Interfacial Microrheology of Biological Films Daniel B. Allan

213 Pages·2014·3.91 MB·English

Interfacial Microrheology of Biological Films
by
Daniel B. Allan
A dissertation submitted to The Johns Hopkins University in conformity with the
requirements for the degree of Doctor of Philosophy.
Baltimore, Maryland
December, 2014
c Daniel B. Allan 2014
(cid:13)
All rights reserved
Abstract
This thesis reports experimental studies of the rheology of biological materials,
with an emphasis on interfacial layers, including protein layers and bacterial biofilms.
Results are presented on layers formed by the proteins lysozyme and Staphylococcal
nuclease at the air–water interface, on biofilms formed by Pseudomonas bacteria at
the oil–water interface, and on the bulk rheological properties of fibrin and cystic
fibrosis mucus with an eye toward its role as an interfacial barrier in the lung. The
evolution of interfacial mechanical response through time is interpreted in terms of
the changing microscopic structure of the layer.
The studies employ interfacial microrheology, which uses the motion of
micrometer-scaleparticlesembeddedintheinterfacetoprobethemechanicalresponse
of the surrounding material and infer its rheology. Passive measurements, which rely
on thermal forces to drive the particles, are complemented by active measurements,
in which ferromagnetic nanowires were rotated using magnetic fields. Additionally,
the study of fibrin and cystic fibrosis mucus employs a novel technique using custom
fluorescent particles that can be selectively “switched on” and used to characterize
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ABSTRACT
rheology and particle mobility over physiologically relevant time and distance scales.
This thesis also presents a software toolkit, developed as part of the thesis work to
meet the demands of this research, that has found applications by other researchers
in other areas.
This work was conducted under the supervision of Professor Robert L. Leheny
and Professor Daniel H. Reich.
Primary Reader: Professor Robert L. Leheny
Secondary Reader: Professor Daniel H. Reich
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Acknowledgments
First, I am grateful to Bob Leheny for his mentorship. He has been encouraging,
patient, generous with his insight and his talent for communicating research, and
unfailingly available and attentive. I am also grateful to Dan Reich for his incisive
guidance. Together, they have given me great opportunities and provided detailed
attention to prepare me to succeed. Additionally, I thank my thesis committee: Prof.
Kate Stebe, Prof. Mark Robbins, Prof. Mike Bevan, and Prof. Joelle Frechette.
Special thanks go to Mark, as well as Prof. Alex Szalay, for helping support and
develop my interest in computing.
Many generous collaborators have enriched my work and brightened my expe-
rience. Kate Stebe welcomed me to her group and inspired me with her vigorous
enthusiasm. I thank Liana Vaccari for sharing her project with me and accompany-
ing me through the demanding rite of the 24-hour measurement. Likewise, I thank
everyone in the Hanes group and in particular Ben Schuster for sharing his broad
knowledge and for inviting me to contribute to his exciting projects. I am indebted
to Marek Cieplak for involving me in his work and exposing me to a different aspect
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ACKNOWLEDGMENTS
of our field. And I am lucky to have found Tom Caswell and Nathan Keim, who have
dwelled with me in the details of particle tracking. I have enjoyed great luck in work-
ing with many talented undergraduates, including Steve Cardinali, Daniel Firester,
Victor Allard, and Bilyana Tzolova, whose dedicated efforts drove much of the work
presented here. I am grateful to my predecessors in the Leheny and Reich labs for the
tools they built and the guidance they provided—especially Myung Han Lee, Stuart
Kirschner, Nate Capallo, Hongyu Guo, and Joel Rovner. Kui Chen, Tristan Sharp,
Dan Richman, Nik Hartman, and Nuala McCullagh also provided insights and deeply
appreciated camaraderie.
Finally, I am grateful to my father for teaching me that childlike curiosity has
no age limit, to my mother for her cheer and encouragement, to my brother for his
loyal friendship and inspiring courage, to Kelly for her unwavering confidence and
loving support, and to all friends and family who have made these years of study and
research enjoyable.
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Contents
Abstract ii
Acknowledgments iv
List of Figures xii
1 Introduction 1
1.1 Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.2 Measurement Techniques . . . . . . . . . . . . . . . . . . . . . 4
1.1.3 Interfacial Rheology . . . . . . . . . . . . . . . . . . . . . . . 5
1.2 Microrheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.1 Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.2 Interfacial Microrheology . . . . . . . . . . . . . . . . . . . . . 9
1.2.3 Translational drag on a sphere at an interface . . . . . . . . . 10
1.2.4 Rotational drag on a rod at an interface . . . . . . . . . . . . 13
1.3 Topics of Thesis Research . . . . . . . . . . . . . . . . . . . . . . . . 14
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CONTENTS
1.3.1 Protein Layers . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Bacterial Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . 16
2 Experimental Methods 19
2.1 Experimental Challenges in Interfacial Particle Tracking . . . . . . . 19
2.1.1 Colloidal Aggregation at the Interface . . . . . . . . . . . . . . 19
2.1.2 Distinguishing Interfacial Particles . . . . . . . . . . . . . . . 21
2.1.3 Minimizing and Correcting for Convective Drift . . . . . . . . 22
2.1.4 Choosing an Appropriate Particle Concentration . . . . . . . . 22
2.2 Experimental Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3 Ferromagnetic Nickel Nanowires . . . . . . . . . . . . . . . . . . . . . 26
2.3.1 Overview of Fabrication and Characteristics . . . . . . . . . . 26
2.3.2 Hydrophobic Functionalization . . . . . . . . . . . . . . . . . 27
2.4 Tracking Nanowire Orientation . . . . . . . . . . . . . . . . . . . . . 28
3 Software Development: A Modern Particle-Tracking Toolkit 31
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2 Review of the Crocker–Grier Particle Tracking Algorithm . . . . . . . 33
3.3 Trackpy: An Enhanced Implementation of Crocker–Grier . . . . . . . 37
3.4 Subpixel precision and accuracy . . . . . . . . . . . . . . . . . . . . . 39
3.4.1 Static Error . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4.2 Motion Blur . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
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CONTENTS
3.4.3 Experimental Best Practices to Minimize Error . . . . . . . . 46
3.5 Prediction Framework . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.5.1 Prescribed predictors . . . . . . . . . . . . . . . . . . . . . . . 49
3.5.2 Dynamic predictors . . . . . . . . . . . . . . . . . . . . . . . . 51
3.5.2.1 Channel flow prediction . . . . . . . . . . . . . . . . 55
3.5.2.2 Drift prediction . . . . . . . . . . . . . . . . . . . . . 55
3.6 Testing & Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . 58
3.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.7.1 “A Modern Approach” . . . . . . . . . . . . . . . . . . . . . . 59
3.7.2 Measuring Success . . . . . . . . . . . . . . . . . . . . . . . . 60
3.7.3 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . 61
4 Linear and Nonlinear Microrheology of Lysozyme Layers Forming
at the Air–Water Interface 62
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.3 Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.3.1 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . 67
4.3.2 Passive (Brownian) Microrheology . . . . . . . . . . . . . . . . 69
4.3.3 Active Microrheology . . . . . . . . . . . . . . . . . . . . . . . 71
4.3.3.1 Magnetic Nanowire Probes. . . . . . . . . . . . . . . 71
4.3.3.2 Microscopy with in situ Magnetic Fields. . . . . . . . 71
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CONTENTS
4.3.3.3 Measurement Procedures and Analysis. . . . . . . . . 72
4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.4.1 Linear Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . 76
4.4.2 Nonlinear Rheology . . . . . . . . . . . . . . . . . . . . . . . . 77
4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.5.1 Nature of the Viscoelastic Transition . . . . . . . . . . . . . . 89
4.5.1.1 Soft Glassy Rheology Model . . . . . . . . . . . . . . 89
4.5.1.2 Critical Behavior of Gelation . . . . . . . . . . . . . 91
4.5.1.3 Possibility of a Yield Stress at Late Ages . . . . . . . 92
4.5.2 Comparison with Other Protein Layers . . . . . . . . . . . . . 93
4.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
5 Mechanical Evolution of Interfacial Layers of SNase: The Role of
Protein Conformation in Layer Formation 96
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
5.2 Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 98
5.2.1 Protein Fabrication . . . . . . . . . . . . . . . . . . . . . . . . 98
5.2.2 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . 100
5.2.3 Active Microrheology . . . . . . . . . . . . . . . . . . . . . . . 100
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.3.1 Wild-Type SNase Layer Evolution . . . . . . . . . . . . . . . . 101
5.3.2 Disordered SNase Layer Evolution . . . . . . . . . . . . . . . . 103
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CONTENTS
5.4 Discussion & Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . 107
6 Dynamical and Mechanical Evolution of Bacterial Biofilms at the
Oil–Water Interface 110
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.2 Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 114
6.2.1 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . 114
6.2.2 Particle Tracking . . . . . . . . . . . . . . . . . . . . . . . . . 115
6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
6.3.1 Tracer Motion at an Active Interface . . . . . . . . . . . . . . 117
6.3.2 Viscoelastic Transition . . . . . . . . . . . . . . . . . . . . . . 130
6.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
7 Multiscale Nanoparticle Mobility Measurements in Biological Gels
Using Photoactivatable Fluorescent Probes 140
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.2 Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
7.2.2 Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
7.2.2.1 Labeling of PLGA with caged rhodamine . . . . . . 143
7.2.2.2 Particle formulation: PS-PEG . . . . . . . . . . . . . 144
7.2.2.3 Particle formulation: PLGA-PEG . . . . . . . . . . . 144
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