surface pressure gradient-induced flow methods of estimating viscosity. At 20 C, DPPC monolayers have a coexistence between a liquid expanded (LE) and a liquid condensed (LC) phase that begins at a surface pressure of about 8–10 mN/m. The LE phase is disordered and expected to be rather fluid, while the LC phase has a semicrystalline packing with a rectangular unit cell. The area per chain is 23.3 Å2 and the molecules are tilted with respect to the normal to the interface [32]. As the temperature increases, the surface pressure at which the LE–LC phase transition occurs increases; by 37 C, the phase transition pressure is above 40 mN/m. The magnetic needle viscometer, which does not require a

48


Dynamic Compression (Exhalation)

Dynamic Expansion (Inhalation) Dynamic Respreading

Collapse by Folding at Domain Boundaries

Figure 41. Cartoon of buckling model of lung surfactant collapse. The high rate of surfactant retention at the interface during dynamic compression is explained by continuous folding regions which connect the monolayer to collapsed surfactant. In the buckling model, such folds are most likely to occur at the boundaries between areas of different curvature in the monolayer, so the surfactant peptide’s promotion of phase separation in the monolayer assists the folding process.

surface pressure gradient to operate, has recently been used to determine the steady-state shear viscosity of DPPC over a range of temperatures and surface pressures [37]. DPPC, in addition to achieving a near zero surface tension under compression [29, 32, 33], apparently does not contribute to the flow resistance at physiological temperatures, two factors thought to be essential to a good lung surfactant [195, 253]. This is surprising given that a high shear viscosity is usually invoked to explain pure DPPC’s failure as a replacement lung surfactant [195, 253, 254]. Lowering the temperature of the DPPC monolayer increases the fraction of liquid condensed (LC) phase (Fig. 41). As shown in Figure 42, lowering the temperature also increases the surface viscosity dramatically [29]. An analogous result is achieved by adding palmitic

A

B

50 µm

50 µm C

50 µm Figure 42. Fluorescence micrographs of DPPC monolayer on a pure water subphase. (A) DPPC at a surface pressure of 10 mN/m and a temperature of 20 C showing the dark, chiral liquid condensed domains in a continuous matrix of bright liquid expanded phase; (B) DPPC at the surface pressure of 25 mN/m and a temperature of 30 C, the liquid condensed domains are much smaller. (C) DPPC at the surface pressure of 40 mN/m and the temperature of 37 C. Only the liquid-expanded phase is visible under these conditions. Reprinted with permission from [37], J. Q. Ding et al., Langmuir 18, 2800 (2002). © 2002, American Chemical Society.

49


B Surface Viscosity (P cm)

Normalized Speed

A 1.0 0.8 0.6 20 °C 25 °C 30 °C 37 °C

0.4 0.2

0.1 20 °C 25 °C 30 °C 37 °C

0.01

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Figure 43. (A) Terminal velocity of the magnetic needle on monolayers of DPPC as a function of surface pressure and temperature, normalized to the speed of the needle on a clean subphase–air interface, v1 /v. The constant normalized terminal needle velocity at 37 C shows that there is a minimal change in the subphase drag as a function of the surface pressure. (B) Surface shear viscosity of a monolayer of DPPC as a function of surface pressure and temperature. For the 20 , 25 , and 30 C data, the slopes of the normalized speed and the surface viscosity plots change abruptly at surface pressures that correspond to the LE–LC transition pressure at that temperature. At 37 C, the surface viscosity cannot be resolved with this instrument; essentially all of the drag on the needle is from the subphase rather than from the DPPC monolayer. Reprinted with permission from [37], J. Q. Ding et al., Langmuir 18, 2800 (2002). © 2002, American Chemical Society.

5mN/m

15mN/m

30mN/m

45mN/m

Collapse

20%PA

15%PA

10%PA

5%PA

0%PA

π=

50 µm Figure 44. Fluorescence optical micrographs of monolayers of DPPC/POPG (77:23, wt:wt) with varied amounts of palmitic acid (PA) on a 150 mM NaCl, 5 mM CaCl2 , and 0.2 mM NaHCO3 (pH = 6.9) buffer subphase at 25 C. The PA concentration is labeled in the left of each row, and the top line shows the corresponding surface pressure. Reprinted with permission from [36], J. Q. Ding et al., Phys. Rev. Lett. 88, 168102 (2002). © 2002, American Chemical Society.

50


acid (PA) and hexadecanol (HD) to monolayers containing DPPC and POPG (1-palmitoyl2-oleoylphosphatidylglycerol), a mixture which more closely mimics the composition of commercial replacement lung surfactants. Figures 43 and 44 show the increase in LC area fraction upon addition of PA or HD. Figure 45 shows the corresponding increase in surface viscosity, indicated by the steeper drop in normalized speed at greater surface pressures. These results suggest that an optimal lung surfactant should have a surface viscosity that changes from a relatively low value at low surface pressures to a relatively high value at high surface pressures. In addition, they elucidate the function of PA and HD in the commercial LS Exosurf, whose primary component is DPPC. Later studies using DPPC/POPG mixtures with PA and HD also demonstrated that surface viscosity scales as s /so = 1 − A/Ac −1 in which so is the viscosity of the liquid phase fraction of the monolayer, A is the area fraction of solid phase measured by fluorescence microscopy, and Ac is the critical solid phase fraction (Fig. 46). This scaling relationship is analogous to that derived for three-dimensional colloidal dispersions in a solvent with long-range repulsive interactions between the solids (with area fraction replacing volume fraction). The direct analogy implies that dipole–dipole interactions help determine monolayer morphology and mechanics [180, 181, 255] and provides a straightforward method to adjust surface shear viscosity of lung surfactant. 15mN/m

30mN/m

45mN/m

Collapse

HD 20%

HD 15%

HD 10%

HD 5%

HD 3%

HD 0%

π= 5mN/m

50 µm

Figure 45. Fluorescence optical micrographs of monolayers of DPPC/POPG (77:23, wt:wt) with varied amounts of hexadecanol (HD) on a 150 mM NaCl, 5 mM CaCl2 , and 0.2 mM NaHCO3 (pH = 69) buffer subphase at 30 C. The HD concentration is labeled in the left of each row, and the top line shows the corresponding surface pressure.

51

Nanostructure and Dynamics of Biocompatible Surfactant Monolayers and Bilayers 1.0

1.0

0.8

Normalized Speed

Normalized Speed

0.8

0.6

0.4 PA 0% PA 5% PA 10% PA 15% PA 20%

0.2

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30.0 °C, Buffer

0.2

HL 0% HL 3% HL 5% HL 10% HL 15% HL 20%

25.0 °C, buffered saline

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0.0 0

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30

40

50

60

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Figure 46. Speed of magnetic needle on the monolayer of DPPC/POPG with PA or HD as a function of surface pressure, normalized to the speed of the needle at a clean subphase–air interface, v1 /v. Reprinted with permission from [36], J. Q. Ding et al., Phys. Rev. Lett. 88, 168102 (2002). © 2002, American Physical Society.

Systematic studies have also been undertaken of the effect of cholesterol on lung surfactant shear rheology. In contrast to palmitic acid and hexadecanol, addition of 1 mol % of cholesterol dramatically reduces the surface viscosity of DPPC, DPPE, and DPPA monolayers; 10 mol % cholesterol in any of these films makes the viscosity indistinguishable from that

10% PA/Buffer/25 °C 15% PA/Buffer/25 °C 20% PA/Buffer/25 °C 10% PA/Water/25 °C

100

15% PA/Water/25 °C 20% PA/Water/25 °C 10% HD/Water/30 °C 15% HD/Water/30 °C 20% HD/Water/30 °C

ms/mso

15% HD/Buffer/30 °C 20% HD/Buffer/30 °C

(1-A/Ac)–1 (1-A/Ac)–2

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–0.1

0.0

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0.5

0.6

0.7

0.8

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1.0

1-A/Ac Figure 47. Semilog plot of the reduced surface viscosity (s /so as a function of (1 − A/Ac derived from the data in Figures 8 and 9 for those mixtures that showed a zero needle velocity so that Ac could be evaluated. so is the surface viscosity at zero surface pressure and zero solid phase fraction for a given mixture; the critical area fraction Ac is the solid phase area fraction at which the needle speed goes to zero. While absolute values for s were not determined, the quantity s /so was calculated from the normalized needle velocities. The curves are the theoretical expressions corresponding to an exponent of –1 (solid) and –2 (dashed). Reprinted with permission from [36], J. Q. Ding et al., Phys. Rev. Lett. 88, 168102 (2002). © 2002, American Physical Society.

52


of a bare water surface [256]. These results are consistent with the structural work showing decreased lipid ordering in cholesterol-containing monolayers related in the previous section and with diffraction experiments on bulk cholesterol–LS samples, which show a reduction in the main phase transition temperature of the LS sample of almost 10 deg once physiological concentrations of cholesterol are added to an extracted, purified lung surfactant [257].

4. CONCLUSIONS The optimum surfactant composition cannot be determined until the connection between monolayer physical and physiological properties is more completely understood. This is a complicated, interesting problem, made urgent by the need for satisfactory treatments for acute respiratory distress syndrome. There is a particular need to develop a more robust understanding of the mechanisms of collapse and respreading in surfactant monolayers at the alveolar interface and to develop a larger body of experimental data on surface shear rheology of lung surfactants. Both of these areas will require precise, nanoscale experiments to link structural motifs with required physical performance.

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