<<

Surface Measurements of Various Using the Constrained Sessile

Drop Method

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science

in the Graduate School of The Ohio State University

By

Alyssa A. Robson, B.S.

Graduate Program in Chemistry

The Ohio State University

2014

Master's Examination Committee:

Prof. Dr. Barbara Wyslouzil, Advisor

Prof. Dr. Heather Allen

Copyright by

Alyssa A. Robson

2014

ABSTRACT

Respiratory distress syndromes affects over 150, 000 patients a year, and is the result of injury which typically results in inhibition or inactivation of pulmonary (PS). PS is composed of and proteins, and is located near the lining in the . When the PS becomes inhibited or inactivated becomes difficult. PS lowers the tension in the lungs during breathing, and low is central to any surfactant replacement therapy (SRT) for respiratory distress syndrome. SRTs, which vary in source and composition, do not greatly improve the recovery or mortality of adolescent and adult patients. This is due in part to the lack of knowledge of how PS, and the individual components, perform in the lungs to lower the surface tension. Additionally, SRT is placed in lungs which have inhibited or inactivated PS. For example, if the endogenous PS is inhibited and synthetic surfactant is administered to the lungs, inhibition could happen to the SRT, which does not improve the recovery of the patient, therefore, a superior treatment is needed. Constrained sessile (CSD) is a unique way to study PS components and analogs since the surface tension of a spherical drop is measured. The alveoli in the lungs closely resemble a cluster of bulbous grapes, and the alveoli expand and contract during breathing. A constrained sessile drop is able to mimic an alveolus during by changing the volume of a drop, called cycling, at rates comparable to that of breathing.

ii

In this thesis, was the first liquid that was studied using CSD since the surface tension of water is well documented. Once constant and reliable surface tension values were measured for both static and dynamic water drops, surfactant thin films were studied. Two PS components, dipalmitoylphosphatidylcholine (DPPC) and cholesterol, were studied by way of water drops with different of a thin coating of surfactant. Static measurements were studied with higher concentrations to ensure proper drop and to verify that obtaining surface tension values lower than that of water were possible. Preliminary compression isotherms were also conducted for DPPC and cholesterol. Lipopolysaccharide was a third surfactant studied using CSD; static and dynamic drops were studied. Overall, the CSD setup functions well and is capable of forming pure water drops as well as those with surfactant thin films. Surface tension measurements obtained for water are consistent with those published. The surface tension values for surfactant thin films can be measured and compression isotherms similar to those measured using a Langmuir trough are possible.

iii

DEDICATION

This thesis is dedicated to my family and friends.

iv

ACKNOWLEDGMENTS

This thesis could not have been possible without the support and encouragement from several people. First of all, I would like to express gratitude to my advisor, Dr. Barbara

Wyslouzil, for believing in me, giving me the opportunity to grow as a person and a scientist, and being a true mentor. Additionally, I would like to thank my co-advisor, Dr. Heather Allen, for her guidance and enthusiasm. I would like to express my gratitude to the members of the

Wyslouzil and Allen groups, in particular Dr. Dominique Verreault, Ms. Dana Telesford, and Ms.

Ellen Adams for helpful discussions and useful insights with this project. I would like to thank

Mr. Mike Wilson for his time, perseverance, and assistance in improvements to my setup.

I would like to express my appreciation to my family for their continued and unwavering support throughout my academic career. I am grateful for their willingness to listen and encourage me at each step in this process. Furthermore, I could not have made it through without the support and motivation from my friends. And, finally, I would like to thank God for His strength and continual guidance.

v

VITA

November 19, 1986……………………………………Born – Cleveland, OH

May 2009……………………………………………...B.S. ACS–Certified Chemistry, University

of Southern Mississippi, Hattiesburg, MS,

USA

September 2011……………………………………...Graduate Teaching Associate, Department

of Chemistry and Biochemistry, The Ohio

State University – Columbus, OH, USA

FIELD OF STUDY

Major Field: Chemistry

vi

TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vi

Field of Study ...... vi

Table of Contents ...... vii

List of Figures ...... xi

List of Tables ...... xvi

List of Abbreviations and Symbols ...... xvii

1. Introduction ...... 1

1.1 Overview ...... 1

1.2 , Lungs, and Pulmonary Alveoli ...... 1

1.2.1 Respiratory System and Lung ...... 1

1.2.2 Pulmonary Alveoli and Breathing ...... 4

vii

1.3 ...... 6

1.3.1 Lifecycle of Pulmonary Surfactant ...... 6

1.3.2 Physical Properties of Pulmonary Surfactant ...... 9

1.3.3 Pulmonary Surfactant Components ...... 11

1.3.4 PS Inhibition, Inactivation, and Degradation ...... 15

1.4 Respiratory Syndromes ...... 17

1.4.1 Overview of Respiratory Syndromes ...... 17

1.4.2 Neonatal Respiratory Distress Syndrome ...... 18

1.4.3 Acute Lung Injury ...... 19

1.4.4 Acute Respiratory Distress Syndrome ...... 19

1.5 Replacement Therapy ...... 21

1.5.1 Types of Current Surfactant Replacement Therapy ...... 22

1.6 Goals and Objectives ...... 24

2. of Interfaces ...... 26

2.1 Theory of Surface Tension and the Gibbs Isotherm ...... 26

2.1.1 Surface Tension ...... 35

2.2 Theory of Capillarity ...... 37

2.2.1 Laplace-Young Equation and Axisymmetric Drop Shape Analysis ...... 37

3. Surface Tension Techniques ...... 45

3.1 Overview ...... 45

3.1.1 Langmuir-Wilhelmy Balance ...... 46

viii

3.1.2 Captive Surfactometer ...... 49

3.1.3 Pulsating Bubble Surfactometer ...... 50

3.1.4 Pendant drop ...... 51

3.1.5 Constrained Sessile Drop ...... 52

4. Experimental Setup ...... 54

4.1 Materials ...... 54

4.2 CSD Setup ...... 54

4.2.1 Illumination ...... 56

4.2.2 Pedestal ...... 57

4.2.3 ...... 60

4.2.4 Infusion system ...... 62

4.2.5 Methods ...... 63

4.3 Axisymmetric Drop Shape Analysis and Other Software ...... 65

4.3.1 UC480 ...... 65

4.3.2 VirtualDub ...... 65

4.3.3 Microsoft ...... 65

4.3.4 Axisymmetric Drop Shape Analysis ...... 66

5. Results and Discussion ...... 67

5.1 Nanopure water ...... 67

5.1.1 Preliminary Results ...... 71

5.1.2 Quantifying Results ...... 77

5.1.3 Possible Improvements ...... 80

ix

5.2 Thin Films of Surfactants ...... 82

5.2.1 Preliminary DPPC ...... 83

5.2.3 Quantifying DPPC Results ...... 86

5.2.4 DPPC Isotherms ...... 88

5.2.5 Preliminary Chol Results...... 89

5.2.6 Quantifying Cholesterol Results ...... 94

5.2.7 Cholesterol Isotherms ...... 95

5.2.8 Lipopolysaccharide (LPS) ...... 97

6. Conclusions and Future ...... 102

6.1 Conclusions ...... 102

6.1.1 CSD Setup ...... 102

6.1.2 Surface Tension Measurements ...... 105

6.2 Future Work ...... 111

6.2.1 CSD Setup ...... 111

6.2.2 Future Experiments ...... 113

References ...... 115

Appendix A: Instructions for Using ADSA-CSD and Blueprints for Setup Pieces ...... 126

x

LIST OF FIGURES

Figure 1.1: Details of the human respiratory system.2 ...... 3

Figure 1.2: The structure of the alveoli.5 ...... 5

Figure 1.3: Lifecycle of the pulmonary surfactant.11 ...... 7

Figure 1.4: Details of composition of PS.30 ...... 12

Figure 2.1: System of two contacting bulk phases α (blue) and β (yellow). (a) In an real system, the contact region is represented by and interface, s (green), while in an ideal system, α and β phases are constant upto an ideal interface (dark grey), or Gibbs dividing surface (GDS)...... 27

Figure 2.2: Diagram used to illustrate where R1 and R2 are positioned relative to the top of a flat circle. . 39

Figure 2.3: Coordinate system for Laplace equation of capillarity and ADSA illustrating that R1 and R2 are arbitrarily chosen, but are perpendicular to one another...... 41

Figure 3.1: Tensiometry techniques utilized to study surface tension of PS.14 ...... 46

Figure 4.1: CSD setup. Schematic (a) angled view, (b) top view. Photographs (c) angled view, (d) top view.

Components of the CSD setup include (1) syringe pump, (2) light source, (3) diffuser, (4) microsyringe, (5) pedestal, (6) optical table, (7) base plate, (8) lens, (9) lens holder, (10) 3-axis rollerblock, and (11) CMOS camera...... 55

Figure 4.2: Different generations of pedestals. (a) Original design. (b) New design...... 57

Figure 4.3: SEM images of (a) and (b) a poor quality pedestal as denoted by bent under edges and wavy, non–circular top, and (c) and (d) a good quality pedestal denoted by a clean sharp edge and smooth continuous top...... 59

Figure 5.1: A 23 μL drop of nanopure water constrained to the pedestal. The green line corresponding to the Laplacian fit derived by ADSA. Note the quasi–spherical shape...... 69 xi

Figure 5.2: Surface tension for water droplets of increasing volume. The volume plotted is the volume measured using ADSA, whereas the volume infused falls in the 18 – 25 μL range. All drops are within a ±

3% range of the surface tension at room (72.9 mN/m). The light and darker red shaded areas represetn the ±1% and ±2% ranges, respectively. Trial 1 and 2 correspond to two sets of experiments that were completed on the same day, but for which the syringe was refilled between trials...... 73

Figure 5.3: Surface tension measurements of static water drops with infusion volume of 23 μL. All surface tension values are within ±3% of the theoretical value at room temperature (72.9 mN/m), designated by the black horizontal line. The drops obtained on Day 2 are from two different sets of experiments; all drops on this plot ar freshly infused...... 75

Figure 5.4: Dynamic cycling measurements of a water drop infused at an initial volume of 23 μL followed by 18% volume compression/expansion cycle. This plot starts partially through the first expansion and shows drop volume and as a function of time as well as surface tension. The green and blue peaks and valleys in the plot show that the drop material was conserved. The maximum volume of the green and blue lines parallel the nearly flat valleys of the red line that signifies the most accurate surface tension values for the cycling water...... 76

Figure 5.5: Surface tension of water with respect to time and initial infused volume which shows that as volume and time decrease the surface tension stays mostly constant...... 78

Figure 5.6: Surface tension as a function of time for an experiment where the initial volume was set to 13

μL, 10 μL was infused followed by 10 μL withdrawn. The final volume should be 13 μL, however, that is not the case as is shown. Surface tension values stayed constant until the volume reached very low volumes

(<10 μL)...... 80

Figure 5.7: (a) 23 μL drop of nanopure water with green line that shows the Laplacian fit using ADSA from Fig. 5.1. (b) 23 μL drop of nanopure water with a thin film of DPPC. The green line on the drop profile shows the Laplacian fit using ADSA. Note the flatter, less spherical shape of the drop...... 84

xii

Figure 5.8: DPPC surface tension measurements as a function of droplet volume. A 23 μL drop was formed and 1 μL of DPPC thin film was placed on top of the drop. Each drop was freshly infused, and the majority of the surface tension values measured are between 58–65 mN/m...... 85

Figure 5.9: Surface tension and volume with respect to time for of a 23 μL water drop with a 3

μL DPPC thin film...... 87

Figure 5.10: (a) 3 μL of 83 μM DPPC placed on 23 μL water drop and (b) 1 μL of 83 μM DPPC solution placed on 23 μL water drop...... 89

Figure 5.11: (a) 23 μL drop of nanopure water with green line that shows the Laplacian fit using ADSA from Fig. 5.1. (b) and (c) 23 μL drop of nanopure water with a Chol thin film taken at time t = 0 and 1 min, respectively. The green line on the drop profile shows the Laplacian fit using ADSA. Note the flatter, less spherical shape of the drop, especially as time increases...... 90

Figure 5.12: Three drops which were imaged at t ≈ 0 and 1 of maximum volume. The top drop in each pair was taken at t ≈ 0 min and after one minute it can be clearly seen that the surface tension decreased and that a slight loss of volume occurs. These drops are six of the drops shown in Figure 5.13...... 91

Figure 5.13: Four different sets of surface tension measurements of a Chol thin film on a bulk water drop, all taken on the same day. As can been seen in the plot, if the drop is allowed to rest, the surface tension values decrease...... 93

Figure 5.14: Surface tension and volume with respect to time for evaporation of a 23 μL water drop with 3

μL Chol thin film. Surface tension is fairly constant...... 95

Figure 5.15: (a) 3 μL of 57 μM cholesterol solution placed on 23 μL water drop and (b) 2 μL of 57 μM cholesterol solution placed on 23 μL water drop...... 97

Figure 5.16: (a) 23 μL drop of nanopure water with green line that shows the Laplacian fit using ADSA from Fig. 5.1. (b) 23 μL drop of nanopure water with a thin film of LPS. Note the much flatter, wider

xiii

shape of the drop with the LPS thin film compared to the water drop. The green line on the drop profile shows the Laplacian fit using ADSA...... 98

Figure 5.17: Surface tension measurements of static water drops with a LPS thin film. The surface tension values vary by ~7 mN/m from the lowest value calculated to the highest, with the majority of values falling between 56–58 mN/m...... 99

Figure 5.18: Dynamic cycling plot of a LPS thin film on a bulk water drop that was infused and withdrawn.

The volume and area appear to be smooth and return to the same point after each infusion and withdrawal which signifies little to no overall drop loss. The steady increase of surface tension most likely signifies that the drop did not reach equilibrium before the cycling began...... 100

Figure 6.1: Custom–built components. (a) one–piece pedestal and holder, (b) base plate, and (c) lens holder (top and bottom parts)...... 112

Figure A.1: Instructions for using ADSA-CSD Title page...... 127

Figure A.2: Instructions for using ADSA-CSD page 1 ...... 128

Figure A.3: Instructions for using ADSA-CSD page 2...... 129

Figure A.4: Instructions for using ADSA-CSD page 3...... 130

Figure A.5: Instructions for using ADSA-CSD page 4...... 131

Figure A.6: Instructions for using ADSA-CSD page 5...... 132

Figure A.7: Bottom portion of lens holder...... 133

Figure A.8: Top portion of lens holder.Figure B.2 ...... 134

Figure A.9: Dimensions of current pedestal...... 135

Figure A.10: Dimensions of new pedestal for one-piece pedestal design...... 136

Figure A.11: One-piece pedestal design...... 137

Figure A.12: Main body of new grid holder...... 138

Figure A.13: Second part of new grid holder that secures the grid in place...... 139

xiv

Figure A.14: Baseplate that new grid holder and new one-piece pedestal will be placed on...... 140

xv

LIST OF TABLES

Table 1.1: Four main categories of exogenous surfactant...... 23

Table 5.1: Analysis of data corresponding to Fig. 5.2...... 74

xvi

LIST OF ABBREVIATIONS AND SYMBOLS

Abbreviations

ADSA Axisymmetric Drop Shape Analysis

ALI Acute Lung Injury

ARDS Acute Respiratory Distress Syndrome

CBS Captive Bubble Surfactometer

Chol Cholesterol

CMC Critical

CMOS Complementary Metal–Oxide–Semiconductor

CPAP Continuous Positive Airway

CSD Constrained Sessile Drop

DPPC Dipalmitoylphosphatidylcholine

FFA Free Fatty Acids

GDS Gibbs Dividing Surface

LE Liquid–Expanded

LED Light–Emitting Diode

LPL Lysophopholipid

xvii

LPS Lipopolysaccharide

L/S Lecithin/Sphingomyelin

LWB Langmuir Wilhelmy Balance

MMA Mean Molecular Area nRDS Neonatal Respiratory Distress Syndrome

PBS Pulsating Bubble Surfactometer

PD Pendant Drop

PEEP Positive End-Expiratory Pressure

PG Phosphatidylgycerol

PI Phosphatidylinositol

PL Phospholipid

PS Pulmonary Surfactant

PTFE

SEM Scanning Electron Microscopy

SP Surfactant Protein

SRT Surfactant Replacement Therapy

SS Stainless Steel

TM Tubular Myelin

xviii

Symbols

A Area a Activity b Curvature of the Drop c Capillary Constant

Γ Surface Excess

G Gibbs Free g Gravitational Acceleration

γ Surface Tension

L

ØID Inner Diameter

ØOD Outer Diameter

μ Chemical Potential ni Number of Components

φ Angle of Inclination Relative to the Interface

ΔP Change in Pressure

π Surface Pressure q Heat

xix

R Radius

ρ

S Entropy s Interface, Arc Length of Drop

T Temperature t Time

U Internal Energy

V Volume w Work x Length of Drop z Height of Drop

xx

1. Introduction

1.1 Overview

The goal of this thesis is to develop and use an experimental model system to study the behavior of pulmonary surfactant. Pulmonary surfactant (PS) is found in the lining of the lungs. The low surface tension achieved by the PS allows the alveoli, small air sacs in the lungs, to expand and contract easily, and to prevent their collapse.

Hence, without PS, breathing would not be possible. PS contains several key components that together determine the physical properties of the surfactant, in particular their ability to lower the surface tension. The latter can be investigated using a number of tensiometric techniques.

1.2 Respiratory System, Lungs, and Pulmonary Alveoli

1.2.1 Respiratory System and Lung

Breathing is made possible by the lungs and respiratory tract. As illustrated in

Figure 1.1, air enters the body via the trachea that leads to two bronchi. The bronchi branch into a series of bronchioles that, in turn, lead to the alveoli. There are

1

approximately 20 divisions going from the trachea to the alveoli, and the larger number of alveoli means that they account for the greatest surface area of the lungs. Overall, the lungs of a healthy adult contain more than 2,400 kilometers of airways that include more than 300 million alveoli, corresponding to a surface area of about 70 m; this is about 80 times the surface area of the human skin and is comparable to half of a tennis court for an a 70 kg person.1

2

Figure 1.1: Details of the human respiratory system.2

The lungs can be divided into two “zones”: the conducting and the respiratory.

The conducting zone comprises the trachea, the bronchi, the bronchioles, and the terminal bronchioles. The respiratory zone is composed of the respiratory bronchioles, alveolar ducts, and the alveoli. The conducting zone is reinforced with cartilage, to hold open the

3

airways, and is responsible for humidifying the air. There is no exchange with the in the conducting zone, all occurs in the respiratory zone.

1.2.2 Pulmonary Alveoli and Breathing

Breathing is possible using either negative or positive pressure gradients. During normal breathing, the diaphragm creates an overall negative pressure on the outside of the alveoli and air is pulled into the lungs from outside the body. At normal breathing rates, the average human takes 15-18 breathes per minute and over the course of a minute approximately 500 mL of air is exchanged.3

The lower surface tension (~30 mN/m) of the fluid lung lining, the aqueous layer at the surface of the alveoli allows the diaphragm to use less and energy to facilitate breathing. If the lung surfactant is compromised, the diaphragm requires more energy per breath, taking energy away from other parts of the body.

PS lessens the energy required to inflate the lungs by increasing the pulmonary compliance, i.e. the ratio of the change in lung volume when a distending pressure is applied. During breathing the alveoli undergo a cycle of inflation and deflation with the

4

surface tension of the PS decreasing at the air-aqueous interface during expiration and increasing during inhalation. The lowering in the alveolar surface tension during expiration reduces the likelihood of alveolar collapse during expiration by decreasing the elastic recoil. The lungs are therefore able to maintain patency with a small

4 transpulmonary pressure of less than 10 cm H2O.

Figure 1.2: The structure of the alveoli.5

5

Alveoli range in size from about 75 – 300 μm in diameter in adults.1 Weibel and coworkers used electron microscopy to show that a large amount of the alveolar surface is coated by a thin aqueous layer.6–8 The aqueous layer was determined to have an average thickness of 0.14 μm over the alveolar surface, 0.89 μm in alveoli corners, and an overall area-weighted thickness of 0.2 μm.9 The aqueous layer is significantly thicker than is necessary to accommodate a bulk comparable in size to the surfactant film on top. The aqueous subphase is crucial for PS because it provides a place for the adsorption, desorption, surfactant secretion, morphological transformation, and recycling of the components that make up PS.1 This is commonly referred to as the life cycle or alveolar metabolism of PS.10

1.3 Pulmonary Surfactant

1.3.1 Lifecycle of Pulmonary Surfactant

Pulmonary surfactant is located in the alveoli, bronchioles, and alveolar ducts in the lungs and is synthesized by pulmonary type II epithelial cells. Once PS is formed, it is processed and packed into lamellar bodies which are structures composed of closely

6

packed multiple surfactant bilayers. Lamellar bodies are transformed into a morphological form called tubular myelin (TM) when PS is secreted into the aqueous subphase of the alveoli, which can be seen in Figure 1.3.

Figure 1.3: Lifecycle of the pulmonary surfactant.11

Tubular myelin is comprised of large squared and elongated tubes that range in size from nanometers to microns.12,13 It is primarily composed of phospholipids and

7

proteins, but also requires the presence of apoproteins and calcium.14 The surface– active film at the air-aqueous interface is formed from the surfactant components when they are released from the TM. PS is thought to be a monolayer,15 but various methods16–19 have shown that at least in part, PS has regions that are thicker than a monolayer. One or more lipid bilayers can form these thicker layers that are collectively referred to as the surface– associated “surfactant reservoir”.14 Once the PS has adsorbed at the interface, the surfactant film is intermittently compressed and expanded throughout the course of breathing. After the PS has served its purpose, it is released as small unilamellar vesicles.

The majority of the used surfactant is removed by endocytosis and placed back into the lamellar bodies while a smaller portion is taken up by the alveolar macrophages.

The type II cells recycle some of the surfactant components into the lamellar bodies, and some of the PS is able to flow into the trachea where it is eventually swallowed. The turnover time for PS is fairly short, ranging from about 4 to 11 hours.20

8

1.3.2 Physical Properties of Pulmonary Surfactant

PS are not very soluble in the alveolar aqueous subphase. As the available area shrinks during a breathing cycle, the PS monolayer at the air–aqueous interface becomes denser and the molecules approach each other more closely, thus increasing the surface pressure. The surface pressure π is related to surface tension by π

= γw – γm, where γw and γm are the surface tensions of pure water and PS monolayer, respectively. The surface tension tends to spread the PS molecules out, thereby opening the alveoli up and not allowing them to collapse upon themselves. As this happens enough PS must be at the interface to cover the surface throughout inhalation while maintaining proper surface tension.21

PS has at least three biophysical properties that are considered crucial for normal respiratory physiology: ability to efficiently lower surface tension upon film compression, rapid adsorption, and effective film replenishment upon film expansion.10,22

A competent PS must be able to maintain a near–equilibrium surface tension during inspiration to work properly in the lungs;23,24 this can be made possible by surfactant re- spreading from a surface–associated surfactant reservoir. PS preparations that adsorb

9

proficiently usually function well to reduce surface tension values to low numbers during compression and to re-spread sufficiently during film expansion. A surfactant reservoir likely forms during adsorption, compression, and situations promoting rapid adsorption such as, for example, high surfactant concentrations and lack of inhibitors that favor formation of a multilayer reservoir.

For a PS to function properly it should adsorb to the air–aqueous interface in the lungs quickly i.e., within a few seconds or less; this most likely takes place in two steps.

First, the surfactant aggregates must diffuse to the subphase closely bordering the interface, and this is very dependent upon the surfactant concentration in said subphase.

Second, the surfactant aggregates need to make their way to the surface. Since the surfactant prefers to form aggregates to lessen its contact with water, the aggregates, or vesicles, need to “unzip” in order to penetrate the air–aqueous interface. An energy barrier must be crossed to overcome the (since a fair amount of energy is conserved when vesicles are formed) as well as the weak van der Waals .25,26

10

Limited experimental evidence points to the surface tension lowering to having very small values during expiration, and during compression proficient PS films lower to near zero surface tension values. There should only be a relatively small compression area (area of reduction) of approximately 20-30%, whatever the maximum variation of alveolar surface area is during normal tidal breathing.27,28 The PS film should only marginally increase surface tension and remain close to equilibrium surface tension upon expansion.24 A minimization in film collapse and competent film replenishment during the compression-expansion cycle should be reflected by a lack of hysteresis. No single component of endogenous PS is able to mimic PS in its ability to withstand high surface pressure, correlating to low surface tension. Additionally, no single PS component is able to adsorb quickly in order to avoid alveolar collapse at 37°C while maintain low surface tension.14

1.3.3 Pulmonary Surfactant Components

The composition of human PS is very similar to that of other mammals. PS contains about 90% lipids and 10% proteins by .29 The lipid fraction, in turn, is

11

comprised of ~90-95% phospholipids (PL) with the remainder being neutral lipids.

Figure 1.4 shows the breakdown of the phospholipid components in PS.

1%

3% 3% 9% Phosphatidylcholine 1%

Phosphatidylethanolamine 3% Phosphatidylserine Phosphatidylglycerol Sphingomyelin Phosphatidylinositol Unidentifiable phospholids 80%

Figure 1.4: Details of lipid composition of PS.30

Phospholipids are amphiphilic molecules that partition at the air–aqueous interface by having their hydrophobic fatty acid tail groups into the air and their

12

hydrophilic head groups in the aqueous subphase. For example, when 1,2-dipalmitalyl- sn-glycero-3-phosphocholine (DPPC), a long-chained disaturated, zwitterionic PL, is deposited on an aqueous solution, it will spread out as much as possible to create a one –thick thin film or monolayer. If this monolayer is compressed the surface pressure is increased (surface tension decreased) due to the PL molecules going from dilute gaseous state to a more concentrated liquid-expanded (LE) phase where they can interact. During this the fatty acids remain mobile due to the presence of kinked gauche- gauche or trans-gauche configurations.

Phospholipids tend to form bilayers spontaneously at room temperature. As the temperature is increased, PL bilayers will “melt,” going from an ordered phase to a disordered liquid-crystalline phase. of PL molecules within each leaflet bilayer is possible due to the fatty acid disorder – this property is called “fluidity”. Sterols affect the physical properties of PLs. When a sterol, such as cholesterol (Chol), is added to PL bilayers ordered gel and disordered liquid-crystalline phases transform into liquid- ordered and liquid-disordered, respectively. Since Chol is not overly amphiphilic, it does not follow the usual lipid behavior; therefore it affects each packing differently. Chol has

13

a fluidizing effect on lipids in the gel phase due to it disruption of the lipid packing and a condensing effect on fluid membranes due to the stabilization of the disordered chains.31

This is not always the case since temperature and cholesterol concentration greatly affects the phase behavior of Chol-PL mixtures.32

Cholesterol appears to naturally occur in PS;33–35 most mammalian surfactants contain 5-10 wt% (10-20 mol%) Chol.34–37 The role of Chol in PS is still relatively unknown. It was originally thought that Chol was detrimental to PS and its ability to reach low surface tension.38,39 That conclusion is now believed to be a consequence of the early experimental techniques used to measure PS surface tension – Langmuir trough and pulsating bubble surfactometer.14 Several studies have shown that higher concentrations (also known as supraphysiological levels) of Chol naturally occurring in

PS are disadvantageous.37,40–43 The mechanism by which Chol inhibits the PS has not yet been determined. The ambiguous of the role of Chol in PS forces this begs the question as to whether or not it should be included in the synthetic and partially synthetic PS currently being tried, formulated, or already on the market.

14

Besides PLs, there are 4 PS-specific proteins namely surfactant protein (SP)-A, -

B, -C, and -D. The nomenclature of these proteins is based on that proposed by

Possmayer.44,45 SP-A is the most abundant surfactant apoprotein in PS, moderately hydrophilic, and with a molecular weight of 26–28 kDa. SP-B is mostly hydrophobic and is between 8.5–9 kDa. SP-C is also mostly hydrophobic and is between 3.5–4.2 kDa.

SP-D plays no biophysical role in PS and is between 39–46 kDa.

The proteins are required in the correct amount/concentration. For example, albumin can affect the respreading ability of a model PS,46 this happens at surface tensions greater than 50 mN/m for a mixture of DPPC/POPG/palmitic acid. Some studies have been conducted47–49 which suggest that proteins may be responsible for surfactant inhibition, and additionally, this hints that surfactant interaction/binding may play a role in PS inhibition.

1.3.4 PS Inhibition, Inactivation, and Degradation

Decrease or absence of normal surface activity of PS is called surfactant inhibition or inactivation. Surfactant inhibition may prevent the film from reaching low

15

surface tension upon compression, affect re-spreading of PL during expansion, or interfere with the PL adsorption to form a functional surfactant film. There are several substances that can inhibit PS: including free fatty acids (FFAs), meconium, plasma proteins, supraphysiological levels of Chol, lysophopholipids (LPLs), and unsaturated membrane PL.1,50 Surfactants can also be compromised by reactive species51,52 and by pollutants,53,54 and degradation of SPs by proteases or of surfactant lipids by phospholipases. These degradation agents are already in the lungs in low levels, but can increase due to microbial infections, and through secretations by type II cells and leukocytes with pulmonary inflammation.

Another type of PS inhibition stems from lipids such as LPLs, bile acids, unsaturated membrane PL, unsaturated FFAs (e.g., oleic acid), Chol, and diacylglycerol.41,55–57 Unsaturated amphiphilic lipids and fatty acid molecules becoming inserted or mixed in the PS PL can drastically fluidize the PL monolayers and could prevent a low surface tension from being reached due to early collapse. The inactivation due to lipids penetrating the surfactant film cannot be overcome by means of increasing the surfactant concentration.55,56,58

16

1.4 Respiratory Syndromes

1.4.1 Overview of Respiratory Syndromes

Respiratory distress syndromes can affect people of any age, although neonatal respiratory distress syndrome (nRDS) only affects infants while acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) can affect adolescents and adults. Lung injury that results in respiratory syndromes may result from any number of traumas outside of hospital care, but may also be caused by -induced or ventilator- induced injury while in intensive care. Lung injuries encompass damages to multiple parts of the lungs as well as numerous pulmonary cell types.

Type II pneumocytes are responsible for the formation of PS, and the absence of these cells in premature infants is thought to cause nRDS.1,59 ALI and ARDS have similar symptoms to nRDS, but are acquired instead of due to a lack of PS formation during gestation. A diagnosis of ALI may come, although not necessarily, before ARDS, but all patients with ARDS have clinical ALI. Due in part to surfactant therapy, premature infant moratality rate in the USA attributable to nRDS fell by 24% in 1990 and

17

has continued to decline.60,61 The fatality rate of ARDS is 30-40% and it is thought to affect about 150,000 patients a year in the United States alone.62

1.4.2 Neonatal Respiratory Distress Syndrome

nRDS is the most common PS deficient disease worldwide and it effects prematurely born infants.1 It is thought that nRDS affects roughly 10% of all prematurely born infants in developed countries,63 and in 2002 it affected approximately

24,000 babies in the United States alone.63 Infants born before 29 weeks of gestation are at a greater risk of developing nRDS than those born later because the lung is the last organ to develop during gestation. The maturity of fetal lungs can be monitored by measuring PL and lecithin-sphingomyelin content in the amniotic fluid, since there is an increase of these components as the lungs mature.59

Risk factors that increase the chances of an infant having nRDS include: premature birth, maternal diabetes, male sex, ethnicity, lower birth weight, and multiple gestations (e.g., twins, triplets). Healthy newborns have about 100 mg/kg of PS while

18

PS–deficient infants tend to have less than 5 mg/kg.21 Signs of nRDS tend to appear within the first few hours after birth, usually manifested by impaired breathing.

1.4.3 Acute Lung Injury

ALI is typically associated with physico-chemical effects on lung surfactant.

Most often patients initially present an injury to the lung followed by a diagnosis of edema that quickly turns into respiratory failure. The PS formation can become inhibited or its components can be degraded, altered, or depleted.

The standard therapeutic treatment for patients with ALI is exogenous surfactant replacement therapy in which a modified natural or synthetic PS is delivered to the lungs.64 The most common modified natural treatment comes from bovine or porcine extracted PS.

1.4.4 Acute Respiratory Distress Syndrome

ARDS is an indication of ALI that results from direct lung injuries as well as injuries to other organs. Most often patients have ALI before ARDS so the path is the

19

same – lung injury that leads to respiratory failure. Although several factors tend to exist, the ARDS diagnosis is strictly based on pulmonary criteria.

The pathogenesis of ARDS is still not completely understood, but it is believed that surfactant inhibition is a major cause, often resulting from extensive lung injury, severe pulmonary infection, , near-, trauma, or radiation damage.62 Some of the materials that can leak and cause inhibition are serum proteins, hemoglobins, and certain lipids.

Plasma proteins/surfactant ratio plays a large role in surfactant inhibition;58 it is thought an early event in the pathogenesis of ARDS is leakage of plasma proteins into the alveolar space, commonly caused by an impaired alveolar-capillary barrier.65 The mechanism by which this happens is not completely understood. There are some data that fibrinogen, hemoglobin, and albumin can inhibit PS by means of competitive adsorption since these compounds are also surface active.58,66,67 Proteins are smaller and water soluble so they can travel to the air-water interface more rapidly than vesicle(s) of

PL which must first unzip and then spread out. Once the proteins are adsorbed to the air- water interface they create a steric and/or electrostatic energy barrier68,69 which excludes

20

the PL from entering the interface. Studies in vitro58 have shown that surfactant inhibition from competitive adsorption can be overcome by increasing the surfactant concentration high enough or by adding SP-A70,71 since both approaches enhance adsorption kinetics of PL. Exogenous surfactant replacement therapy has shown limited positive effects for ARDS patients.50,72,73

1.5 Replacement Therapy

Since nRDS and ARDS share several similarities, it was thought they could be sensitive to the same treatment – surfactant replacement therapy (SRT).74 The main course of successful treatment for patients with nRDS is the administration of exogenous lung surfactant. The treatment of nRDS appears to be easier to treat due to infants having healthy lungs; SRT needs to be added as a place keeper until the lungs are able to produce endogenous surfactant on their own, in most cases. When compared to the price of neonatal intensive care, SRT is considered cost–effective, although it is relatively expensive. Breathing is typically improved in ARDS patients who receive SRT, but the overall effect on the disease can vary and is very dependent on the timing, administration,

21

dosing, and surfactant preparation used.75,76 SRT does not typically greatly improve the recovery, or mortality of children and adults affected by ARDS. This being said, there is a great need for “designer” surfactant preparations.

1.5.1 Types of Current Surfactant Replacement Therapy

There are a wide range of exogenous SRTs as illustrated in Table 1. The first type is PS harvested and purified from full–term human amniotic fluid during cesarean sections.77,78 Another common type is animal-derived sources which typically perform better compared to synthetic surfactants, another type of surfactants studied, in both preclinical animal experiments and clinical practice.79–81

22

Table 1.1: Four main categories of exogenous surfactant. Source Type Unique to composition Example Human amniotic Whole Contains hydrophobic and hydrophilic N/A fluid surfactant proteins Bovine/Porcine Modified Contains only hydrophobic proteins SP- BLES natural B and SP-C Curosurf surfactant Infasurf Survanta Synthetic Synthetic Contains simplified peptides or Surfaxin recombinant surfactant protein analogs Venticute Synthetic Protein -free Contains only PLs (mostly DPPC) and N/A synthetic additives such as ALEC and Exosurf

Modified natural surfactants have some drawbacks, chief among which are batch-

to-batch variations in composition and the potential for microbiological infections.

Additionally, the SP contents in naturally derived sources available can be low compared

to endogenous surfactants. The hydrophilic proteins, SP-A and SP-D, are removed from

modified natural surfactants due to immunological considerations. Modified natural

sources differ in neutral lipids (mainly Chol), additives e.g., triacylglycerol supplement

and palmitic acid in source (porcine or bovine), and different production procedures (lung

23

mincing or bronchoalveolar lavage). A drawback of animal-derived surfactants is the cost which is about $500 per dose for nRDS;82 the expense is mostly from quality control of small batches. In comparison to nRDS, when SRT is used for ARDS patients, the cost only increases due to the greater amounts required, increased number of doses, and the need for continuous supply.

The first type of synthetic surfactant mentioned in Table 1 may contain peptides83 or recombinant surfactant analogs84 modeled from surfactant proteins. An additional type of synthetic surfactant mainly consists of PLs and spreading agent such as, for example, hexadecanol and pharmaceutical additives.85

1.6 Goals and Objectives

The expense and low success rates of current exogenous SRTs are the motivation behind studying the surface tension of PS and its components. The primary goal of this study is to design and construct an instrument capable of measuring the surface tensions of various surfactants related to lung surfactants. Once an optimal setup is achieved physical properties of PS components and PS inhibiting materials will be measured.

24

25

2. Thermodynamics of Interfaces

2.1 Theory of Surface Tension and the Gibbs Adsorption Isotherm

An understanding of interfacial dynamics is required to comprehend the partitioning of PS at the air-aqueous interface and the effect this partitioning has on the surface tension. An interface is formed whenever two separate, distinct phases meet.86 In the following discussion two bulk phases, e.g. a gaseous (α) and a liquid (β) phase, meet at an interface, as is illustrated in Fig. 2.1.

26

Figure 2.1: System of two contacting bulk phases α (blue) and β (yellow). (a) In an real system, the contact region is represented by and interface, s (green), while in an ideal system, α and β phases are constant upto an ideal interface (dark grey), or Gibbs dividing surface (GDS).

The two systems illustrated in Fig. 2.1 are the “real” and the “ideal” systems commonly used to describe an interface between two bulk phases α and β. For the real system, the α phase is separated from the β phase by an interface, denoted s, which is not infinitely thin, so it has some volume, and contains a mixture of the α and β phases. The details of the transition in this interfacial region or interface, s, are not so well known, therefore the system is often simplified to an ideal case. The ideal system has the α phase separated from the β phase by an infinitely thin, arbitrarily placed mathematical plane

27

called the Gibbs dividing surface (GDS). In this ideal system, the α phase is constant up to the GDS, at which there is sharp change to the β phase.

There are two common approaches used to explain surface tension effects, namely, the Gibbs approach and the Guggenheim approach. In this chapter, the former approach will be discussed. The Gibbs’ approach defines the surface properties of the real interface, (s), with respect to an ideally thin reference system, (GDS). The surface excess is the difference between the real and ideal, or reference, . Starting from the ideal case, the volume of the interface would then be and therefore, the total volume of the system is given by

. (2.1)

Other extensive properties of the system can then be written in terms in the α and β phases and the interface s as follows

(2.2) (2.3)

(2.4)

28

where U is internal energy, ni are the components, and S is the entropy.

The internal energy is an important part of this approach so the discussion should start there. Using the first law of thermodynamics the reversible change in internal energy is expressed as

(2.5)

where δq and δw are the change in heat and work respectively. If the system is of fixed composition then the change in internal energy for the system in Fig. 2.1 is governed by

. (2.6)

where TdS represents the heat flow for the entire system, -PdV is the volume work of the bulk phases (α and β, in this case), and γdA is the surface work of the interface, s. Here T is the absolute temperature, P is the pressure, γ is the surface tension, and A is the area of the dividing surface. Additionally, when looked at with respect to constant volume, Eqn.

29

(2.6) illustrates that internal energy changes when the area is varied, which can be represented by γ. Also, if the area is held constant, internal energy changes with respect to changes in volume and can be defined as -P. In a multicomponent system,87 the two

bulk phases, α and β, may each contain one or more components (ni), and therefore the interface, s, may as well. The internal energy relation in this case would be written as

(2.7)

where μi is the chemical potential of the ith substance. The μidni terms take into account the energy change due to an alteration in composition.

Up until now, the entire system has been considered. To obtain the surface excess, only surface terms are of importance. The internal energy of the surface is

(2.8)

30

where the -PdV term does not exist due to the interface not having a volume. Eqn. (2.8) may be integrated while keeping all the intensive variables constant. This gives to the physical interpretation that it is possible to increase the size of the system by increasing the surface area. This is accomplished in such a manner that the change in components is the same in the original system, i.e, the volume and number of components of the bulk phases. Integration of Eqn. (2.8) gives

(2.9)

Differentiation of Eqn. (2.9) yields

(2.10)

Setting Eqn. (2.10) equal to Eqn. (2.8) gives

31

(2.11)

At constant temperature Eqn. (2.11) can be simplified to

(2.12)

Rearranging and solving for dγ gives

(2.12)

(2.13)

where Γi denotes the surface excess which is

(2.14)

and Eqn. (2.13) can then be rewritten as

32

(2.15)

The location of the arbitrary GDS determines the values of . The GDS can be

chosen to give a more physical meaning. For instance, consider a binary system with

components n1 (solvent) and n2 (solute) in, for example, the β phase. The GDS can be

positioned so that , which means the surface excess of the solvent vanishes.

Rearrangement and simplification of Eqn. (2.15) yields

(2.16)

where denotes the relative surface excess of the second component, and the superscript (1) indicates the choice of the dividing surface.88 Furthermore, the value of surface excess can be less than, greater than, or equal to zero.

The chemical potential is related to the activity, a, by

33

(2.17)

where R is the and is the chemical potential of the standard state of the solute, which depends only on temperature and pressure. Differentiating Eqn. (2.17) at constant T yields

(2.18)

Substituting Eqn. (2.18) into Eqn. (2.16) gives

(2.19)

while rearranging and solving for yields the Gibbs adsorption equation,

(2.20)

34

This equation shows the relationship between the surface excess of the second component with the activity of second component in the β phase.

2.1.1 Surface Tension

For a one component system, molecules in the bulk are symmetrically surrounded by other molecules and have a perfect force balance. However, at the surface the up- down symmetry is broken and this interrupts the perfect force balance. In the direction parallel to the interface, the symmetry is still present, therefore guaranteeing a force balance parallel to the surface. Additionally, the attractive forces along the direction parallel to the interface are stronger in magnitude than the repulsive forces. In actuality, a positive surface tension force comes from this stronger attractive forces magnitude being larger than the repulsive forces. This can be seen from Eqn. (2.21) where only the surface terms matter:

(2.21)

35

Eqn. (2.21) can additionally be written as a sum of partial differentials

(2.22)

A comparison of Eqns. (2.21) and (2.22) shows that surface tension can be defined as the work required to increase the area of a surface reversibly and isothermally by a unit amount.86

(2.23)

At room temperature the surface tension of most organic and ranges between 10-30 mN/m.86 In comparison to most liquids, water at room temperature exhibits a rather high surface tension of approximately 72 mN/m89 because of its strong hydrogen bonding ability.

36

2.2 Theory of Capillarity

2.2.1 Laplace-Young Equation and Axisymmetric Drop Shape Analysis

An interface can be thought of as a ribbon held firmly over an open end of a hollow, double ended open tube. If the pressure on both sides of the ribbon is equal then the ribbon is level and constant. If the pressure, which is a force per unit area, on one side of the ribbon held over the tube changes then ribbon will be concave on the side which has a greater pressure. Similarly, a pendant or sessile drop can be thought of in the same manner. The drops are influenced by two competing force – gravity and surface tension. Gravity will pull down on the pendant drop elongating it while the sessile drop will be flattened by this force. Surface tension, which is a force parallel to the interface, will make the drop more spherical. Sessile and pendant drops tend to be axisymmetric in nature. In the case of a drop, axisymmetric signifies that if the drop was bisected each half would be identical. Additionally, if a drop is small enough gravity and surface tension can be assumed to be the only external forces working on the drop. The Laplace equation of capillarity allows for a mathematical way to relate the pressure difference

37

across a curved interface separating two homogeneous , for example, the drop and surrounding gas phase. The Laplace equation is

, (2.24)

where R1 and R2 are the two main radii of curvature, γ is the surface tension, and ΔP is the pressure difference across the interface.

38

Figure 2.2: Diagram used to illustrate where R1 and R2 are positioned relative to the top of a flat circle.

Illustrated in Figure 2.2 is a central point, X, which can be thought of as the origin, around which a circle has been drawn, this circle is equal distance from this central point at all positions. Intersecting at the central point are two lines which are perpendicular to

each other. These lines that meet at the top of the circle represent the radii, R1 and R2.

The surface tension can be thought of as an inward pull of a small segment of a radius of this circle.

39

As mentioned earlier, if the drop is small enough gravity can be assumed to be the only external force; the hydrostatic approach to capillarity will be considered in this case.

When this is the case the hydrostatic pressure difference can be rewritten as a linear function of the elevation

(2.25)

where ΔP0 is the pressure difference at a reference plane, Δρ is the density difference between the two bulk phases, z is the vertical height measured from the same reference plane, and g is the gravitational acceleration. Based on these equations, the shape of a drop is readily determined if the surface tension and principle radii of curvature are known. In contrast, it is more challenging to obtain the surface tension from the shape of the drop.

Since sessile and pendant drops are usually axisymmetric in nature this allows for numerical procedures to be used in tandem with Eqn. (2.25). The coordinate system for the two homogeneous systems separated by a curved interface is defined as in Fig. 2.3 is

40

the basis for the axisymmetric drop shape analysis (ADSA) program used in this thesis to obtain surface tension measurements.90

Figure 2.3: Coordinate system for Laplace equation of capillarity and ADSA illustrating that R1 and R2 are arbitrarily chosen, but are perpendicular to one another.

The drop is drawn on an xz-coordinate system with the arc length, s, evaluated along the curve of the drop. The angle of inclination relative to the interfacial plane, φ, is

at the point (xi, zi) and R1 and R2 are the main radii of curvature. The main axis of

41

curvature R1 is correlated to the arc length s as well as the angle φ due to the assumption that the drop is axisymmetric about the z-axis as seen in Fig. 2.3:

(2.26)

and the second main radius of curvature R2 is

(2.27)

Using simple trigonometric relations, one can obtain from Fig. 2.3 that

(2.28)

(2.29)

At the apex, the intersection of R1 and R2 is considered to be constant in all in directions because of axial symmetry. Therefore the two main radii of curvature are deemed equal:

42

(2.30)

when where b is the curvature at the origin and R0 is the radius of curvature. The difference in pressure at the origin can be restated, starting from Eqn. (2.24), as

Δ . (2.31)

Combining Eqns. (2.24) and (2.25) yields

Δ Δ (2.32)

then substituting Eqns. (2.26), (2.27), and (2.31) into Eqn. (2.32) gives

(2.33)

where

Δ (2.34)

Additionally, at Eqn. (2.33) becomes

43

(2.35)

For pendant drops the capillary constant, c, is negative and for sessile drops it is positive. A set of first–order differential equations can be formed for x, z, and φ as functions of the arc length s by Eqns. (2.28), (2.29), and (2.33) with boundary conditions

(2.36)

If the values of b and c are known, using simultaneous integration of the above set of equations, the complete shape of the Laplacian axisymmetric interface between two homogeneous fluids can be determined. This means that gravity, change in density, and the curvature at the apex must be known as well as an estimated surface tension in order to initiate the system of equations above used to determine the surface tension of a drop based on the drop profile.

44

3. Surface Tension Techniques

3.1 Overview

Several methods have been developed to study PS, in particular to measure the surface tension of PS as a function of composition and concentration. These methods include the: Langmuir trough, captive bubble surfactometer, pendant drop, pulsating bubble surfactometer, and constrained sessile drop apparatus. These techniques are schematically illustrated in Figure 4 and are discussed in more detail below.

45

Figure 3.1: Tensiometry techniques utilized to study surface tension of PS.14

3.1.1 Langmuir-Wilhelmy Balance

The Langmuir trough was first used in in the beginning of the 20th century.91 In a Langmuir trough experiment, a trough is filled with an aqueous solution and an insoluble surfactant monolayer is carefully placed on the aqueous subphase. The film is slowly compressed, and expanded, using one or two barriers that move inward

46

across the top of the material in the trough. There can be one fixed barrier and one that moves, or two movable barriers that move simultaneously toward one another. Surface pressure can be determined with a horizontal force transducer measuring the force acting on the floating barrier. To measure surface tension directly, a Wilhelmy plate is added and surface tension is determined based on the change in vertical pull on the plate.92–94

The combined experimental apparatus is called the Langmuir-Wilhelmy balance

(LWB). The LWB is still widely used in the study of PS since the surface area per molecule can be precisely known; it is commonly used to determine surface area isotherms. It is a very versatile technique in that other microscopic and spectroscopic techniques can be combined with LWB to gather more information; these techniques include Brewster angle microscopy,95,96 atomic force microscopy,18,49 sum frequency generation spectroscopy,97,98 fluorescence microscopy,18,95,96 and infrared spectroscopy99,100. Some of the aspects of PS that have been studied using a combination of LWB along with some of the spectroscopic and microscopic methods listed above are domain formation, localized chemical composition of surfactant films at the air-water interface, orientation, molecular structure, and electrical surface potential.

47

One of the downsides to the Langmuir trough or LWB is that it requires a relatively large amount of material, usually tens of milliliters, and the film can leak at the barriers and trough walls. The barriers may experience film leakage at or above the water level, i.e., at the air- interface, or below the water at the liquid-solid interface, respectively.101 Pulmonary surfactant has a high wettability and that enables it to “leak” from the trough whereas most materials do not have as high as a wettability.

The normal breathing rate of an adult human is 15-18 breaths per minute, 3-4 seconds per cycle.1 Since the cycling time of a Langmuir trough is roughly 5 minutes this device cannot readily simulate breathing rates. Langmuir trough experiments also require a zero degree and this cannot be maintained during rapid cycling. If the cycling is too rapid waves form at the air-water interface and negatively impact the surface tension measurements. Finally, the temperature and humidity are very difficult to control – these are important to mimic the environment of the lung.

48

3.1.2 Captive Bubble Surfactometer

In a captive bubble surfactometer (CBS) an air bubble is established in a chamber that has a smooth and hydrophilic ceiling. The latter is achieved by using a stainless steel chamber102 or by coating the ceiling with 1% agar gel.14 and eliminates film leakage.

Once the surfactant film is formed, the bubble is compressed and expanded by changing the hydraulic pressure in the chamber; this can accomplished by adjusting the liquid flow from the chamber to an external reservoir103 or by changing the volume of the chamber.14

Due to its relatively large size, typically 2-7 mm in diameter, the bubble is unlikely to be spherical. The bubble shape depends on two competing forces: surface tension and local gravity. The CBS is ideal for studying film stability and collapse at extreme surface pressure as well as film compressibility. Additionally, it is an excellent technique to study surfactant films at physiological and higher . The surface tension and surface area are coupled in this technique since when the volume of the drop decreases so does the surface area and the shape of the drop changes which affects the surface tension.38 But this is not overly significant since both changes are consistent. The maximum surfactant concentration able to be studied with this technique is ~3 mg/mL16

49

because at higher concentrations surfactant becoming cloudy making the bubble challenging or impossible to see. It is difficult to ensure full humidity using this technique which is a chief concern when studying PS.38

3.1.3 Pulsating Bubble Surfactometer

The pulsating bubble surfactometer (PBS) works by filling a chamber, usually made of polyacrylamide, with the sample material, for example PS.104 The material is then immersed in a temperature-controlled bath. A bubble is formed using a capillary tube; the pressure difference across this bubble is measured for 10 seconds and recorded as the surface tension. After equilibrium is achieved, the bubble is oscillated between two set radii, a maximum and minimum, at a set number of cycles per minute. The pressure gradient across the bubble is measured by a pressure transducer while the maximum and minimum radii are observed by a microscope. This technique is commonly used to ensure quality control of clinical surfactants. PBS requires relatively small amounts of sample, ~20 μL, and a single measurement can be taken in 5 minutes.

50

This technique is very useful for comparing surface activity. A key advantage PBS has is that is it able to mimic breathing rates.

Film leakage is, however, a major issue with this technique. There are two places film leakage can occur at low surface tension: the air-solid interface and the liquid-solid interface. The time it takes for equilibrium to be reached, 10 seconds, is very difficult to change, and this long of an equilibrium time is one of the reasons for film leakage.

Additionally, the surface tension is usually only recorded at the maximum and minimum bubble radii. The surface tension measurements at low surface tension are unreliable so

PBS is not the best technique to use to acquire this kind of data.105 But it is acceptable to use when comparing different PS in a clinical trial situation.

3.1.4 Pendant drop

A pendant drop (PD) system has been designed by Neumann et al106 to measure the surface activity of PS.107,108 Traditionally, a pendant drop is formed at the end of a capillary tube or a blunt hypodermic needle,14 although the Neumann group has also used

51

an inverted CSD pedestal to form the drop. Drawbacks associated with pendant drop method include film leakage or droplet detachment if the drop becomes too large.

3.1.5 Constrained Sessile Drop

In this method, a drop is formed on a flat, circular pedestal with a knife sharp edge which has an angle between 4560°. This sharp edge prevents film leakage. The optimal diameter of the flat top of the pedestal varies depending on the surface tension of the material being measured.109 The drop is formed using a programmable motor-driven syringe pump connected to the surfactant reservoir, and it is possible to expand and contract the drop using the motor driven syringe. There have been several iterations of

CSD setups by the Neumann group from a very basic pedestal to a pedestal with a cuvette over it to control humidity, to an environmental chamber encompassing the pedestal which can control temperature and humidity. Yu et al.110 modified the constrained sessile drop (CSD) to measure the surface tension of PS. No serious limitations have been discovered with this setup.14

52

CSD requires a relatively small amount of material, in the microliter range. There have been several types of materials studied employing CSD, for example, the effect of humidity on film cycling.111,112

53

4. Experimental Setup

4.1 Materials

DPPC and Chol (> 99% purity; Avanti Polar Lipids, Alabaster, AL) and lipopolysaccharide (LPS) (L2630, Aldrich) were used without further purification.

The methanol and (spectrometric grades) were purchased from Fisher

Scientific. Ultrapure water (not purged of CO2) with a resistivity of 18.2 MΩ·cm and a measured pH of 5.5 was obtained from a Barnstead Nanopure system (model D4741,

Thermolyne Corporation) equipped with additional cartridges for organic removal

(D5026 Type I ORGANICfree Cartridge kit; Pretreat Feed). 2 mM cholesterol and I mM

DPPC solutions were prepared in chloroform. 1 mg/mL LPS solution was prepared in ultrapure water.

4.2 CSD Setup

The CSD setup described here is custom-built and its design was inspired by the work of Neumann and coworkers14,110 as well as Lamourand and Hamraoui.110,113 The setup consists of four main sections: (1) the illumination source and diffuser, (2) the

54

pedestal, (3) the imaging system, and (4) the liquid infusion system as shown in Fig. 4.1.

Each of these sections is briefly described below. The light source, pedestal, and imaging system are mounted on a vibration-free optical table and are aligned along the same optical axis approximately 133 mm above the table surface.

a (3) b (9) (8) (1) (2) (4) (11)

(5)

(7) (6) (10)

c d

Figure 4.1: CSD setup. Schematic (a) angled view, (b) top view. Photographs (c) angled view, (d) top view. Components of the CSD setup include (1) syringe pump, (2) light source, (3) diffuser, (4) microsyringe, (5) pedestal, (6) optical table, (7) base plate, (8) lens, (9) lens holder, (10) 3-axis rollerblock, and (11) CMOS camera.

55

4.2.1 Illumination

The white light illumination of the sessile drop was provided by a light-emitting diode (LED) flashlight (mini Eco-I-Lite Lithium-ion 4). The flashlight is placed in an iris diaphragm holder (ID-1.0, Newport) held by a 75 mm post in a 75 mm post holder fixed to the optical table by a mounting base (BA2, Thorlabs) that allows for lateral (left/right) movement. Several other light sources were tried (e.g., Maglite flashlights and desk lamps), but most were too bright or too centrally focused to evenly distribute the drop profile.

A diffuser is required soften and evenly distribute the light. The diffuser used here is a simple thin acrylic plastic sheet (21.5 cm × 28 cm × 0.5 cm); a white paper sheet had also been used previously, but was found to be insufficient to reduce the light intensity. The plastic diffuser is mounted on a small plastic clamp and positioned approximately 4 cm from the flashlight.

56

4.2.2 Pedestal

The pedestal, where the sessile drop is formed, was a very challenging part to design and have machined. The pedestal is a small cone–shaped piece with a very small neck at the base. It is made of 316 stainless steel (SS); a material chosen for its corrosion resistance and cost effectiveness. SS is also fairly easy to machine and will hold a sharp knife edge. The knife edge is crucial to prevent film leakage.

Figure 4.2: Different generations of pedestals. (a) Original design. (b) New design.

57

The first generation of pedestals was made from two separate pieces, the pedestal itself and an inverted T-shaped holder. A slot was milled in the front of the holder to allow for the insertion of a plastic, then later a square–angled piece of SS tubing, connected to the pedestal (see below). The first problem encountered with this design was getting the proper degree of edge sharpness to the pedestal itself which is a crucial to the principle of the technique. The first pedestal machined had blunt, bent under edges and a somewhat elliptical shape, and as a consequence when the drop diameter became too large film leakage occurred. The second attempt of this design yielded a sharper and more circular edge; this was achieved by machining the piece at higher RPMs. The edge sharpness and top shape were verified by scanning electron microscopy (SEM).

58

Figure 4.3: SEM images of (a) and (b) a poor quality pedestal as denoted by bent under edges and wavy, non–circular top, and (c) and (d) a good quality pedestal denoted by a clean sharp edge and smooth continuous top.

The main drawback to this pedestal design is that the pedestal was set in a holder and its horizontal position was not necessarily level. To ensure the levelness of the pedestal, a roll and pitch tilt platform (AMA027/M, Thorlabs) was placed under the holder for fine adjustment. A vertical rotation stage (RP01/M, Thorlabs) was also positioned under the roll and pitch platform to ensure that the pedestal was level on all sides, not just the side visible from the camera. These additions greatly helped with the reproducibility of the results and allowed for on-the-fly minor adjustments. An R10 grid (Electron Microscopy

59

Sciences) is used for calibration, i.e. to translate between number of pixels and physical dimensions. The grid is placed in a filter holder and is attached to the vertical rotation stage once the roll and pitch tilt platform is removed.

4.2.3 Optics

The imaging system also underwent several improvements during the course of this work. The first imaging system used to view the drop was made up of a Kodak

Easyshare (C643) on close-up setting and an optical lens (200mm focal length, Newport) placed between the camera and the pedestal. Although it was possible to view the drop and demonstrate that the setup was working, this imaging system did not allow for the pedestal to completely fill the field of view.

Later on, a compact high resolution color compltementary metal–oxide– semiconductor (CMOS) camera (DCC1645C, Thorlabs; 4.035 cm × 3.2 cm × 3.85 cm, frame rate: 25 fps) with a resolution of 1280 × 1024 pixels was used together with a

Soligor lens 35-105 mm focal length and 1:3.5 aperture to image the drop. The camera and lens combination allowed for closer images of the drop and better edge definition.

60

Additionally, this combination allowed for a live feed of the drop and the possibility of video recording. This was instrumental in determining if the drop was level, and if not, which direction the pedestal needed to be tilted to achieve levelness. Unfortunately, the color camera generated a blue ring around the drop profile which made it difficult to distinguish the true edge of the drop. Moreover, the first Soligor lens was slightly damaged and caused a chromatic aberration on left half of the drop image which in turn made the edge drop profile challenging to determine.

To circumvent these issues, a Soligor lens with the same focal length and aperture was used and a monochrome version of the CMOS camera (DCC1545M, Thorlabs; 4.035 cm × 3.2 cm × 3.85 cm, frame rate: 25 fps, ½" optical sensor) with the same resolution were purchased. This imaging system allows for capture of a clearer and more distinct drop profile. The Soligor lens and camera are spaced (~14 cm) by a series of lens tubes

(SM1 type, Thorlabs; L 0.5–2", ØOD 1") and adapters that aid in the magnification.

The lens assembly is much heavier than the camera and thus needs to be supported. The lens is held in place by a custom–built, two–part aluminum holder (top:

100 mm × 40 mm × 52 mm, bottom: 100 mm × 40 mm × 40 mm) as can be seen in Fig.

61

6.1 and Appendix B contains the drawings. The lens holder is mounted on a 3–axis rollerblock with micrometric drives (RB13/M, Thorlabs) via a flexure stage adapter plate that gives the lens height as well as allows for the lens to be moved in all three directions in the xyz-plane for fine focusing adjustments. Additional height is provided by a set up kinematic (magnetic) base (KB3X3, Thorlabs) onto which the rollerblock is fixed.

4.2.4 Infusion system

The injection system includes a syringe pump, a microsyringe, the tubing from the microsyringe to the pedestal, and the pedestal tubing itself. The syringe pump has been improved as the project developed from the water being delivered by hand to two different syringe pumps. The first one was a syringe pump (KDS100, KD Scientific) that only allowed for infusion. However, in order to perform dynamic and static cycling measurements which require infusion/withdrawal of very small, precise volumes of liquid at various flow rates, a programmable syringe pump (Legato 180, KDS Scientific) was later purchased. For the infusion, and infusion/withdrawal, a 500 μL gastight syringe

(1700 series, Hamilton) with a 21 gauge blunt SS needle was used. For experiments that

62

involved drops covered with surfactants, a 10 μL Gilson micropipette was used to deposit the surfactant on the surface of the preformed drop.

Various types of tubing have been used to deliver the water from the syringe to the pedestal. First, tubing that connects the pedestal tubing to the 21 gauge blunt SS needle have included, for example, Masterflex tubing, 21 gauge regular–walled polytetrafluoroethylene (PTFE), and 21 gauge light–walled PTFE tubing, the last of which is currently used. The tubing that goes through the pedestal and is level with the top of the pedestal is PTFE tubing (21 gauge, Component Supply Company) and 90° bent

304 SS tubing (21 gauge, ØID: 0.05 cm, ØOD: 0.08 cm, 1" legs on both sides of bend,

Component Supply Company) have been used with the SS being the one currently employed.

4.2.5 Methods

Preparation of a Sessile Drop

Prior to and between each experiment, the pedestal is cleaned with a drop of methanol and three drops of water. To begin an experiment the tubing is placed in the

63

pedestal and connected to the liquid filled microsyringe. The syringe is placed and secured in the syringe pump. Additionally, the light source is placed in its holder. The camera is turned on through the uc480 viewer software (see below). The syringe pump is placed in the infusion mode and a couple of drops are formed on the pedestal to ensure that the focus is clear. For drops with surfactants, a surfactant film is deposited on the drop using a handheld micropipette.

Drop Imaging and Calibration

Depending on the experiment to be performed on the drop, different steps may be conducted. If the experiment to be performed is a cycling experiment the video feature is setup, and the syringe pump is programmed to infuse and withdraw for a series of time intervals. To calibrate the drop images, the pedestal is removed and a grid is placed in the same spot; the grid is then imaged at the same focus. The image of the grid may be taken before or after the drop images have been taken.

64

4.3 Axisymmetric Drop Shape Analysis and Other Software

4.3.1 UC480

The uc480 software (Thorlabs, May, 10, 2013) interfaces the camera to the computer. This software is capable of live feed, video, and still image taking capabilities.

Additionally, this software allows the pedestal and drop to be viewed in real–time and focus them to achieve the clearest view.

4.3.2 VirtualDub

VirtualDub, (downloaded on May 1st, 2013) is a video frame grabber software that allows still images to be extracted from video and is used to take still images from the video of the drops during the dynamic cycling experiments. It allows for the multiple images to be taken per second of video.

4.3.3 Microsoft Paint

An image of the pedestal is opened in Microsoft Paint and the left and right coordinates are determined. These coordinates are used in the input file to calculate the physical properties using axisymmetric drop shape analysis.

65

4.3.4 Axisymmetric Drop Shape Analysis

The Axisymmetric Drop Shape Analysis (ADSA) software was purchased from the Neumann group at the University of Toronto. To use this software drop images need to be taken where the drop fills the majority of the field of view. The drop profile has to be in focus and have clear edge definition. An input file needs to be written which is specific to the drop(s) to be analyzed. The instructions on how to fill in the input file can be found in Appendix A.

66

5. Results and Discussion

5.1 Nanopure water

The surface tension of water has been widely studied using various methods.

Some techniques are better than others, but all obtain the same results. Where drop shape techniques are used in concert with ADSA, the PD is superior to the CSD because the pendant drop shape is more deformed as a pendant, and deformation is a key requirement for accurate surface tension measurements. Additionally, the diameter of the pedestal used in this thesis is not optimal for high surface tensions, as will be discussed later.

Nevertheless, water is a good test sample to verify that the CSD design and setup can achieve reasonable surface tension measurements. Published surface tension measurements for water were free of impurities, therefore nanopure water was used in the

following experiments. The water was not purged to remove CO2 since the drop was exposed to ambient air.

Surface tension has also been measured as a function of temperature.89 For example, the surface tension values for water at 15, 20, and 25°C are 73.50, 72.75, and

71.99 mN/m, respectively.89 The temperature in the lab during the preliminary

67

experiments was 18.9 ± 0.5°C so the surface tension is calculated to be 72.9 ± 0.8 mN/m.

The temperature in the lab during the later experiments was 19.8 ± 0.5°C, therefore the calculated value is 72.7 ± 0.8 mN/m.

There is an optimal range of pedestal size to use which depends on the surface tension and density of the material to be tested.109 If a maximal error of ± 0.1 mN/m is desired a pedestal with diameter of 4 mm is good for smaller surface tension values, i.e., in the range of 0.5–38 mN/m, which encompasses the surface tension of several surfactants and many organic compounds. In contrast, a pedestal with a diameter of 6 mm is better for higher surface tension values, i.e., 15-72 mN/m, the high end of which is very close to the surface tension of water. The size of the pedestal used in this thesis, 2.5 mm diameter, is better suited for low surface tension systems and surfactants, but reasonable values were still obtained for water.

68

Figure 5.1: A 23 μL drop of nanopure water constrained to the pedestal. The green line corresponding to the Laplacian fit derived by ADSA. Note the quasi–spherical shape.

The water drops were formed using a programmable syringe pump, a syringe full of nanopure water, and tubing that goes up through the bottom of the pedestal. The syringe pump is programmed to deliver a selected volume, for example, 23 μL, at a given flow rate, for example 30 μL/min. For the static studies of water droplets with different volumes, the input volume was manually changed between each experiment. The static studies at a constant volume (23 μL) were programmed once and only required the press of the start button for each new drop. For the dynamic cycling experiments, each step was programmed in sequence so that the drop was filled to the maximum volume and then immediately began to decrease by reversing the pump without interference. The

69

droplet volume was enlarged and reduced by ~18% several times. Drop images were taken at the end of each static experiment, and for the dynamic studies video were recorded for the time of interest and still images were pulled from the video prior to analysis.

There is an optimal drop volume to achieve accurate surface tension values; an ideal drop needs to be well deformed and have a balance of surface tension and gravity.

If one force is more prominent than the other, the surface tension measurements based on the drop profile will not be accurate. The water drop in Fig. 5.1 is an example of an appropriately sized drop for a 2.5 mm diameter pedestal. If a drop is too small, the edge of the maximum diameter of drop, the widest part of the drop, would be even with the edge of the pedestal, instead of the desired case where the maximum diameter of the drop is approximately one and a half times wider than the diameter of the pedestal.

Conversely, if a drop is too large, the maximum drop diameter would be two or three times that of the pedestal and the drop “hangs over” the edge of the pedestal; the ADSA software cannot analyze this situation. Even if the droplet is of an appropriate size for the given pedestal, accurate surface tension measurements are only possible if gravity

70

deforms the shape enough. If the droplet is too spherical, accurately determining surface tension is difficult.

During the calculation of surface tension using ADSA, there is an option to have

ADSA draw a green line on the output drop image, which represents the best fit of the

Laplace equation. The fit of this green line to the profile of the drop correlates to the accuracy of the surface tension measurement for that drop. A good Laplacian fit starts at, for example, where the drop and pedestal intersect on the left, follows the drop profile around, and end at the drop and pedestal intersection on the right. If the green line does not begin and end at the intersection of the drop and pedestal on the left and the right, or if the green line bisects the drop at any point, the surface tension measurements are incorrect. Figure 5.1 illustrates of good Laplacian fit on a drop profile.

5.1.1 Preliminary Results

A series of static water drops ranging in volume, 18–25 μL, were measured to determine the best volume to use. Based on the drops measured, of which 3 different days are shown in Fig. 5.2, the surface tension depends more on the day than the drop

71

volume for a reasonable range in volume. The surface tension for each day, as shown in

Fig. 5.2, is very consistent for a given day, but tends to vary when different days are compared. The surface tension values for water on Days 1 and 2 are in good agreement with the theoretical value (72.9 mN/m). Day 3 Trial 1 and 2 have consistently lower surface tension values which may signify some contamination of the water sample.

There is more variation in the second trial than the first which may additionally indicate that the contamination increased over the day. Possible sources of contamination include not using fresh nanopure water and previously contaminated water.

72

Figure 5.2: Surface tension for water droplets of increasing volume. The volume plotted is the volume measured using ADSA, whereas the volume infused falls in the 18 – 25 μL range. All drops are within a ± 3% range of the surface tension at room temperature (72.9 mN/m). The light and darker red shaded areas represetn the ±1% and ±2% ranges, respectively. Trial 1 and 2 correspond to two sets of experiments that were completed on the same day, but for which the syringe was refilled between trials.

The surface tension measurements, as shown in Fig. 5.2, all fall within ±3% of the calculated surface tension value at room temperature (72.9 mN/m). The results from Fig.

5.2 are tabulated in Table 5.1 to illustrate the relative variation from the theoretical surface tension value as well as to show how much the variance was per set of

73

experiments. These values are quite good considering the small diameter of the pedestal, which is not suited for high surface tension measurements.

Table 5.1: Analysis of data corresponding to Fig. 5.2. Date Relative variation (%) Variance per day (mN/m) Oct 8 +0.1 ~0.4 Oct 5 ±0.05 ~0.9 Oct 3 Trial 1 –1.5 ~0.2 Oct 3 Trial 2 –2.5 ~0.9

Once the optimal drop volume was determined several static water drops were measured to verify the surface tension value of water. Multiple measurements must be performed to achieve reliable surface tension data. A representative survey of several drops can be seen in Fig. 5.3 which has data from different days and times on the same plot to show that an infusion volume of 23 μL yields consistent surface tension values regardless of the day; this is why a 23–25 μL volume range was chosen as ideal. A

74

representative survey of the results can be seen in Fig 5.7 where several drops of approximately the same volume have very close surface tension measurements.

Figure 5.3: Surface tension measurements of static water drops with infusion volume of 23 μL. All surface tension values are within ±3% of the theoretical value at room temperature (72.9 mN/m), designated by the black horizontal line. The drops obtained on Day 2 are from two different sets of experiments; all drops on this plot ar freshly infused.

75

Dynamic water drops were subsequently measured. The drop was filled to a maximum volume of 23 μL and decreased to a minimum volume of 19 μL; this was repeated 1.5 more times. Fig 5.4 does not show the entire cycle.

Figure 5.4: Dynamic cycling measurements of a water drop infused at an initial volume of 23 μL followed by 18% volume compression/expansion cycle. This plot starts partially through the first expansion and shows drop volume and area as a function of time as well as surface tension. The green and blue peaks and valleys in the plot show that the drop material was conserved. The maximum volume of the green and blue lines parallel the nearly flat valleys of the red line that signifies the most accurate surface tension values for the cycling water.

76

The volume and area plots show that little to no drop volume was lost over the time the cycling took place, and that the infusion and withdrawal was without interruption. The surface tension measurements for cycled water were all within ± 3% of 72.9 mN/m, and agree with the previously measured surface tension values of static drops shown in Fig.

5.4. The consistency of the area and volume during infusion and withdrawal as well as the mostly smooth and continuous surface tension curve in Fig. 5.4 demonstrates that cycling is feasible and gives accurate surface tension measurements. The flow from the syringe pump during dynamic cycling may create a slight raise at the top of the drop profile making the drop appear more spherically shaped, but this does not appear to be the case in Fig. 5.4 since the surface tension does not sharply increase when water is infused. If infusion influences the shape of the drop the surface tension output based on the drop profile may be affected, but the effect should be minimal.

5.1.2 Quantifying Results

A series of water evaporation experiments were carried out at a later date than the

Preliminary Results. A total of three different water drops of initial infused volume of 23

77

μL were formed, a set of data that exemplifies the results is shown in this section. Each drop was allowed to form at a rate of 30 μL/min; once formed a video was recorded for a period of approximately 15 minutes. All three trials showed the same trend with an evaporation rate of ~0.4 μL/min. Fig. 5.5 shows the time evolution of the surface tension as well as its dependency on the initial infused volume.

Figure 5.5: Surface tension of water with respect to time and initial infused volume which shows that as volume and time decrease the surface tension stays mostly constant.

78

The γ vs volume plot does not indicate a linear progression of surface tension since the volume gradually increases before gradually decreasing, but it does show that drop profiles of smaller volumes of pure water were able to be measured and that good surface tension values could be obtained. In an ideal system, the infusion rate of water, i.e. 1 μL/min would have lead to the desired maximum volume; however, this was not the case. When taking into account the evaporation rate measured in the evaporation studies, the overall increase of volume per minute for the drop is actually ~0.6 μL/min. This agrees with the data for both experiments performed. The two water trials were used to measure addition and withdrawal of water in a stepwise fashion. The trials were performed in the same manner including infusing and withdrawing the same volume of water, but one trial started at 12 μL and the other started at 13 μL to see if this initial volume affected the outcome. Fig. 5.5 shows that surface tension is constant over a wide range of volumes, until very low volumes values are obtained.

79

Figure 5.6: Surface tension as a function of time for an experiment where the initial volume was set to 13 μL, 10 μL was infused followed by 10 μL withdrawn. The final volume should be 13 μL, however, that is not the case as is shown. Surface tension values stayed constant until the volume reached very low volumes (<10 μL).

5.1.3 Possible Improvements Areas

There are a few areas where improvement of the setup could greatly enhance the measured output based on the drop profile. An area of improvement that would directly affect the measurements is the levelness of the pedestal. Since the pedestal is set in a holder at the beginning of each setup, and the pedestal is so small, there is no guarantee that the pedestal is level or placed at the same position each time – height, tilt and inclination angles. The measured volume is typically 1 μL less than the infused volume;

80

it is probable that the slight tilt of the pedestal is in part responsible for this difference since the ADSA software assumes that the pedestal is level, and the grid used to calibrate is more than likely straighter than the grid is level. There is no way to fix this with the current design. However, a solution would be in an alternate design provided that a machine shop is found that is capable of machining the edge to appropriate sharpness, such that this issue should be overcome or minimized greatly (see the Future Work

Section for more details).

The temperature of the environment in which the drop is formed in could be better controlled. Typically, the ambient temperature fluctuates around 19°C, far from the physiological temperature necessary in the study of pulmonary surfactants. The relative humidity in the lab fluctuates as well, depending on the season. It is typically around 30–

50%, which is not close to physiological relative humidity of approximately 90%.

Additionally, the drop is open to the air, and therefore any air current in the lab which includes currents from air conditioning or heating as well as people walking by the setup. All of these problems could affect the rate at which the drop evaporates and even the shape of the drop. These problems could be reduced by enclosing the pedestal in an

81

environmental chamber. Another possible reason explaining the discrepancy between infused and measured volumes could be that the material in the tubing may not be flush with the top of the pedestal at the time of infusion.

5.2 Thin Films of Surfactants

To form a surfactant layer on a droplet, the droplet was first formed following the procedure described above. A thin film of surfactant dissolved in solvent, chloroform for DPPC and cholesterol and water for LPS, was then placed on the water drop via a micropipette. Initially, the thin film was placed on a fully formed drop, i.e., once the drop had reached maximum volume. However, more than half of the drops were knocked off the pedestal by the surfactant drop. The thin film is now placed on the drop once the drop has reached approximately 50–75% of its final volume; this time was chosen because at this point the pedestal is fully covered by the bulk, but the water drop is not large enough to be knocked off the pedestal.

The pedestal is cleaned thoroughly between each drop formed. First, the drop is wiped off of the pedestal carefully, then a few drops of methanol are applied to the

82

pedestal, and finally a few water drops are placed on the pedestal. At each step the material is removed by a Kimwipe. During the measurements of DPPC, Chol, and LPS thin films, pure water drops were formed and imaged intermittently to ensure the pedestal is cleaned properly before and after each drop. Figure 5.3 shows pure water drops that were formed during DPPC and LPS experiments; the surface tension values of these water drops fell within ±3% of 72.9 mN/m, demonstrating that the pedestal is being cleaned thoroughly between each newly formed drop. Possible reasons for the variations present in the data from day-to-day measurements, and even to some extent on the same day measurements will be discussed at the end of this Section.

5.2.1 Preliminary DPPC

DPPC was studied because of its prevalence and importance in PS, as mentioned in detail in Chapter 1. The data in this section is obtained from images of static drops covered with 1 μL of 1 mM DPPC thin film. The drop images were taken shortly after the drop reached the maximum volume of 23 μL. As can be seen in Fig. 5.7 the drop

83

with a DPPC thin film has a different shape than a 23 μL drop of pure water. The drop with DPPC is flatter, wider, and less spherical than the pure water droplet.

Figure 5.7: (a) 23 μL drop of nanopure water with green line that shows the Laplacian fit using ADSA from Fig. 5.1. (b) 23 μL drop of nanopure water with a thin film of DPPC. The green line on the drop profile shows the Laplacian fit using ADSA. Note the flatter, less spherical shape of the drop.

Static experiments of different drops with thin films of DPPC were conducted on various occasions over several days and each drop was imaged; all drops formed had the same parameters and conditions. Figure 5.8 shows a typical distribution of surface tension values of static drops using CSD. The average surface tension range measured

84

based on the DPPC thin film drop profiles is 60–62 mN/m. Since DPPC is dissolved in chloroform; the distribution of surface tension values is more than likely due to the chloroform evaporating since there was less than a minute between the addition of the surfactant and the measurement. This may not be enough time to reach equilibrium.

Figure 5.8: DPPC surface tension measurements as a function of droplet volume. A 23 μL drop was formed and 1 μL of DPPC thin film was placed on top of the drop. Each drop was freshly infused, and the majority of the surface tension values measured are between 58–65 mN/m.

85

The estimated surface (thin film) coverage of 1 μL DPPC on 23 μL of water is

0.248 2 per molecule; this does not correspond to a range that is comparable to the one found in a DPPC isotherm obtained using a Langmuir trough.

5.2.3 Quantifying DPPC Results

A series of evaporation studies were conducted on DPPC as well, but using a much lower concentration of 83 μM. A 1 mM concentration was used earlier to ensure deformation of the drop, but now a lower concentration is instead preferred not perturb too much the small drop volume. Three different evaporation studies were conducted; all with a bulk water drop of 23 μL and with increasing volumes of thin films from 1–3 μL.

Although the thin film volume increased from 1 to 3 μL in 1 μL increments, a new drop was formed each time and a new thin film was applied for each experiment. To perform these evaporation studies, the water drop was infused to 23 μL where video recording began. The drop volume was then decreased to 13 μL for deposition of surfactant, and the drop was re-infused to bring final volume to 23 μL. The drop was monitored over a

86

period of approximately 10 minutes, the first three of which were taken to allow the drop to come to equilibrium (Fig. 5.9). One trial of each thin film volume was measured, and the average evaporation rate was calculated to be 0.3 μL/min. Increasing the volume of the surfactant thin film did not make a significant difference to the volume loss of the drop. However, the volume loss per minute was decreased from 0.4 μL/min for pure water to 0.3 μL/min for a pure water drop with a surfactant thin film.

Figure 5.9: Surface tension and volume with respect to time for evaporation of a 23 μL water drop with a 3 μL DPPC thin film.

87

5.2.4 DPPC Isotherms

The lower concentration of the DPPC, 83 μM, was chosen to correlate with

Langmuir isotherm experiments. A drop of water was infused to 23 μL where video recording began, decreased to 13 μL for the surfactant drop to be deposited, and finally re-infused to 23 μL where the drop rested for 3 minutes to allow equilibrium to be reached. After the 3 minutes the drop was decreased at a rate of 6.5 μL/min until the final volume was 10 μL. The drop video was analyzed as usual. The isotherms shown below have the same basic shape as those obtained using Langmuir trough. The 1 and 3

μL thin films correspond to calculated mean molecular areas (MMA) of 22.7 and 68

2/molecules, respectivley. Fig. 5.10 presents isotherms measured on two different days with (a) two separate drops with 3 μL placed on each 23 μL drop and (b) four separate drops with 1 μL deposited on each 23 μL water drop. In Fig 5.10 (a) surface pressure of

~65 mN/m may correspond to the collapse point and is difficult to observe using traditional methods. In contrast, in Fig 5.10 (b) Isotherms 3 and 4 are comparable to that achieved by Neumann et al114 in the corresponding surface pressure range shown below.

88

Figure 5.10: (a) 3 μL of 83 μM DPPC solution placed on 23 μL water drop and (b) 1 μL of 83 μM DPPC solution placed on 23 μL water drop.

5.2.5 Preliminary Chol Results

Chol was studied due its presence in PS, as described in Chapter 1. A 23 μL water drop was formed and a 2 μL Chol thin film was spread on it. The Chol thin film alters the surface tension of water which can be seen in Fig 5.11. The drop with Chol has a flatter shape, one more similar to a sideways oval than a ; this is due to Chol having a lower surface tension than water.

89

Figure 5.11: (a) 23 μL drop of nanopure water with green line that shows the Laplacian fit using ADSA from Fig. 5.1. (b) and (c) 23 μL drop of nanopure water with a Chol thin film taken at time t = 0 and 1 min, respectively. The green line on the drop profile shows the Laplacian fit using ADSA. Note the flatter, less spherical shape of the drop, especially as time increases.

During the Chol thin film experiments drop images were taken directly of drops once maximum volume was reached as well as from drops that were allowed to rest for approximately a minute before the image was taken; not all drops were imaged at both times. It is clear based on the shape of the drops at approximately t ≈ 0 and 1 minute that the surface tension decreases as the drop is allowed to rest. Figure 5.12 shows three drops that were imaged at zero and one minute after the drop reached maximum volume,

90

and illustrates that surface tension decreases significantly over one minute, and that there is a slight volume loss.

Figure 5.12: Three drops which were imaged at t ≈ 0 and 1 of maximum volume. The top drop in each pair was taken at t ≈ 0 min and after one minute it can be clearly seen that the surface tension decreased and that a slight loss of volume occurs. These drops are six of the drops shown in Figure 5.13.

This decrease in surface tension may be due to the chloroform in which the Chol was dissolved still evaporating. For the drops measured directly after the maximum

91

volume of 23 μL was reached, the average surface tension measured is 55 mN/m; for the drops measured approximately 1 min after maximum volume was obtained, the average surface tension is 40 mN/m, which can be seen in Fig. 5.13. The data points in Figure

5.13 are the mean volume of one Chol thin film experiment which was analyzed using two different sets of coordinates where the drop and pedestal meet; this demonstrates that the surface tension measurements are the same regardless of the specific pedestal coordinates entered, as long as the coordinates are as close to the true value as possible.

Clearly, there should not be this wide a range in surface tension values, again, this may be caused by the chloroform not evaporating off complete before the image was taken.

92

Figure 5.13: Four different sets of surface tension measurements of a Chol thin film on a bulk water drop, all taken on the same day. As can been seen in the plot, if the drop is allowed to rest, the surface tension values decrease.

The estimated surface coverage for 2 μL of 2mM Chol thin film on a 23 μL is 0.123

2/molecules; this is not comparable to the measurements obtained using the Langmuir trough.

93

5.2.6 Quantifying Cholesterol Results

A series of evaporation studies were conducted on Chol as well, but using a lower concentration, 57 μM, than the 2 mM used in preliminary results. The initial 2 mM concentration was used to ensure deformation of the drop. Three different evaporation studies were conducted; all with a bulk water drop of 23 μL and with increasing volumes of thin films from 1–3 μL. Although the thin film volume increased for each evaporation study, a new drop was formed each time and a new thin film was applied. The water drop was infused to 23 μL where video recording began, drop volume was decreased to

13 μL for deposition of surfactant, and the drop was re-infused to bring final volume to

23 μL. The drop was observed for a total of 10 minutes, the first three of which were taken to allow the drop to come to equilibrium. One trial of each thin film volume was measured, and the average evaporation rate was calculated to be 0.3 μL/min. Increasing the volume of the surfactant thin film did not make a significant different to the volume loss of the drop. However, the volume loss per minute was decreased from 0.4 μL/min for pure water to 0.3 μL/min for a pure water drop with a surfactant thin film.

94

Figure 5.14: Surface tension and volume with respect to time for evaporation of a 23 μL water drop with 3 μL Chol thin film. Surface tension is fairly constant.

5.2.7 Cholesterol Isotherms

The lower concentration of the Chol, 57 μM, was chosen to correlate with

Langmuir isotherm experiments. A drop of water was infused to 23 μL where video recording began, decreased to 13 μL for the surfactant drop to be deposited, and finally re-infused to 23 μL where the drop rested for 3 minutes to allow equilibrium to be reached. After the 3 minutes the drop was decreased at a volume of 6.5 μL/min until the

95

final volume was 10 μL. The drop video was analyzed as usual. The isotherms shown below have the same basic shape of isotherms obtained using Langmuir trough. The 2 and 3 μL thin films corresponds to calculated MMAs of 33.3 and 50 2/molecule, respectively. Fig. 5.15 are isotherms measured on two different days with (a) three separate drops with 3 μL placed on each 23 μL drop and (b) three separate drops with 2

μL deposited on each 23 μL water drop. Fig 5.15 (a) and (b) both have good minimum surface pressure. In Fig. 5.15 (a) Isotherm 1 has a good corresponding maximum surface pressure to Chol isotherms obtained using Langmuir trough although the MMA for 40 mN/m is off by approximately 16 MMA since the one below is at ~18 MMA instead of

34 MMA.

96

Figure 5.15: (a) 3 μL of 57 μM cholesterol solution placed on 23 μL water drop and (b) 2 μL of 57 μM cholesterol solution placed on 23 μL water drop.

5.2.8 Lipopolysaccharide (LPS)

The physical properties of LPS have not been studied in nearly as much detail as

DPPC and Chol, therefore any surface tension measurements are critical to understanding the role of this biomolecule. Figure 5.16 shows a pure water drop next to a drop with an

LPS thin film. Note the flatter, wider appearance of the drop coated in LPS.

97

Figure 5.16: (a) 23 μL drop of nanopure water with green line that shows the Laplacian fit using ADSA from Fig. 5.1. (b) 23 μL drop of nanopure water with a thin film of LPS. Note the much flatter, wider shape of the drop with the LPS thin film compared to the water drop. The green line on the drop profile shows the Laplacian fit using ADSA.

A series of static drop measurements of drops with LPS thin films were taken first. 2 μL of 1 mg/mL LPS solution was placed on a 23 μL drop of water via micropipette. The LPS thin film drop images were taken directly after the infusion drop volume reached 23 μL. The total drop volume was approximately 25 μL since the LPS was dissolved in water, not chloroform. This could account for the LPS thin film drop appearing larger than the pure water drop in Fig. 5.16. The surface tension range for static LPS thin film drops was measured to be 56–58 mN/m, as can be seen in Fig. 5.17.

98

Figure 5.17: Surface tension measurements of static water drops with a LPS thin film. The surface tension values vary by ~7 mN/m from the lowest value calculated to the highest, with the majority of values falling between 56–58 mN/m.

Dynamic cycling experiments of LPS were also conducted. The water drop was infused to maximum volume, 23 μL, with the 2 μL thin film applied during the initial infusion. The drop with the LPS thin film was then allowed to rest for 5 seconds before being decreased to 21 μL; infusion and withdrawal were repeated once, and the drop was returned to 25 μL. This process proceeded without interference since the steps were programmed in the syringe pump.

99

Figure 5.18: Dynamic cycling plot of a LPS thin film on a bulk water drop that was infused and withdrawn. The volume and area appear to be smooth and return to the same point after each infusion and withdrawal which signifies little to no overall drop loss. The steady increase of surface tension most likely signifies that the drop did not reach equilibrium before the cycling began.

Shown in Fig. 5.18 are volume, area, and surface tension measurements with respect to time formed in the manner explained above. The volume and area overlap very well and show that little or no drop material was lost over this time frame. The surface tension of LPS thin film appears to have the opposite trend of pure water in cycling

100

experiments where the correlation to the volume and area are concerned. The steady increase of surface tension most likely signifies that the drop did not reach equilibrium before the cycling began. This may be due to LPS being above the critical micelle concentration (CMC), which is around 14 μg/mL. As the drop becomes smaller and then increases the LPS molecules have a chance to form therefore decreasing their presence at the interface. Additionally, LPS tends to form micelles more rapidly when agitated. The surface tension measurements in the dynamic cycling experiments, 56–58 mN/m, agree with the values obtained during static measurements. The estimated surface coverage of LPS is 2.39 2/ molecule assuming all the LPS is at the surface; this is not comparable to what has been obtained using the Langmuir trough.

101

6. Conclusions and Future Work

6.1 Conclusions

The conclusions for this thesis will be divided into two sections, firstly, discussing the outcome of design and application of the CSD setup and secondly, presentation of the results obtained using the CSD setup.

6.1.1 CSD Setup

The CSD setup underwent several iterations to reach the current design used to obtain the data presented in this thesis. An overview will be given followed by a more detailed description for the more challenging parts. In the initial stages of the project, the objective was to make a pedestal that had a sharp enough edge to hold a water drop. The next step was to determine an appropriate combination of lenses and camera to obtain a magnified image of good quality. Those two goals took the majority of the time to get the setup to the point where it is today. After obtaining a well focused drop image it was time to test the quality of the pedestal by using the ADSA software to determine surface tension measurements of water drops based on the images. A syringe pump that could

102

deliver small volumes of liquid as well as infuse and withdraw liquids was the next major improvement of this setup. Other pieces were added to the setup to make slight improvements such as grid holders, various stages, and a micrometer stage for the imaging system.

The main hurdles in this project so far have been machining a pedestal of good quality, i.e., that has a circular top and knife sharp edges. Once the initial hurdle of machining the desired shape and size of the pedestal was completed, the edge had to be sharpened. The size, design, and sharpness of the pedestal was challenging to machine and required a precise, high powered machine and a well thought out set of steps to achieve. The edge of the pedestal had a tendency to bend under when the tools used to sharpen the edge and then polish the top were used; this was unfortunate as the sharp edge was imperative to accurately measure surface tension. The sharp edge was verified by looking at the edge under a SEM. Once a proper pedestal was machined the next major hurdle was to acquire an imaging system that would allow the drop on the pedestal to be imaged at a close distance and have a clear resolution to where the profile of the drop is sharp. The imaging system went through several iterations before settling on the

103

current CMOS camera and macro lens. The liquid delivery system was the third key aspect to getting this system up and running. The choice of syringe pump was important because it needed to be able to handle a range of experimental parameters. The syringe pump chosen was programmable in addition to being able to infuse and withdraw at several different flow rates.

The current setup is fully capable of obtaining good surface tension measurements, but there is always room for improvement. The improvements to the setup will be discussed in greater detail in the Future Work Section. Firstly, a pedestal which is permanently attached to the holder would improve the repeatability in placing the pedestal at the same position and eliminating as much of a tilt and angle as possible.

Additionally, it would reduce the amount of time the pedestal is handled, and therefore, reduce the possibility of the edge being dulled. A pedestal with a slightly larger outer diameter would be better suited to measure surfactant surface tension and allow for a greater compression and expansion cycle. Also, a larger pedestal may make it easier to deposit a surfactant drop on a larger water droplet. More on this design will be discussed in the Future Work.

104

6.1.2 Surface Tension Measurements

All results in this thesis have been collected using the current version of the setup, however, drop images were taken and surface tension values have been measured using each version of the CSD setup. Water is a good baseline measurement since the surface tension is well documented. Once feasible surface tension and volume measurements were achieved measurements of the drops with surfactant were performed.

Figures 5.2 and 5.3 show water drops as a function of surface tension versus time for a range of droplet volumes. As mentioned, the volume calculated using ADSA is lower than the measured input volume. A possible reason for this may be that the water may not be flush with the top of the tubing. An additional reason may be that the pedestal and grid were not both perpendicular to the table, meaning the grid might have been perpendicular to the table but the top of the pedestal may not be parallel to the table.

However, the volume measured is typically 1 μL or less than the infused volume.

Surface tension measurements tend to be more precise for a given day than day–to–day, although the difference from day–to–day is 3% or less than the published values. The

105

cycling experiment shown in Fig. 5.4 was performed to demonstrate that cycling is possible with the setup and to verify that the surface tension calculations for water would still be that of water regardless of the motion, cycling versus static. The area and volume plots were smooth and consistent which shows the infusion and withdrawal were constant and without interruption. The surface tension values did fluctuates a little, around 1 mN/m overall, but that may be due the speed of the cycling since one entire cycling was completed in approximately 15 sec.

For the water evaporation studies example plots shown in Fig. 5.5 a water drop was formed and left untouched for a given amount of time. All water drops studied evaporated at a rate of ~0.4 μL/min. Fig. 5.6 shows a water drop with a series of 1 μL infusions and withdrawals at one minute intervals. The rate at which the drop actually increased, and then decreased by, was ~0.6 μL/min due to the evaporation. The infusions were very consistent, but the withdrawal, although programmed as the reverse of the infusions, appeared to have been more of a steady decrease than in a stepwise manner; this is more than likely due to evaporation combined withdrawing the liquid. Fig. 5.6

106

shows that the surface tension measurements were pretty constant except at very low volumes.

Surfactant thin films were also studied using CSD. To ensure the pedestal was being properly cleaned between each new surfactant thin film drop, pure water drops were formed and imaged intermittently, some of which are shown in Fig. 5.7. If the pure water drops evaluated had a surface tension of around 72 mN/m then the pedestal was cleaned sufficiently and the drops with surfactant thin films also had reliable surface tension measurements.

DPPC was a studied as a thin film using CSD. Originally, 1 μL of 1 mM DPPC in chloroform was placed on water drop and an image of the drop was taken immediately once the drop infusion volume reached 23 μL. This was a poor choice of timing as the chloroform more than likely did not have enough time to evaporate and, therefore, the drop with surfactant did not have ample time to reach equilibrium. Fig. 5.9 shows water drops with 1 μL DPPC thin films formed in this manner, and this is more likely the reason the surface tension values are so variable. The concentration of 1 mM was used initially to ensure the drop would be deformed in shape, i.e., not spherical. Evaporation

107

studies were also conducted of drops with thin films of DPPC, although at a much lower concentration of 83 μM. Three evaporations trials, one each of 1, 2, and 3 μL of DPPC thin film, were conducted and an example plot is shown in Fig. 5.10. The evaporation in all three trials was ~0.3 μL/min, which is 0.1 μL/min less than of a pure water drop of the same volume. Finally, compression isotherms were conducted with 83 μM DPPC thin films. DPPC isotherms have a typical lift–off of around 100 MMA and reach collapsed phase around 43 MMA. The lift–off for Fig. 5.11 (a) is around 18 MMA which is about

80 MMA removed from the typical lift–off point. Isotherm 2 reached a higher final surface pressure and appears to have reached a collapse point around 65 mN/m before decreasing. Fig. 5.11 (b) the second and third have liftoff points around 53 MMA and the fourth isotherm has a liftoff point around 47 MMA so all approximately 50 MMA removed from where they should be. All isotherms finish at about 14 mN/m, well below the collapse point at around 55 mN/m.

Chol was an additional surfactant thin film to measured using CSD. When Chol was first used, 2 μL of a 2 mM in chloroform solution deposited on a 23 μL water drop was used to ensure deformation of the drop. Images were taken of drops immediately

108

once a drop with a Chol thin film reached 23 μL, and for a three of those drops an image was taken approximately a minute later to see if the shape of the changed, and therefore, the surface tension. Fig. 5.13 shows how the surface decreases over the span of about a minute; the top most of each set of points, the top point is for time zero and lower point of same color is for one minute later. Although, the time zero drops did not have the same initial surface tension values, or the same surface tension values one minute later, all three decreased approximately 15 mN/m in surface tension over one minute. Fig. 5.14 has the drops in Fig. 5.13 in addition to some drops that were imaged at only time zero or time one minute. Also, Fig. 5.14 has drops all from the same series of experiments measured at two slightly different coordinate assignments of the right and left edge of the pedestal. The ends of the error bars are the original data and the point in the middle is the standard deviation. This serves to illustrate that deviations from the input pedestal coordinates were okay as long as they are very small; only the measured volume differed, the surface tension measurements were the same.

Additionally, evaporation studies were conducted for 23 μL drops of water with 1,

2, and 3 μL thin films of 57 μM Chol in chloroform; an example plot is shown in Fig.

109

5.15. The evaporation of drops were ~0.3 μL/min, same as those for DPPC.

Compression isotherms were also conducted for the lower concentration of Chol, 5.6 x

10-2 mM. Chol isotherms typically have a liftoff of around 38 MMA and attain a maximum surface pressure of approximately 45 mN/m which corresponds to approximately 26 MMA. In Fig. 5.16 (a) the lift–off is removed by about 12 MMA. The slope was not as steep as those published using Langmuir trough which reaches maximum surface pressure of 45 mN/m over the span of 4 MMA. Isotherm 3 reaches a maximum surface pressure of approximately 45 mN/m which corresponds to the maximum surface pressure attained by Langmuir trough. In Fig. 5.16 (b) the first and third Isotherms have a liftoff around 33 MMA and Isotherm 4 has a slightly higher liftoff around 36 MMA. The isotherms for this concentration did not reach a surface pressure greater than 20 mN/m, therefore only a partial isotherm was obtained.

LPS was the last surfactant measured on the current CSD setup. The concentration of LPS used was 1 mg/mL dissolved in water. 2 μL of LPS was deposited on a 23 μL pure water drop; images were taken once the drop reached an infused volume of 23 μL. The volumes measured, as shown in Fig. 5.18 were greater than 23 μL due to

110

the fact that LPS was dissolved in water and therefore, the surfactant drop volume did not evaporate, but instead added to the total drop volume. The surface tension values measured varied about 7 mN/m and this may be due the surfactant concentration being higher than the CMC of LPS, but again, a higher concentration than necessary was used to ensure drop deformation. Fig. 5.19 is of a 23 μL pure water drop with a 2 μL drop of

LPS thin film. The area and volume during the compression and expansion are smooth and continuous, with a little evaporation present over the time the cycles took place. The surface tension increased overall during the two cycles which may be due to LPS forming aggregates.

6.2 Future Work

The future work is divided into two sections; firstly, improvements for the CSD setup and secondly, experiments to be conducted.

6.2.1 CSD Setup

As mentioned earlier in this section, a couple of new pieces are currently underway to being implemented, as can be seen in Fig. 6.1. The pedestal design is the

111

same, except that the holder is taller and that the slot for the tubing has been brought to the side. A couple of versions have already been machined, but not being able to attain a sharp edge and circular top have not allowed this version to be fully tested and implemented.

a b c

Figure 6.1: Custom–built components. (a) one–piece pedestal and holder, (b) base plate, and (c) lens holder (top and bottom parts).

A custom–made grid holder has been designed to center an R10 grid to the same spot as the center of the pedestal; the grid is used for calibration, i.e. to translate between number of pixels and physical dimensions. In order to ensure the same position of the pedestal

112

and the grid for each experiment as well as the same position relative to each other, a custom–made base plate (2.54 cm × 10.1 cm × 1.25 cm) was designed. This base plate was mounted on a smaller anodized aluminum breadboard (25.4 cm × 13 cm × 1.3 cm, ¼

- 20 mounting holes) raised by 2.5 cm post holders (PH11, Thorlabs).

The imaging system could benefit from additional braces or a new overall holder to ensure the camera and lens are both level at all times. Improvements that could help improve the parameters of the experiments would be a temperature and humidity controlled chamber. This should help lessen or eliminate the evaporation of the drop as well as allow for experiments to be conducted at physiologically relevant conditions.

6.2.2 Future Experiments

Additional experiments to be conducted include repeating experiments discussed in this thesis to ensure repeatability. For the compression isotherm experiments of Chol and DPPC varying the compression speed as well as the concentration to see how that affects the shape of the isotherm. Also, for Chol and DPPC to conduct dynamic cycling experiments which entails compressing and expanding the drop with surfactant thin film

113

and measuring the surface tension over several cycles. For LPS to conduct compression isotherm studies at various concentrations of LPS solutions, not thin films since LPS is dissolved in water.

114

References

(1) Notter, Robert H. Lung Surfactants: Basic Science and Clinical Applications; Marcel Dekker, New York, 2000; Vol. 149. (2) Breath Matters http://www.breathmatters.org/what-is-copd-lung-disease.php. (3) The Human Respiratory System http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Pulmonary.html (accessed Oct 23, 2013). (4) A.B. Lumb, J.F. Nunn. Nunn’s Applied Respiratory Physiology; Elsevier/Butterworth Heinemann: Edinburgh, 2005. (5) Marieb, E. N. Essential Anatomy and Physiology; Pearson Education, Inc publishing as Benjamin Cummings, 2003. (6) Weibel, E. R.; Gil, J. Electron Microscopic Demonstration of an Extracellular Duplex Lining Layer of Alveoli. Respir. Res. 1968, 4, 42–57. (7) Untersee, P.; Gil, J.; Weibel, E. R. Visualization of Extracellular Lining Layer of Lung Alveoli by Freeze-etching. Resp. Physiol. 1971, 13, 171–185. (8) Gil, J.; Weibel, E. R. Improvements in Demonstration of Lining Layer of Lung Alveoli by Electron Microscopy. Respir. Physiol. 1969, 8, 13–36. (9) Bastacky, J.; Lee, C. Y.; Goerke, J.; Koushafar, H.; Yager, D.; Kenaga, L.; Speed, T. P.; Chen, Y.; Clements, J. A. Alveolar Lining Layer Is Thin and Continuous: Low-temperature Scanning Electron Microscopy of Rat Lung. J. Appl. Physiol. 1995, 79, 1615–1628. (10) Goerke, J. Pulmonary Surfactant: Functions and Molecular Composition. Biochim. Biophys. Acta - Mol. Basis Dis. 1998, 1408, 79–89. (11) Perez-Gil, J.; Weaver, T. E. Pulmonary Surfactant Pathophysiology: Current Models and Open Questions. Physiology 2010, 25, 132–141. (12) Williams, M. C. Freeze-fracture Studies of Tubular Myelin and Lamellar Bodies in Fetal and Adult Rat Lungs. J. Ultra. Mol. Struct. R. 1978, 64, 352–361. (13) Nag, K.; Munro, J. G.; Hearn, S. A.; Rasmusson, J.; Petersen, N. O.; Possmayer, F. Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant. J. Struct. Biol. 1999, 126, 1–15.

115

(14) Zuo, Y. Y.; Veldhuizen, R. A. W.; Neumann, A. W.; Petersen, N. O.; Possmayer, F. Current Perspectives in Pulmonary Surfactant - Inhibition, Enhancement and Evaluation. Biochem. Biophys. Acta - Biomembranes 2008, 1778, 1947–1977. (15) Pattle, R. E. Surface Lining of Lung Alveoli. Physiol. Rev. 1965, 45, 48–79. (16) Schurch, S.; Green, F. H. Y.; Bachofen, H. Formation and Structure of Surface Films: Captive Bubble Surfactometry. Biochim. Biophys. Acta - Mol. Basis Dis. 1998, 1408, 180–202. (17) Bachofen, H.; Gerber, U.; Gehr, P.; Amrein, M.; Schurch, S. Structures of Pulmonary Surfactant Films Adsorbed to an Air-liquid Interface in Vitro. Biochim. Biophys. Acta - Biomembranes 2005, 1720, 59–72. (18) Amrein, M.; von Nahmen, A.; Sieber, M. A Scanning Force- and Fluorescence Light Microscopy Study of the Structure and Function of a Model Pulmonary Surfactant. Eur. Biophys. J. 1997, 26, 349 – 357. (19) Follows, D.; Tiberg, F.; Thomas, R. K.; Larsson, M. Multilayers at the Surface of Solutions of Exogenous Lung Surfactant: Direct Observation by Neutron Reflection. BBA - Biomembranes 2007, 1768, 228–235. (20) Baritussio, A. G.; Magoon, M. W.; Goerke, J.; Clements, J. A. Precursor-product Relationship Between Rabbit Type II Cell Lamellar Bodies and Alveolar Surface- active Material: Surfactant Turnover Time. Biochim. Biophys. Acta - Lipid Lipid Met. 1981, 666, 382–393. (21) Zasadzinski, J. A.; Stenger, P. C.; Shieh, I.; Dhar, P. Overcoming Rapid Inactivation of Lung Surfactant: Analogies Between Competitive Adsorption and Colloid Stability. Biochim. Biophys. Acta - Biomembranes 2010, 1798, 801–828. (22) Serrano, A. G.; Perez-Gil, J. Protein-lipid Interactions and Surface Activity in the Pulmonary Surfactant System. Chem. Phys. Lipids 2006, 141, 105–118. (23) Schürch, S.; Goerke, J.; Clements, J. A. Direct Determination of Surface Tension in the Lung. P. Natl. Acad. Sci. Usa 1976, 73, 4698–4702. (24) Schürch, S.; Goerke, J.; Clements, J. A. Direct Determination of Volume- and Time-dependence of Alveolar Surface Tension in Excised Lungs. P. Natl. Acad. Sci. Usa 1978, 75, 3417–3421. (25) King, R. J.; Clements, J. A. Surface-Active Materials from Dog Lung .2. Composition and Physiological Correlations. Am. J. Physiol. 1972, 223, 715–&.

116

(26) Walters, R. W.; Jenq, R. R.; Hall, S. B. Distinct Steps in the Adsorption of Pulmonary Surfactant to an Air-Liquid Interface. Biophys. J. 2000, 78, 257–266. (27) Bachofen, H.; Schurch, S.; Urbinelli, M.; Weibel, E. Relations Among Alveolar Surface-Tension, Surface-Area, Volume, and Recoil Pressure. J. Appl. Physiol. 1987, 62, 1878–1887. (28) Bachofen, H.; Schur, S. Alveolar Surface Forces and Lung Architecture. Comp. Biochem. Phys. A. 2001, 129, 183–193. (29) Postle, A. D.; Heeley, E. L.; Wilton, D. C. A Comparison of the Molecular Species Compositions of Mammalian Lung Surfactant Phospholipids. Comp. Biochem. Phys. A. 2001, 129, 65–73. (30) Shelley, S. A.; Balis, J. U.; Paciga, J. E.; Espinoza, C. G.; Richman, A. V. Biochemical Composition of Adult Human Lung Surfactant. Lung 1982, 160, 195–206. (31) Sankaram, M. B.; Thompson, T. E. Cholesterol-Induced Fluid-Phase Immiscibility in Membranes. P. Natl. Acad. Sci. Usa 1991, 88, 8686–8690. (32) Hjort Ipsen, J.; Karlström, G.; Mourtisen, O. G.; Wennerström, H.; Zuckermann, M. J. Phase Equilibria in the Phosphatidylcholine-cholesterol System. Biochim. Biophys. Acta - Biomembranes 1987, 905, 162–172. (33) Veldhuizen, R.; Nag, K.; Orgeig, S.; Possmayer, F. The Role of Lipids in Pulmonary Surfactant. Biochimica et Biophysica Acta (BBA)/Molecular Basis of Disease 1998, 1408, 90–108. (34) Orgeig, S.; Daniels, C. B. The Roles of Cholesterol in Pulmonary Surfactant: Insights from Comparative and Evolutionary Studies. Comp. Biochem. Phys. A. 2001, 129, 75–89. (35) Ormond, C. J. Thermal Acclimation of Surfactant Secretion and Its Regulation by Adrenergic and Cholinergic Agonists in Type II Cells Isolated from Warm-active and Torpid Golden-mantled Ground Squirrels, Spermophilus Lateralis. J. Exp. Bot. 2003, 206, 3031–3041. (36) Diemel, R. V. Multilayer Formation Upon Compression of Surfactant Monolayers Depends on Protein Concentration as Well as Lipid Composition. An Atomic Force Microscopy Study. J. Biol. Chem. 2002, 277, 21179–21188.

117

(37) Casals, C.; Arias-Diaz, J.; Valino, F.; Saenz, A.; Garcia, C.; Balibrea, J. L.; Vara, E. Surfactant Strengthens the Inhibitory Effect of C-reactive Protein on Human Lung Macrophage Cytokine Release. Am. J. Physiol.-Lung Cell. Mol. Physiol. 2003, 284, L466–L472. (38) Zuo, Y. Y.; Acosta, E.; Policova, Z.; Cox, P. N.; Hair, M. L.; Neumann, A. W. Effect of Humidity on the Stability of Lung Surfactant Films Adsorbed at Air- water Interfaces. Biochim. Biophys. Acta - Biomembranes 2006, 1758, 1609– 1620. (39) Yu, S.; Harding, P. G. R.; Smith, N.; Possmayer, F. Bovine Pulmonary Surfactant: Chemical Composition and Physical Properties. Lipids 1983, 18, 522 – 529. (40) Sibug-Aga, R.; Dunn, R. C. High-resolution Studies of Lung Surfactant Collapse. Photochem. Photobiol. 2004, 80, 471–476. (41) Gunasekara, L.; Schü rch, S.; Schoel, W. M.; Nag, K.; Leonenko, Z.; Haufs, M.; Amrein, M. Pulmonary Surfactant Function Is Abolished by an Elevated Proportion of Cholesterol. Biochim. Biophys. Acta - Mol. Cell Biol. L. 2005, 1737, 27–35. (42) Keating, E.; Rahman, L.; Francis, J.; Petersen, A.; Possmayer, F.; Veldhuizen, R.; Petersen, N. O. Effect of Cholesterol on the Biophysical and Physiological Properties of a Clinical Pulmonary Surfactant. Biophys. J. 2007, 93, 1391–1401. (43) Malcharek, S.; Hinz, A.; Hilterhaus, L.; Galla, H.-J. Multilayer Structures in Lipid Monolayer Films Containing Surfactant Protein C: Effects of Cholesterol and POPE. Biophys. J. 2005, 88, 2638–2649. (44) Possmayer, F. A Proposed Nomenclature for Pulmonary Surfactant-associated Proteins. Am. Rev. Respir. Dis. 1988, 138, 990–998. (45) Possmayer, F. The Role of Surfactant-associated Proteins. Am. Rev. Respir. Dis. 1990, 142, 749–752. (46) Warriner, H. E.; Ding, J.; Waring, A. J.; Zasadzinski, J. A. A Concentration- Dependent Mechanism by Which Serum Albumin Inactivates Replacement Lung Surfactants. Biophys. J. 2002, 82, 835–842.

118

(47) Larsson, M.; Nylander, T.; Keough, K. M. W.; Nag, K. An X-ray Diffraction Study of Alterations in Bovine Lung Surfactant Bilayer Structures Induced by Albumin. Chem. Phys. Lipids 2006, 144, 137–145. (48) Nag, K.; Hillier, A.; Parsons, K.; Garcia, M. F. Interactions of Serum with Lung Surfactant Extract in the Bronchiolar and Alveolar Airway Models. Respir. Physiol. Neuro. 2007, 157, 411–424. (49) Zuo, Y. Y.; Tadayyon, S. M.; Keating, E.; Zhao, L.; Veldhuizen, R. A. W.; Petersen, N. O.; Amrein, M. W.; Possmayer, F. Atomic Force Microscopy Studies of Functional and Dysfunctional Pulmonary Surfactant Films, II: Albumin-Inhibited Pulmonary Surfactant Films and the Effect of SP-A. Biophys. J. 2008, 95, 2779–2791. (50) Frerking, I.; Gunther, A.; Seeger, W.; Pison, U. Pulmonary Surfactant: Functions, Abnormalities and Therapeutic Options. Intens. Care Med. 2001, 27, 1699 – 1717. (51) Manzanares, D.; Rodriguez-Capote, K.; Liu, S.; Haines, T.; Ramos, Y.; Zhao, L.; Doherty-Kirby, A.; Lajoie, G.; Possmayer, F. Modification of Tryptophan and Methionine Residues Is Implicated in the Oxidative Inactivation of Surfactant Protein B. Biochemistry-US 2007, 46, 5604–5615. (52) Rodriguez-Capote, K.; Manzanares, D.; Haines, T.; Possmayer, F. Reactive Oxygen Species Inactivation of Surfactant Involves Structural and Functional Alterations to Surfactant Proteins SP-B and SP-C. Biophys. J. 2006, 90, 2808– 2821. (53) Bringezu, F.; Pinkerton, K. E.; Zasadzinski, J. A. Environmental Tobacco Smoke Effects on the Primary Lipids of Lung Surfactant. Langmuir 2003, 19, 2900– 2907. (54) Bakshi, M. S.; Zhao, L.; Smith, R.; Possmayer, F.; Petersen, N. O. Metal Nanoparticle Pollutants Interfere with Pulmonary Surfactant Function In Vitro. Biophys. J. 2008, 94, 855–868. (55) Hall, S. B.; Lu, R. Z.; Venkitaraman, A. R.; Hyde, R. W.; Notter, R. H. Inhibition of Pulmonary Surfactant by Oleic-Acid - Mechanisms and Characteristics. J. Appl. Physiol. 1992, 72, 1708–1716.

119

(56) Cockshutt, A.; Possmayer, F. Lysophosphatidylcholine Sensitizes Lipid Extracts of Pulmonary Surfactant to Inhibition by Serum-Proteins. Biochim. Biophys. Acta - Biomembranes 1991, 1086, 63–71. (57) Clark, D. A.; Nieman, G. F.; Thompson, J. E.; Paskanik, A. M.; Rokhar, J. E.; Bredenberg, C. E. Surfactant Displacement by Meconium Free Fatty Acids: An Alternative Explanation for Atelectasis in Meconium Aspiration Syndrome. J. Pediatr. 1987, 110, 765–770. (58) Holm, B. A.; Wang, W. D.; Notter, R. H. Multiple Mechanisms of Lung Surfactant Inhibition. Pediatr. Res. 1999, 46, 85–93. (59) Dalence, C. R.; Bowie, L. J.; Dohnal, J. C.; Farrell, E. E.; Neerhof, M. G. Amniotic Fluid Lamellar Body Count: a Rapid and Reliable Fetal Lung Maturity Test. Obstet. Gynecol. 1995, 86, 235–239. (60) Guyer, B.; Freedman, M. A.; Strobino, D. M.; Sondik, E. J. Annual Summary of Vital Statistics: Trends in the Health of Americans During the 20th Century. Pediatrics 2000, 106, 1307–1317. (61) Schoendorf, K. C.; Kiely, J. L. Birth Weight and Age-specific Analysis of the 1990 US Infant Mortality Drop - Was It Surfactant? Arch. Pediatr. Adolesc. Med. 1997, 151, 129–134. (62) McIntyre, R. C.; Pulido, E. J.; Bensard, D. D.; Shames, B. D.; Abraham, E. Thirty Years of Clinical Trials in Acute Respiratory Distress Syndrome. Crit. Care Med. 2000, 28, 3314–3331. (63) Martin, Joyce A; Hamilton, Brady E.; Sutton, Paul D.; Ventura, Stephanie J.; Menacker, Fay; Munson, Martha L. Births: Final Data for 2002. Natl. Vital. Stat. Rep. 2003, 25, 1–113. (64) Engle, W. A.; Stark, A. R. Surfactant-Replacement Therapy for Respiratory Distress Syndrome in the Preterm and Term Neonate: Congratulations and Corrections: In Reply. Pediatrics 2008, 121, 1291–1292. (65) Gunther, A.; Ruppert, C.; Schmidt, R.; Markart, P.; Grimminger, F.; Walmrath, D.; Seeger, W. Surfactant Alteration and Replacement in Acute Respiratory Distress Syndrome. Respir. Res. 2001, 2, 353–U2.

120

(66) Tabak, S.; Notter, R. Effect of Plasma Proteins on the Dynamic Pi-A Characteristics of Saturated Phospholipid Films. J. Colloid Interface Sci. 1977, 59, 293–300. (67) Holm, B. A.; Notter, R. H.; Finkelstein, J. N. Surface Property Changes from Interactions of Albumin with Natural Lung Surfactant and Extracted Lung Lipids. Chem. Phys. Lipids 1985, 38, 287–298. (68) Taeusch, H. W.; de la Serna, J. B.; Perez-Gil, J.; Alonso, C.; Zasadzinski, J. A. Inactivation of Pulmonary Surfactant Due to Serum-Inhibited Adsorption and Reversal by Hydrophilic Polymers: Experimental. Biophys. J. 2005, 89, 1769– 1779. (69) Zasadzinski, J. A.; Alig, T. F.; Alonso, C.; de la Serna, J. B.; Perez-Gil, J.; Taeusch, H. W. Inhibition of Pulmonary Surfactant Adsorption by Serum and the Mechanisms of Reversal by Hydrophilic Polymers: Theory. Biophys. J. 2005, 89, 1621–1629. (70) Venkitaraman, A. R.; Hall, S. B.; Whitsett, J. A.; Notter, R. H. Enhancement of Biophysical Activity of Lung Surfactant Extracts and Phospholipid-apoprotein Mixtures by Surfactant Protein A. Chem. Phys. Lipids 1990, 56, 185–193. (71) Cockshutt, A. M.; Weitz, J.; Possmayer, F. Pulmonary Surfactant-associated Protein A Enhances the Surface Activity of Lipid Extract Surfactant and Reverses Inhibition by Blood Proteins in Vitro. Biochemistry-US 1990, 29, 8424–8429. (72) Lewis, J. F.; Veldhuizen, R. The Role of Exogenous Surfactant in the Treatment of Acute Lung Injury. Annu. Rev. Physiol. 2003, 65, 613–642. (73) Lewis, J. F.; Veldhuizen, R. A. W. The Future of Surfactant Therapy During ALI/ARDS. Sem. Resp. Crit. Care. M. 2006, 27, 377–388. (74) Gregory, T. J.; Steinberg, K. P.; Spragg, R.; Gadek, J. E.; Hyers, T. M.; Longmore, W. J.; Moxley, M. A.; Cai, G. Z.; Hite, R. D.; Smith, R. M.; et al. Bovine Surfactant Therapy for Patients with Acute Respiratory Distress Syndrome. Am. J. Respir. Crit. Care Med. 1997, 155, 1309–1315. (75) Baudouin, S. V. Exogenous Surfactant Replacement in ARDS — One Day, Someday, or Never? New Engl. J. Med. 2004, 351, 853–855.

121

(76) Davidson, W. J.; Dorscheid, D.; Spragg, R.; Schulzer, M.; Mak, E.; Ayas, N. T. Exogenous Pulmonary Surfactant for the Treatment of Adult Patients with Acute Respiratory Distress Syndrome: Results of a Meta-analysis. Crit. Care 2006, 10. (77) Hailman, M.; Merritt, T. A.; Schneider, H.; Epstein, B. L.; Mannino, F.; Edwards, D. K.; Gluck, L. Isolation of Human Surfactant from Amniotic Fluid and a Pilot Study of Its Efficacy in Respiratory Distress Syndrome. Pediatrics 1983, 71, 473–482. (78) Hallman, M.; Merritt, T. A.; Jarvenpaa, A.-L.; Boynton, B.; Mannino, F.; Gluck, L.; Moore, T.; Edwards, D. Exogenous Human Surfactant for Treatment of Severe Respiratory Distress Syndrome: A Randomized Prospective Clinical Trial. J. Pediatr. 1985, 106, 963–969. (79) Halliday, H. Overview of Clinical-Trials Comparing Natural and Synthetic Surfactants. Biol. Neonate 1995, 67, 32–47. (80) Moya, F.; Maturana, A. Animal-derived Surfactants Versus Past and Current Synthetic Surfactants: Current Status. Clin. Perinatol. 2007, 34, 145–+. (81) Been, J. V.; Zimmermann, L. J. I. What’s New in Surfactant? Eur. J. Pediatr. 2007, 166, 889 – 899. (82) Robertson, B.; Halliday, H. L. Principles of Surfactant Replacement. Biochim. Biophys. Acta - Mol. Basis Dis. 1998, 1408, 346–361. (83) Revak, S. D.; Merritt, T. A.; Cochrane, C. G.; Heldt, G. P.; Alberts, M. S.; Anderson, D. W.; Kheiter, A. Efficacy of Synthetic Peptide-Containing Surfactant in the Treatment of Respiratory Distress Syndrome in Preterm Infant Rhesus Monkeys. Pediatr. Res. 1996, 39, 715–724. (84) Spragg, R. G.; Lewis, J. F.; Walmrath, H.-D.; Johannigman, J.; Bellingan, G.; Laterre, P.-F.; Witte, M. C.; Richards, G. A.; Rippin, G.; Rathgeb, F.; et al. Effect of Recombinant Surfactant Protein C–Based Surfactant on the Acute Respiratory Distress Syndrome. New Engl. J. Med. 2004, 351, 884–892. (85) Durand, D. J.; Clyman, R. I.; Heymann, M. A.; Clements, J. A.; Mauray, F.; Kitterman, J.; Ballard, P. Effects of a Protein-free, Synthetic Surfactant on Survival and Pulmonary Function in Preterm Lambs. J. Pediatr. 1985, 107, 775– 780.

122

(86) Butt, H.-J.; Kappl, M.; Graf, K. Physics and Chemistry of Interfaces; Second, Revised, and Enlarged.; Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim, 2006. (87) J.S. Rowlinson; B. Widom. Molecular Theory of Capillarity; International Series of Monographs on Chemistry. (88) Gaines Jr, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishers, 1966. (89) Vargaftik, N. B.; Volkov, B. N.; Voljak, L. D. International Tables of the Surface-Tension of Water. J. Phys. Chem. Ref. Data 1983, 12, 817–820. (90) Neumann, A. W.; David, R.; Zuo, Y. Applied Surface Thermodynamics; Surfactant Science Series 151; Second.; CRC Press Taylor and Francis Group. (91) Langmuir, I. The Constitution and Fundamental Properties of and Liquids. II. Liquids. J. Am. Chem. Soc. 1917, 39, 1848–1906. (92) Clements, J. A. Surface Tension of Lung Extracts. Exp. Biol. Med. 1957, 95, 170– 172. (93) Clements, J. A.; Hustead, R. F.; Johnson, R. P.; Gribetz, I. Pulmonary Surface Tension and Alveolar Stability. J. Appl. Physiol. 1961, 16, 444–450. (94) Ja, C. Surface Phenomena in Relation to Pulmonary Function. Physiologist 1962, 5, 11–28. (95) Lipp, M. M.; Lee, K. Y.; Waring, A.; Zasadzinski, J. A. Fluorescence, Polarized Fluorescence, and Brewster Angle Microscopy of Palmitic Acid and Lung Surfactant Protein B Monolayers. Biophys. J. 1997, 72, 2783–2804. (96) Piknova, B.; Schram, V.; Hall, S. Pulmonary Surfactant: Phase Behavior and Function. Curr. Opin. Struc. Biol. 2002, 12, 487–494. (97) Ma, G.; Allen, H. C. Real-Time Investigation of Lung Surfactant Respreading with Surface Vibrational Spectroscopy. Langmuir 2006, 22, 11267–11274. (98) Ma, G.; Allen, H. C. New Insights into Lung Surfactant Monolayers Using Vibrational Sum Frequency Generation Spectroscopy. Photochem. Photobiol. 2006, 82, 1517–1529. (99) Brockman, J. M.; Wang, Z.; Notter, R. H.; Dluhy, R. A. Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on Binary Phospholipid Monolayers. Biophys. J. 2003, 84, 326–340.

123

(100) Phang, T.-L.; McClellan, S. J.; Franses, E. I. Displacement of Fibrinogen from the Air/Aqueous Interface by Dilauroylphosphatidylcholine Lipid. Langmuir 2005, 21, 10140–10147. (101) Prokop, R. M.; Neumann, A. W. Measurement of the Interfacial Properties of Lung Surfactant. Curr. Opin. Colloid Interface 1996, 1, 677–681. (102) Prokop, R. M.; Jyoti, A.; Eslamian, M.; Garg, A.; Mihaila, M.; del Rio, O. I.; Susnar, S. S.; Policova, Z.; Neumann, A. W. A Study of Captive Bubbles with Axisymmetric Drop Shape Analysis. Colloid Surf. A-Physicochem. Eng. Asp. 1998, 131, 231–247. (103) Putz, G.; Goerke, J.; Schurch, S.; Clements, J. Evaluation of Pressure-Driven Captive Bubble Surfactometer. J. Appl. Physiol. 1994, 76, 1417–1424. (104) Enhorning, G. Pulsating Bubble Technique for Evaluating Pulmonary Surfactant. J. Appl. Physiol. 1977, 43, 198–203. (105) Hall, S.; Bermel, M.; Ko, Y.; Palmer, H.; Enhorning, G.; Notter, R. Approximations in the Measurement of Surface-Tension on the Oscillating Bubble Surfactometer. J. Appl. Physiol. 1993, 75, 468–477. (106) Kwok, D. Y.; Vollhardt, D.; Miller, R.; Li, D.; Neumann, A. W. Axisymmetric Drop Shape Analysis as a Film Balance. Colloid Surf. A-Physicochem. Eng. Asp. 1994, 88, 51–58. (107) Yu, L. M. Y.; Lu, J. J.; Chiu, I. W. Y.; Leung, K. S.; Chan, Y. W.; Zhang, L.; Policova, Z.; Hair, M. L.; Neumann, A. W. Poly(ethylene Glycol) Enhances the Surface Activity of a Pulmonary Surfactant. Colloids and Surfaces B: Biointerfaces 2004, 36, 167–176. (108) Wustneck, R.; Wustneck, N.; Moser, B.; Karageorgieva, V.; Pison, U. Surface Dilatational Behavior of Pulmonary Surfactant Components Spread on the Surface of a Pendant Drop. 1. Dipalmitoyl Phosphatidylcholine and Surfactant Protein C. Langmuir 2002, 18, 1119–1124. (109) Saad, S. M. I.; Policova, Z.; Acosta, E. J.; Neumann, A. W. Range of Validity of Drop Shape Techniques for Surface Tension Measurement. Langmuir 2010, 26, 14004–14013. (110) Yu, L. M. Y.; Lu, J. J.; Chan, Y. W.; Ng, A.; Zhang, L.; Hoorfar, M.; Policova, Z.; Grundke, K.; Neumann, A. W. Constrained Sessile Drop as a New

124

Configuration to Measure Low Surface Tension in Lung Surfactant Systems. J. Appl. Physiol. 2004, 97, 704–715. (111) Zuo, Y. Y.; Gitiafroz, R.; Acosta, E.; Policova, Z.; Cox, P. N.; Hair, M. L.; Neumann, A. W. Effect of Humidity on the Adsorption Kinetics of Lung Surfactant at Air−Water Interfaces. Langmuir 2005, 21, 10593–10601. (112) Acosta, E. J.; Gitiafroz, R.; Zuo, Y. Y.; Policova, Z.; Cox, P. N.; Hair, M. L.; Neumann, A. W. Effect of Humidity on Lung Surfactant Films Subjected to Dynamic Compression/expansion Cycles. Respir. Physiol. Neuro. 2007, 155, 255–267. (113) Lamour, G.; Hamraoui, A.; Buvailo, A.; Xing, Y.; Keuleyan, S.; Prakash, V.; Eftekhari-Bafrooei, A.; Borguet, E. Contact Angle Measurements Using a Simplified Experimental Setup. J. Chem. Educ. 2010, 87, 1403–1407. (114) Saad, S. M. I.; Policova, Z.; Acosta, E. J.; Hair, M. L.; Neumann, A. W. Mixed DPPC/DPPG Monolayers at Very High Film Compression. Langmuir 2009, 25, 10907–10912.

125

APPENDIX A: INSTRUCTIONS FOR USING ADSA-CSD AND BLUEPRINTS

FOR SETUP PIECES

126

Figure A.1: Instructions for using ADSA-CSD Title page.

127

Figure A.2: Instructions for using ADSA-CSD page 1

128

Figure A.3: Instructions for using ADSA-CSD page 2.

129

Figure A.4: Instructions for using ADSA-CSD page 3.

130

Figure A.5: Instructions for using ADSA-CSD page 4.

131

Figure A.6: Instructions for using ADSA-CSD page 5.

132

Figure A.7: Bottom portion of lens holder.

133

Figure A.8: Top portion of lens holder.Figure B.2

134

Figure A.9: Dimensions of current pedestal.

135

Figure A.10: Dimensions of new pedestal for one-piece pedestal design.

136

Figure A.11: One-piece pedestal design.

137

Figure A.12: Main body of new grid holder.

138

Figure A.13: Second part of new grid holder that secures the grid in place.

139

Figure A.14: Baseplate that new grid holder and new one-piece pedestal will be placed on.

140