Integrated Raman and Angular Scattering of Single Biological Cells

250 Pages·2009·2.91 MB·English

Integrated Raman and Angular
Scattering of Single Biological Cells
by
Zachary J. Smith
Submitted in Partial Fulfillment
of the
Requirements for the Degree
Doctor of Philosophy
Supervised by
Professor Andrew J. Berger
The Institute of Optics
Arts, Sciences and Engineering
School of Engineering and Applied Sciences
University of Rochester
Rochester, New York
2009
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Curriculum Vitae
Zachary Smith was born in Fairport, NY in 1979. As a National Merit Scholar,
he received his Bachelor of Science degree in Optics with highest distinction from
the University of Rochester in 2002. In the same year he was accepted into
the graduate program at the Institute of Optics at the University of Rochester.
In 2003 he joined the research group of Prof. Andrew J. Berger, where he has
worked on several projects centered around Raman spectroscopic applications
to cell biology. During his tenure at the Institute he was elected as the senior
graduate representative to the Institute graduate committee, as well as being the
chair for 2007 and 2008 of a series of summer talks given by Institute students.
CURRICULUM VITAE iii
Refereed Publications
Z. J. Smith and A. J. Berger, “Construction of an Integrated Raman and An-
gular Scattering Microscope,” Review of Scientific Instruments, in press.
Z. J. Smith and A. J. Berger, “Validation of an Integrated Raman- and Angular-
Scattering Microscopy system on heterogeneous bead mixtures and single human
immune cells,” Applied Optics 48 (10), D109-D120 (2008).
Z. J. Smith and A. J. Berger, “Integrated Raman and Angular Scattering Mi-
croscopy,” Optics Letters, 33 (7), 714-716 (2008).
Z. J. Smith and A. J. Berger, “Surface-sensitive polarized Raman spectroscopy of
biological tissue,” Optics Letters, 30 (11) (2005).
Talks
Z. J. Smith and A. J. Berger, “Investigation of Single Human Immune Cells by
Integrated Raman and Angular-Scattering Microscopy,” SPIE Photonics West,
BiOS Conference, San Jose, CA (2009).
Z. J. Smith and A. J. Berger, “Integrated Raman and Angular Scattering Mi-
croscopy (IRAM),” SPIE Photonics West, BiOS Conference San Jose, CA (2008).
Z. J. Smith, S. Mitra, T. H. Foster, and A. J. Berger, “Raman Spectroscopy
of Single PDT-Treated Cells,” OSA Annual Meeting, Rochester, NY (2006).
Poster Presentations
Z. J. Smith and A. J. Berger, “Construction of an Integrated Raman and An-
gular Scattering Microscope,” SPEC Conference, S˜ao Paolo, Brazil (2008).
Z. J. Smith, B. D. Chuzles, and A. J. Berger, “Integrated Raman and Angu-
lar Scattering Microscopy,” Engineering Conferences International, Advances in
Optics for Biotechnology, Medicine And Surgery, Naples, FL (2007).
Z. J. Smith, S. Mitra, T. H. Foster, and A. J. Berger, “Raman Spectroscopy
of Single PDT-Treated Cells,” SPIE Photonics West, BiOS Conference San Jose,
CA (2006).
Z. J. Smith and A. J. Berger, “Surface-Sensitive Polarized Raman Spectroscopy,”
OSA Annual Meeting, Rochester, NY (2004).
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Acknowledgments
I would like to acknowledge the contributions of several people and institutions
who have helped make the work presented in this thesis possible.
Firstly, I would like to thank my advisor, Professor Andrew J. Berger. I have
been extremely fortunate to have Andrew as my advisor. His devotion to his
students, both in his group and in his classroom, has served as an inspiration to
me personally and professionally. His thinking is clearer and his grasp of physics
is stronger than anyone else I have had the privilege of knowing, qualities that,
duringourdiscussions,enablehimtozeroinontheshakiestpartsofmyknowledge
and ask the one question I was dreading being asked. These research discussions
and his thoughtful suggestions and advice have been invaluable in my successfully
conducting this research.
I would also like to thank the members of my committee, Prof. Miguel A.
Alonso, Prof. Thomas G. Brown, and Prof. Thomas H. Foster, for their commit-
ment to my success and their helpful suggestions throughout the course of this
project. In particular, Professor Foster’s kind encouragement and support, begun
ACKNOWLEDGEMENTS v
during a period of collaboration and continued throughout my graduate career,
has had a profound impact on me both scientifically and personally.
I would like to sincerely express my gratitude to our collaborators in this the-
sis from the Human Immunology Center at the University of Rochester Medical
Center. In particular, Dr. Sally A. Quataert and Dr. Ernest Wang have been great
sources of both encouragement and aid in providing a biological context through
which our instrument has been able to make a positive contribution. Additionally,
I would like to thank technicians Matthew R. Cochran, Kelli M. Fudella, Shelly
Secor-Socha, and Terry Wightman for their kind and untiring assistance both in
preparing samples for me as well as teaching me the fundamentals of cell prepara-
tion and cell culture. Without this collaboration the work presented in this thesis
would have been impossible.
I would also like to thank all members of the Biomedical Spectroscopy group
that I have had the pleasure of working with: Dr. Qingyuan Zhu, Dr. Dahu
Qi, Dr. Rolf Saager, Jason Maher, Brooke Beier, and Captain Jack DeLong. In
particular Qingyuan and Dahu helped to teach me the finer details of constructing
and aligning Raman instruments when I was just starting out in the field. All of
my colleagues in Andrew’s group have helped to make my tenure both rewarding
and enjoyable. From road trips to plane trips to trips to a new building, their
discussions, assistance, inspiration, and friendship are gratefully appreciated.
ACKNOWLEDGEMENTS vi
I would like to acknowledge the financial support this work has received, with-
out which none of this work would be possible. Funding from the National Insti-
tutes of Health and the National Science Foundation came through grant numbers
1-R21-DE016111-01A1 and CBET-0754698, respectively. Additionally, I would
like to thank the Human Immunology Center at the University of Rochester Med-
ical Center for their financial support.
Finally, I would like to acknowledge my family and friends for helping to bring
me to this point. I would like to thank Kaiqin Chu for her constant and brilliant
help and support during my brightest times and darkest hours. I would like to
thank my parents, James and Bonnie Smith, for their constant encouragement
throughout my many years of academic studies. I would like to thank my siblings
Shannon Smith and Tyler Smith for their support and commiseration. And finally
I would like to thank my peerless friends both at the Institute and beyond, par-
ticularly Ross Campbell, Mark Sterling, Daniel Rozman, Prof. Wanli Chi, Prof.
Robert Newcomb, Andrey Gordienko, and Dr. Yanqiao Huang.
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Abstract
Raman, or inelastic, scattering and angle-resolved elastic scattering are two
optical processes that have found wide use in the study of biological systems. Ra-
man scattering quantitatively reports on the chemical composition of a sample by
probing molecular vibrations, while elastic scattering reports on the morphology
of a sample by detecting structure-induced coherent interference between incident
and scattered light. We present the construction of a multimodal microscope plat-
form capable of gathering both elastically and inelastically scattered light from
a 38 µm2 region in both epi- and trans-illumination geometries. Simultaneous
monitoring of elastic and inelastic scattering from a microscopic region allows
noninvasive characterization of a living sample without the need for exogenous
dyes or labels. A sample is illuminated either from above or below with a focused
785 nm TEM mode laser beam, with elastic and inelastic scattering collected
00
by two separate measurement arms. The measurements may be made either si-
multaneously, if identical illumination geometries are used, or sequentially, if the
two modalities utilize opposing illumination paths. In the inelastic arm, Stokes-
shifted light is dispersed by a spectrograph onto a CCD array. In the elastic
ABSTRACT viii
scattering collection arm, a relay system images the microscope’s back aperture
onto a CCD detector array to yield an angle-resolved elastic scattering pattern.
Post-processing of the inelastic scattering to remove fluorescence signals yields
high quality Raman spectra that report on the sample’s chemical makeup. Com-
parison of the elastically scattered pupil images to generalized Lorenz-Mie theory
yields estimated size distributions of scatterers within the sample.
In this thesis we will present validations of the IRAM instrument through
measurements performed on single beads of a few microns in size, as well as on
ensembles of sub-micron particles of known size distributions. The benefits and
drawbacks of the epi- and trans-illumination modalities are also discussed. In ad-
dition, transilluminated Raman and elastic-scattering spectra were obtained from
several biological test-cases, including Streptococcus pneumoniae, baker’s yeast,
and single human immune cells. Both the Raman and elastic-scattering channels
extract information from these samples that are well in line with their known
characteristics from the literature. Finally, we report on an experiment in which
CD8+ T lymphocytes were stimulated by exposure to the antigens staphylococcal
enterotoxin B and phorbol myristate acetate. Clear chemical and morphological
differences were observed between the activated and unactivated cells, with the
results correlating well to analysis performed on parallel samples using fluorescent
stains and a flow cytometer.
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Table of Contents
Curriculum Vitae ii
Acknowledgments iv
Abstract vii
List of Tables xv
List of Figures xvi
1 Introduction 1
1.1 Biological Background . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Overview of the Cell . . . . . . . . . . . . . . . . . . . . . 2
1.1.2 The Human Immune System . . . . . . . . . . . . . . . . . 5
1.2 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.2.1 Raman Spectroscopy of Biological Samples . . . . . . . . . 11
1.2.2 Elastic Scattering of Biological Samples . . . . . . . . . . . 13
TABLE OF CONTENTS x
1.2.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.3 Outline of the Thesis . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2 Theoretical Background 25
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2 Raman Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.3 Elastic Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.1 Statement of the Scattering Problem . . . . . . . . . . . . 34
2.3.2 Construction of the Vector Spherical Harmonics . . . . . . 38
2.3.3 Solution for Planar Illumination - Mie Theory . . . . . . . 42
2.3.4 Mie Theory for a Coated Sphere . . . . . . . . . . . . . . . 47
2.3.5 Solution for a Gaussian beam - Generalized Lorenz-Mie
Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.4 Background for Data Analysis . . . . . . . . . . . . . . . . . . . . 61
2.4.1 The Savitzky-Golay filter . . . . . . . . . . . . . . . . . . . 61
2.4.2 Downhill Simplex Searching . . . . . . . . . . . . . . . . . 64
2.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3 Integrated Raman and Angular Scattering Microscope Design
and Construction 70