Novel Endoscopes for Microscopic Assessment of
Airway Clearance using Micro-Optical Coherence
ARCHIES
Tomography
MASSACHUSETTS INSTITUTE
OF TECHNOLOLGY
by
APR 14 2015
Carolin Isabella Unglert, PhD
LIBRARIES
Submitted to the Harvard-MIT Division of Health Sciences and
Technology
in partial fulfillment of the requirements for the degree of
Master of Science
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 2015
Massachusetts Institute of Technology 2015. All rights reserved.
Signature redacted
A u th or ............
...................
Harvard-MIT Divi~s'6 Health Sciences and Technology
Signature redacted
C ertified by ..........
Accepted by ....
February 2, 2015
..................
Guillerm4. Tearney, MD, PhD
Professor of Pathology, Harvard Medical School
Thesis Supervisor
Signature redacted..............
Emery N. Brown, MD, PhD
Direc or, Harvard-MIT Program in Health Sciences and
Technology/Professor of Computational Neuroscience and Health
Sciences and Technology
Novel Endoscopes for Microscopic Assessment of Airway
Clearance using Micro-Optical Coherence Tomography
by
Carolin Isabella Unglert, PhD
Submitted to the Harvard-MIT Division of Health Sciences and Technology
on February 2, 2015, in partial fulfillment of the
requirements for the degree of
Master of Science
Abstract
The health of the human respiratory system depends critically on airway clearance via
motile hair-like structures (cilia), which transport and eliminate unwanted particles
trapped within mucus. Impairment of mucociliary clearance (MCC) can lead to lifethreatening airway narrowing and lung infections, and is a major cause of morbidity
and mortality in patients with cystic fibrosis, primary ciliary dyskinesia and chronic
obstructive lung disease. However, no tool for microscopic in-vivo visualization of
ciliary function is currently available, limiting studies of disease pathogenesis, refined
diagnosis and phenotyping, and the development of novel therapeutics.
In this thesis, a novel, 1-pm resolution, optical interferometric imaging technique
termed Micro-OCT was incorporated into miniaturized common-path endoscopes and
mucociliary transport was visualized in vivo for the first time.
The first-generation Micro-OCT probe had a rigid design with outer diameter
of 4 mm and a two-prism configuration providing beam splitting and sample beam
shaping into an annular profile. Image quality of the probe allowed visualization of
the periodic pattern of ciliary beating, measurement of airway surface liquid depth
(ASL) and visualization of mucociliary transport. Unaltered ciliary function was
demonstrated in a living, spontaneously breathing swine model. Newer generation
common-path endoscope designs were demonstrated that improve, among other limitations, the stability of the reference reflector position and provide greater potential
for miniaturization.
The presented work opens unprecedented avenues for studying MCC and the
effect of novel therapeutics within the complexity of a living organism. Further, it
lays the groundwork for the development of a human probe with the potential to
revolutionize diagnosis, phenotyping, and therapy management for all patients with
respiratory disease involving the mucociliary escalator.
Thesis Supervisor: Guillermo J. Tearney, MD, PhD
Title: Professor of Pathology, Harvard Medical School
3
4
Acknowledgments
First and foremost I would like to thank my advisor Prof.
Gary Tearney for the
opportunity to work in his unique lab and for his continuous and generous support,
which has enabled me to grow as a researcher and to pursue the work that I was
passionate about. Not only have I found my calling as a translational scientist here,
but also a second family and I would like to express my sincerest thank you to everyone
I have had the chance to work and interact with.
I am deeply indebted to Dr. Ken Chu for his most generous support. It was
a true honor to work with Ken and his patient teaching, immense knowledge, and
great sense of humor really enabled this thesis. I would also like to thank the other
members of the Micro-OCT pulmonary group, Drs. Tim Ford and Kanwarpal Singh,
for their teaching and support and for making the animal studies and lunch breaks
most enjoyable. A critical work load of this project was achieved in collaboration with
our fantastic engineering team. I would like to wholeheartedly thank Rob Carruth
and Weina Lu for sharing their skills and for making the impossible possible on a
regular basis.
I could not be more grateful to have had the unique chance to work with and
learn from Dr. Mireille Rosenberg and her clinical team. I owe Mireille everything
I know about translational science and the regulatory landscape, and I am tremendously grateful for her mentorship, and career and life guidance. I would also like
to particularly thank Drs. Michalina Gora and Aubrey Tiernan, who have been a
critical help with understanding and advancing in the medical device design process.
Absolutely critical to the success of my work in the lab and of this thesis were our
assistants Valerie Madden and Princess Cruz. Val, my sincerest thank you for your
smile and patience during all these years of scheduling and rescheduling meetings.
Your organizational superpowers and personal warmth made the whole difference!
Many, many others in the lab also made a big difference in my daily work and
life and every contribution was absolutely essential. To just name a few: Dr. Mohini
Lutchman continuously offered an open ear and generous treats to make life sweeter.
5
And I consider myself fortunate to have had the chance to be an advisor and mentor
to Diana Mojahed, whose enthusiasm and scientific talent are truly enjoyable.
In addition to the lab, I have had the chance to receive invaluable support from
my department and fellow students. I would like to thank the HST faculty and HST
academic office and in particular Profs. Elazer Edelman, Elfar Adalsteinsson, Martha
Gray, Brett Brouma, and Julie Greenberg, as well as Laurie Ward, Traci Anderson,
and Joe Stein. I have been continuously impressed by their open ears, helpful advice,
and strong support.
Last, but not least, I would like to express my sincerest thanks to the funding
sources that have enabled my work and study, namely a generous gift from Air Liquide, a 9-month fellowship from HST, RA support from Prof. Gary Tearney and the
NIH, and the SPIE Optics and Photonics Education Scholarship.
6
Contents
1
15
Introduction
21
2 Background
Mucociliary clearance . . . . . . . . . . . . . . . . . . . . . . . . . .
.
2.1
2.1.1
Spatial and temporal requirements for visualization of mucocil.
iary function . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1
Primary ciliary dyskinesia
24
2.2.2
Cystic fibrosis . . . . . . . .
26
2.2.3
Chronic obstructive pulmonary disea se . . . .
27
Current methods characterizing ciliary func tion . . .
29
.
.
29
2.3.2
Fluorescence confocal microscopy
29
2.3.3
High-speed video microscopy . . .
30
2.3.4
Radioaerosol clearance . . . . . .
30
2.3.5
Micro-OCT . . . . . . . . . . . .
30
2.3.6
Summary
. . . . . . . . . . . . .
31
.
31
Micro-Optical Coherence Tomography
2.4.1
.
.
.
.
.
Electron microscopy
.
. . . . . . .
2.3.1
Introduction to Optical Coherence Tomography (OCT) and
Micro-OCT . . . . . . . . . . . .
31
2.4.2
Axial resolution . . . . . . . . . .
33
2.4.3
Tranverse resolution versus depth of focus in OCT
.
7
. . . . .
.
2.4
24
.
2.3
23
Diseases of mucociliary clearance
.
2.2
22
34
Annular sample beam and aperture improve both lateral resolution and depth of focus in Micro-OCT
2.4.5
. . . . . . . . . . . .
35
Design requirements for a miniaturized Micro-OCT probe to
visualize MCC in vivo
. . . . . . . . . . . . . . . . . . . . . .
37
3 Miniaturized Micro-OCT probe for real-time visualization of mu41
3.1
M otivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
41
3.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
.
.
cociliary function in vivo
3.2.1
Common-path optical probe design with annular sample beam
profile
3.2.2
Probe scanning . . . . . . . . . . . . . . . . . . . . . . . . .
43
3.2.3
Outer housing design characteristics . . . . . . . . . . . . . .
43
3.2.4
Fabrication of prototype . . . . . . . . . . . . . . . . . . . .
44
3.2.5
Experimental design of animal study . . . . . . . . . . . . .
45
3.2.6
Data processing . . . . . . . . . . . . . . . . . . . . . . . . .
46
R esults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
46
3.3.1
Miniaturized rigid probe . . . . . . . . . . . . . . . . . . . .
46
3.3.2
Lessons from the animal study . . . . . . . . . . . . . . . . .
47
3.3.3
Images of MCC in swine trachea
48
.
.
.
.
.
.
.
.
.
42
. . . . . . . . . . . . . . .
.
3.3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D iscussion . . . . . . . . . . . . . . . . . . . .
48
3.5
C onclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
.
3.4
57
4.1
M otivation . . . . . . . . . . . . . . . . . . . . .
57
4.2
M ethods . . . . . . . . . . . . . . . . . . . . . .
58
4.2.1
Integrated reference reflector design . . .
58
4.2.2
Semi-flexible probe design . . . . . . . .
59
4.2.3
Improvements to the outer housing . . .
59
4.2.4
Animal study . . . . . . . . . . . . . . .
60
Results . . . . . . . . . . . . . . . . . . . . . . .
60
4.3
.
.
.
.
.
.
4 Novel common-path Micro-OCT probe designs
.
............
2.4.4
8
5
4.3.1
Second-generation Micro-OCT probe . . . . . . . . . . . . . .
60
4.3.2
First image of tracheal epithelium in living swine model . . . .
61
4.4
Limitations and future work . . . . . . . . . . . . . . . . . . . . . . .
61
4.5
Summary
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
67
Summary and Outlook
5.1
Thesis summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
68
5.2
N ext steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
69
5.3
Im pact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71
9
10
List of Figures
1-1
Schematic illustrating common techniques for studying mucociliary
clearance and the advantages of Micro-OCT. . . . . . . . . . . . . . .
17
1-2
Thesis summary schematic and outlook.
19
2-1
Qualitative comparison of axial intensities from the focus for a circular
. . . . . . . . . . . . . . . .
and annular apertures of different inner diameters. . . . . . . . . . . .
2-2
Qualitative comparison of radial intensities at the focus for a circular
and annular apertures of different inner diameters. . . . . . . . . . . .
3-1
. . . . . . . . . . . . . . . . . . . . . . . .
52
Schematic illustration of main assembly steps of Micro-OCT optical
probe subassembly. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3-3
37
Schematic and pictures of common-path configuration and components
of first Micro-OCT probe.
3-2
36
53
Picture of a bronchoscopic image during balloon inflation for stabilization of the Micro-OCT probe during image acquisition with respect to
the tissue region of interest.
3-4
. . . . . . . . . . . . . . . . . . . . . . .
Picture of the optical probe subsystem, which is the scanning portion
of the probe, and the donut-shaped beam profile.
3-5
. . . . . . . . . . .
54
Image of mucociliary clearance in freshly excised swine trachea obtained with first-generation Micro-OCT probe. . . . . . . . . . . . . .
3-6
54
55
Image of mucociliary clearance in vivo obtained with first-generation
Micro-OCT probe in swine trachea. . . . . . . . . . . . . . . . . . . .
11
55
4-1
Schematic and pictures of common-path configuration and components
of second-generation Micro-OCT probe. . . . . . . . . . . . . . . . . .
4-2
64
Schematic illustrating the knife edge test for evaluation of transverse
resolution achieved with second generation Micro-OCT probe. ....
65
4-3
First in-vivo image obtained with second-generation Micro-OCT probe. 65
4-4
Schematic illustrating the three generations of Micro-OCT probe designs. 66
12
List of Tables
2.1
Design requirements of an imaging technique for visualization of mucociliary function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2
Comparison of governing parameters of spatial resolution and depth of
focus in confocal microscopy, OCT, and Micro-OCT.
2.3
23
. . . . . . . . .
35
Design requirements for a miniaturized Micro-OCT probe to visualize
mucociliary function in living swine trachea. . . . . . . . . . . . . . .
13
39
14
Chapter 1
Introduction
The health of the human respiratory system depends critically on airway clearance via
motile hair-like structures (cilia), which transport unwanted particles and infectious
agents trapped within secreted mucus so that they can be swallowed or expectorated.
Impairment of mucociliary clearance (MCC) can lead to life-threatening airway narrowing and lung infections, which are the primary cause of morbidity and mortality
in patients with cystic fibrosis (CF) or primary ciliary dyskinesia (PCD) and potentially other diseases such as chronic obstructive pulmonary disease as well as in
patients with acute infections [1, 2]. Great differences exist in patient presentation
and likely in the exact defect and disease progression of any given patient, but no tool
currently exists to evaluate mucociliary function on the ciliary level in a given patient
or even in vivo. Diagnosing the precise mucociliary defect can therefore be difficult
and even once a diagnosis has been made, the therapeutic management is often not
targeted, but comparably uniform due to limited phenotyping and therapeutic options. In addition, researching defects of mucociliary function in animal models and
the development of novel therapies is hindered by the inability to visualize MCC and
the physiologic cause-effect relationships in these models in vivo.
A tool that could visualize the micro-structure and function of MCC in vivo and
in real-time could therefore significantly increase our understanding of disease pathogenesis and accelerate the development of new therapies, for example, in combination
with the recently developed CF pig. In the clinic, such a tool could provide completely
15
new venues for diagnosis, phenotyping, and personalized therapeutic management.
Currently available techniques to study MCC, however, are all limited to investigating excised tissue or cells, and typically provide only a single parameter of interest
instead of a comprehensive picture of the interaction between mucus properties and
ciliary function. Key parameters of mucociliary function include the periciliary layer
(PCL), the airway surface liquid (ASL) depth, which is the combined PCL and mucus
layer, ciliary beat frequency (CBF) and mucociliary transport (MCT) rate. Those
main techniques, illustrated in Figure 1-1, include:
1. Electron microscopy, which can illuminate ciliary ultrastructure, periciliary, and
airway surface liquid depth (PCL and ASL), but is limited to providing static
images of processed cells and tissue [3, 4, 5].
2. Fluorescence confocal microscopy, which also provides static images and measures of PCL and ASL in excised tissue and cell cultures [5].
3. High-speed videomicroscopy, which allows dynamic, qualitative assessment of
ciliary beating patterns and quantitative assessment of ciliary beat frequency
(CBF), but is also limited to analyzing excised tissue specimen or cell cultures
[6, 7].
4. Radioaerosol clearance [8] and CT particle tracking [9], which provide information about the global efficiency and localized pattern of mucociliary transport
(MCT), respectively, but require exogenous contrast and do not illuminate the
mechanism itself or further correlate MCT to mucus properties.
Micro-OCT [10] is a novel, 1- tim resolution optical imaging technique, which was
recently developed in our lab and is capable of simultaneous visualization of several
key parameters governing the function of the respiratory epithelium including PCL,
ASL, CBF and MCT [11, 12]. Thus, Micro-OCT is unique as it provides intrinsic
registration in time and location of those parameters. Even though Micro-OCT can
in principle be utilized in vivo, all studies with this technology to date have been
limited to imaging excised tissue and cultured cells on a bench-top system.
16
1. Electron
microscopy
2. Confocal
microscopy
3. Video
microscopy
<KV
Excised tissue
or
Cell cultures
4. Radioaerosol
clearance test
CT particle tracking
&
Alm
Nasal brushing
or
Biopsy
X
-N
0- ASL
1 PCL
10 ASL
0 PCL
5. MicroOCT
P Ciliary
beat pattern
P CBF
l MCT
D ASL
P PCL
o Ciliary
beat pattern
o CBF
o Local MCT
Figure 1-1: Schematic illustrating common techniques for studying mucociliary clearance
and the advantages of Micro-OCT. Current techniques for studying ciliary function are
limited to the study of cell cultures or excised tissue and most provide only a few parameters
of ciliary behavior each. Micro-OCT is capable of providing several key parameters of
ciliary function for a more comprehensive assessment of ciliary function. Example images
illustrating electron microscopy [5], fluorescence confocal microscopy [5], video microscopy
[6], radioaersol clearance [8], CT particle tracking [9], and Micro-OCT [11] are reproduced
from the respective references.
The goal of this thesis project was to build a miniaturized Micro-OCT probe and
to demonstrate visualization of MCC in vivo.
A 4-mm outer diameter (OD), 50-
cm length rigid probe was built and ciliary activity and mucociliary transport were
visualized for the first time within a living swine trachea.
The swine model was
chosen for the resemblance of its pulmonary anatomy to humans and to establish the
technique as a, novel research tool in combination with the CF pig.
A second objective of this thesis work was to lay the groundwork for the development of a clinical tool for two different applications: First, we envision to use our rigid
17
probe design for assessment of MCC in the human nasal cavity. Used similarly to currently used procedures, such as nasal brushings, access to the respiratory epithelium
of the nasal cavity could provide insights into mucociliary function non-invasively and
allow real-time assessment of a therapeutic response. Therefore, the probe for in-vivo
use has to be stable, safe, and small enough to be comfortable. Secondly, we hope
to extend our reach for assessment of MCC in lower airway generations. Therefore,
the probe needs to be flexible and small enough to be inserted through the accessory
channel of a commonly used bronchoscope.
The scope of this thesis therefore encompasses co-development and fabrication of
the first generation Micro-OCT probe, an experimental setup for in-vivo imaging of
MCC, and development of a second-generation semi-flexible Micro-OCT probe design
with improved stability and potential for further miniaturization.
Figure 1-2 summarizes the project schematically.
Chapter 2 provides a brief background on the clinical motivation, current state of
the art in research methods of mucociliary clearance, an introduction to Micro-OCT,
and design requirements for a miniaturized probe. The design and fabrication of the
first-generation Micro-OCT endoscopes is explained in Chapter 3 and images obtained
with the probe in swine trachea ex vivo and in vivo are shown. Limitations of the
first-generation probe design are discussed. Chapter 4 illustrates improved designs
incorporated into second- and third-generation probes as the groundwork for human
studies and assessment of lower airway generations. Finally, Chapter 5 summarizes
the work conducted to date and presents an outlook for future technology development
and clinical investigations.
18
*
PC
Benchtop setup
A
Achieved Milestones
cipp
-
Co-development and
l Fabrication of miniaturized,
hand-held Micro-OCT probe
4N
EC
---
--
WI4
l Real-time visualization of
MCC in living swine
~IZIZThLI1
TZI
Outlook
Novel probe designs:
- semi-flexible
- improved reference reflector stability
- potential for further miniaturization
4
lo Navigation into smaller airways within accessory channel of
bronchoscope
N Non-invasive assessment of MCC in human nasal cavity
Figure 1-2: Thesis summary schematic and outlook: A miniaturized, hand-held, commonpath Micro-OCT probe was built that incorporates beam splitting, shaping, and focusing as
well as scanning functionality. The probe was demonstrated for visualization of mucociliary
clearance (MCC) in a swine model in vivo. Lastly, novel probe designs were developed and
tested with improved stability, flexibility, and potential for further miniaturization. The
achieved milestones lay the groundwork for further studies in the lower airways as well as
in the human nasal cavity. MCC: Mucociliary clearance.
19
20
Chapter 2
Background
The importance of mucociliary clearance (MCC) as a defense mechanism of the lung
is easily understood by noting the life expectancy of cystic fibrosis patients with
severely impaired MCC, which remains 30 or 40 years even with the most current
therapies. Many other diseases are associated with impaired MCC as well, such as
primary ciliary dyskinesia or COPD, a diagnosis with very high prevalence that affects
an estimated 24 million people in the US alone . Current methods of studying MCC,
however, are limited to the assessment of few isolated parameters each and to the
evaluation of ex-vivo tissue and cells, which hinders effective research into the pathogenesis of the disease and the development of new therapies, and targeted therapeutic
management based on clinical phenotype. Addressing the first limitation, our lab has
recently developed a novel imaging technique, termed Micro-OCT, for improved and
comprehensive characterization of MCC ex vivo. This thesis describes the integration
of the Micro-OCT imaging technology into miniaturized imaging probes and the first
visualization of MCC in large animal airways in vivo.
This chapter outlines the background and motivation of this thesis work and
summarizes: MCC and associated diseases, currently available techniques and their
limitations, an introduction into Micro-OCT, and design requirements for the in-vivo
probe.
21
2.1
Mucociliary clearance
The mucociliary escalator is an important defense mechanisms of the lung and especially critical for clearing the smaller airways, where coughing is less effective than in
the first few airway generations. It is comprised of motile hair-like structures (cilia),
which transport unwanted particles and infectious agents trapped within secreted
mucus so that they can be swallowed or expectorated. About 80% of the respiratory,
pseudostratified columnar epithelium are ciliated cells (with the remaining 20% being
goblet cells that produce mucus).
Each ciliated cell has about 200 cilia to propel mucus and each cilium has a length
of 5 to 7 Am in the trachea and 2 to 3 1 am in the seventh airway generation, at a
diameter of 0.25 to 0.33 pm [2]. The cilia are surrounded by the so-called periciliary
liquid (PCL), which is just about as thick as the length of the cilia and allows them
to beat effectively. The thickness of the mucus layer can vary significantly in health
and disease and is generally given as being on the order of 2 to 10 Am for healthy
airways [13]. Together, the PCL and mucus layer thickness are often described and
measured as the airway surface liquid depth (ASL).
The ciliary stroke is comprised of two phases: the effective stroke, which propels
the mucus and the recovery stroke, where the cilia return to their initial position by
almost bending to their side. The frequency of one full stroke is a commonly measured
parameter and named ciliary beat frequency (CBF). The CBF is on the order of 4 to
12 Hz in healthy humans. Patients with PCD have generally lower CBF, but higher
frequencies have also been observed depending on the genetic mutation. Except for
one mutation, where the maximum CBF value was found to be almost 25 HZ, other
maxima were all below 13Hz [14]. CBF in other mammalian species, including the
pig, were slightly higher and found to be in the range of 11 to 17 Hz at different
locations within the respiratory tract [15].
22
2.1.1
Spatial and temporal requirements for visualization of
mucociliary function
A cross-sectional imaging technology is the most advantageous for visualizing and
measuring PCL and ASL as well as small changes in both disease and therapy. The
technology should also provide best possible axial resolution, ideally better than 2
pm.
Further, in order to resolve the complex ciliary beating pattern of effective
stroke and recovery stroke, the lateral resolution should be finer than half the length
of the cilium, at around 2 to 3 pm in the upper airways and as low as 1 pm in the 7th
airway generation. Using Nyquist sampling as a metric for optimal data acquisition
rates, in order to correctly quantify CBF, a frame rate above 34 Hz is sufficient for
most cases and a frame rate above 50 Hz should capture all possible cases.
Table 2.1 summarizes the design requirements of an imaging technique to visualize
key parameters of mucociliary function.
Table 2.1: Design requirements of an imaging technique for visualization of mucociliary
function.
Parameter of Interest
Size/Frequency
Design Requirement
5 to 7 /Lm (trachea) [2]
Spatial resolution < 2.5 pim
2 to 3 ,um (7th generation)
Spatial resolution < 1 pm
~ Ciliary length
Axial resolution < 2.5/1 pm
2 to 10 pum
Axial resolution < 1 pm
= PCL + Mucus layer
Axial resolution < 2 pm
4 to 12 Hz (human)
Frame rate > 24 Hz
11 to 17 Hz (swine)
Frame rate > 34 Hz
Ciliary length
PCL
Mucus layer
ASL
CBF
23
2.2
Diseases of mucociliary clearance
The classical diseases of mucociliary clearance (MCC) are primary ciliary dyskinesia
(PCD) and cystic fibrosis (CF). However, MCC is also impaired in many others,
such as chronic obstructive pulmonary disease (COPD), acute infections, or following
exposure to hairspray and cigarette smoke [2]. Further, patients within each disease
category present with a wide variety of symptoms and impairment in daily activities,
which seems in contrast to the typically uniform therapeutic management.
This
discrepancy indicates that:
* the pathogenesis of disease is insufficiently understood and patients are insufficiently phenotyped,
e currently available therapies are limited and of limited efficiency, and
* where therapies exist, their exact mechanism remains unclear.
For example, only recently has there been evidence that different antibiotics, a cornerstone of the therapeutic management for many of these patient may influence ciliary
function in different ways and is dependent on the patient's characteristics [16, 17].
An imaging tool that can visualize mucociliary function in vivo and in real time
therefore has the potential to increase our understanding of pathogenesis, to allow
better phenotyping of patients, to provide novel insights into the effect of therapeutic
agents for accelerated development of new therapies and personalized therapeutic
management.
2.2.1
Primary ciliary dyskinesia
Primary ciliary dyskinesia (PCD) is a rare genetic (autosomal recessive) disease characterized by immotile or inefficiently functioning cilia. A typical patient may suffer
from neonatal respiratory distress in the first days of his life and go on to having recurrent respiratory and ear infections leading to bronchiectasis and progressive decline of
lung function. Males are often infertile and females subfertile with an increased risk
24
of ectopic pregnancies. Other associated conditions are situs inversus, polysplenia,
congenital heart defects, and hydrocephalus. While the disease progression cannot
be stopped today, an early diagnosis is the best predictor for sustained lung function
and therefore crucial to the disease management [18, 19].
However, diagnosing PCD is difficult due to the wide variety of symptomatic presentations, likely resulting from a wide variety of structural and functional defects
underlying the disease [20, 21]. Today, a definitive diagnosis can be made on the
basis of genetic testing, which is still at an early stage and does not yet provide sufficient information on phenotype. The most important diagnostics remain electron
microscopy, ciliary beat pattern and frequency analysis using high-speed video microscopy [6, 22, 19]. Slow CBF raises suspicion for PCD, but normal CBF does not
preclude a diagnosis of PCD. Electron microscopy may reveal a lack of dynein arms
indicating a diagnosis of PCD, but many sections need to be looked at, subtle defects
may be missed, and the patient can still suffer from inefficient MCC in spite of normal
ultrastructure. Further, all of these techniques are limited to excised tissue or cell
samples and therapeutic effects cannot be monitored in real time. Other methods for
diagnosing PCD are therefore intensely studied such as measuring the levels of nitric
oxide produced by the respiratory epithelium [22]. While all methods in addition to
the patients medical history are helpful in establishing a diagnosis, none elucidate the
exact functional defect on a ciliary level in an individual patient. As a result, PCD is
believed to be largely underdiagnosed, diagnosed too late, and as a result, the disease
is undertreated [23].
In addition, there are currently no therapies specifically targeted at PCD or at
the individual defect with which a patient may present [1].
Current therapeutic
options are typically extrapolated from other diseases, such as CF, and are limited to
antibiotics, chest physical therapy, exercise, mucolytics, immunisation, and reduction
of exposure to cigarette smoke and environmental pollutants. The effects of some
therapies, such as nebulised rhDNase, normal or hypertonic saline, and prophylactic
antibiotics remain controversial [19].
A method to visualize ciliary microfunction non-invasively and in real-time such as
25
developed in this thesis could therefore contribute to better diagnosis, phenotyping,
and therapeutic management of patients with PCD.
2.2.2
Cystic fibrosis
Cystic Fibrosis (CF) is an autosomal recessive genetic disease currently affecting
about 30, 000 people in the US. It is caused by mutations of an anion transporter of
chloride and bicarbonate named cystic fibrosis transmembrane conductance regulator
(CFTR). This ion transport defect most severely affects the respiratory and digestive
system and results in thick, sticky mucus build-up in the lungs and obstruction of the
pancreas. A patient with CF is often diagnosed shortly after birth or within the first
few years of life. At birth, he/she may for example be unable to pass meconium and
require surgical removal thereof. Later, he/she is likely to present with shortness of
breath, recurrent lung infections or a failure to grow. The compromised respiratory
system is typically the reason for major morbidity and early mortality in CF patients.
Most US patients are now diagnosed at birth if they present with meconium ileum, or
through a newborn screening including a sweat test and genetic test. None of these
diagnostic criteria, however, allow precise assessment of the MCC defect at the ciliary
and mucus level, which in turn could help guide a finer phenotyping of patients for
improved therapeutic management.
Similarly, monitoring options throughout life are limited to indirect and unspecific
measures, such as lung function (spirometry) and oxygen saturation tests, which do
not provide information about the mechanistic defect necessary for optimal therapeutic management - with the exception of microbiologic surveillance of respiratory
secretions enabling targeted and if needed chronic antibiotic treatment.
In addition to unsatisfactory phenotyping and monitoring of CF patients, current
therapeutic options are insufficient. Most therapies do not treat the underlying cause
of CF, such as is the case for chest physical therapy, inhaled mucolytics and/or hypertonic saline or are effective in only a minority of patients, as is the case for the
recently developed CFTR potentiator named ivacaftor (brand name Kalydeco). Ivacaftor rescues some of the functionality of a defective CFTR if the CFTR is present
26
at the cell surface. This is the case for only about
5% of CF patients with very
specific mutations.
Thus, optimal CF diagnosis and monitoring as well as therapeutic management
and the development of novel therapies is hindered by the lack of a tool that allows
assessment of mucociliary clearance and any therapeutic effects on the level of ciliary
micro-anatomy and function in vivo.
Further, such a tool would provide new avenues for studying CF pathogenesis and
novel therapeutics in animal models and humans and could be particularly valuable
to assess the early pathogenesis before secondary changes due to lung infections occur.
A method to visualize ciliary micro-function non-invasively and in real-time has
therefore the potential to significantly increase our understanding of CF pathogenesis
- in particular if it can be adapted to the study of the lower airways of young animal models and small children, and would be uniquely well suited to quantitatively
assess the therapeutic effect of any new compound in a given patient or patient population for accelerated development of new therapies and personalized therapeutic
management.
2.2.3
Chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD) is characterized by a fixed (nonepisodic and irreversible despite bronchodilator use) airflow limitation, currently affecting about 6 % of the US population.
COPD usually develops as a result of
cumulative exposure to direct or indirect cigarette smoke, occupational dust or chemicals, and air pollution, being additionally influenced by genetic effects. The diagnosis
of COPD is currently based on a patient's medical history and lung function tests,
which do not provide any insights into a specific pathogenesis and are only slightly
correlated with the impact of the disease for the patient, which in turn is predictive of
mortality. Further, the current literature distinguishes in general between emphysema
and chronic bronchitis as the primary, not-mutually exclusive, causes for the symptoms in these patients - a distinction without predictive value and little consequence
for therapeutic management. Thus, COPD is an umbrella diagnosis encompassing
27
very different patients with varying pathogenesis. A patient with COPD is typically
beyond his/her 40s complaining about shortness of breath during daily activities such
as climbing stairs or holding a shower head above his/her head. The patient may be
hypoxemic and present with cough, productive sputum, and distinct breath sounds,
or at the other extreme may have normal blood gases at rest, no cough, and only
distant breath sounds with possibly an overinflated, "barrel" chest.
Despite large differences in patient presentation, state-of-the-art therapeutic management is fairly uniform and includes smoking cessation, bronchodilators, inhaled or
oral corticosteroids, and supplemental oxygen in severely hypoxemic patients. The
mismatch between the complexity of the disease and the uniformity of therapy is suboptimal. Better phenotyping of patients and novel therapies are needed to decrease
morbidity and mortality in COPD. For example, it has recently been hypothesized
that COPD patients associated with chronic bronchitis may have an acquired CFTR
dysfunction caused by cigarette exposure leading to reduced ASL, increased mucus
expression, and reduced MCT and thus could benefit from a CFTR potentiator such
as ivacaftor [24]. So far, studies to assess the efficacy of ivacaftor for COPD have only
been performed in cell cultures.
A method to visualize and quantify ASL and MCT non-invasively, over time and
in real-time in a given patient could then provide new insights into individual disease
pathogenesis, allow refined patient selection prior to administration of ivacaftor or any
novel therapy targeting mucus stasis in COPD, and illustrate the functional efficacy
of such therapy at the ciliary and mucus level.
Such an imaging advance could
therefore contribute to better phenotyping and therapeutic management of patients
with COPD.
28
2.3
Current methods characterizing ciliary function
Current techniques for studying mucociliary clearance are all limited to studying
excised tissue or cells and most provide only a few parameters of interest each. (Figure
1-1 illustrates the main techniques schematically.)
2.3.1
Electron microscopy
Electron microscopy is indispensible in the study of ciliary ultrastructure [3, 4] and
remains the "gold" standard for diagnosing PCD. However, it is limited to providing
static images and prone to tissue processing artifacts. It may therefore provide measures of ASL and PCL within the limits of these artifacts, but is unsuitable for the
evaluation of dynamic ciliary function.
2.3.2
Fluorescence confocal microscopy
Fluorescence confocal microscopy has been used previously to measure PCL and ASL
in cell cultures [5]. Due to the point-by-point sampling of the technique, however, it
would be technically challenging to provide real-time visualization of ciliary function
within a miniaturized endoscope. Further, the fluorescent dye remains an exogenous
contrast and it is unclear how its introduction affects the native fluids. Newer generations of regular confocal microscopy, such as the high-speed technique spectrally
encoded confocal microscopy (SECM) [25], have recently been demonstrated within
human imaging probes by our group, and could potentially provide sufficient contrast for the visualization of respiratory cilia. However, at this time, even the best
compromise between probe size, depth of field, spatial and temporal resolution for
SECM would be inferior to the high, isotropic resolution and cross-sectional imaging
capabilities of Micro-OCT.
29
2.3.3
High-speed video microscopy
High-speed video microscopy (HSVM) is the current gold standard for the evaluation
of ciliary function. Both qualitative parameters, such as ciliary beating pattern and
coordination can be studied, as well the quantitative ciliary beat frequency (CBF)
[6, 7].
However, videomicroscopy does not allow optical depth sectioning and is
therefore not suitable to study mucociliary clearance in vivo. This technique therefore
remains limited to analyzing excised tissue specimen or cell cultures.
2.3.4
Radioaerosol clearance
The radioaerosol clearance technique requires the research subject to inhale a radioactively labeled aerosol, the clearance of which is then observed over the following
hours. It can therefore be used to investigate the efficiency of mucociliary transport
(MCT) but due its global character, cannot further illuminate the mechanism itself
[8]. In a similar technique, the saccharin test, a saccharin tablet is placed in the nasal
cavity of the subject and the time is recorded until the subject reports its sweet taste.
Subject to the same limitations as the radioaerosol clearance test, the saccharin test is
in addition prone to artifacts due to the patients behavior, therefore even less suitable
to studying mucociliary function and should be considered obsolete in the diagnosis
of PCD [22].
2.3.5
Micro-OCT
Micro-OCT is the only currently available technique to meet all of the design requirements of an imaging technique for the evaluation of MCC as summarized in table 2.1.
It has been shown to provide co-localized, simultaneous, quantitative characterization
of ASL, PCL, CBF, MCT and the full ciliary stroke pattern [11] within cell cultures
and excised swine trachea using a bench-top system.
30
2.3.6
Summary
Commonly used techniques to study mucociliary function are incapable of measuring
four key features of MCC (ASL, PCL, CBF, MCT) or ciliary beating patterns within
a same test or reasonable correlation between several necessary tests. Micro-OCT is
a novel technique and overcomes this limitation. However, all previous Micro-OCT
applications have been limited to studying excised tissue or cell cultures.
2.4
Micro-Optical Coherence Tomography
Noninvasive imaging techniques such as X-ray computed tomography (CT), magnetic
resonance tomography (MRI), and ultrasound are not capable of directly visualizing
ciliary motion and mucociliary microfunction due to their relatively coarse spatial
resolution (100 pm - 1mm). Optical reflectance techniques can provide spatial resolution on the order of 1 - 10 pm and do not rely on transmission of the signal through
the sample, but detect the backscattered light, such that their spatial resolution and
its decay with increasing penetration depth in the tissue is independent of the sample
size. Micro-Optical Coherence Tomography (Micro-OCT) is a novel high-resolution
optical reflectance technique capable to meet the previously discussed requirements
of spatial (<2pm) and temporal (>40 fps) resolutions for visualization of mucociliary function as demonstrated recently on a bench-top system [11]. The following
paragraphs give some background on the technique, which is based on conventional
(spectral-domain) OCT.
2.4.1
Introduction to Optical Coherence Tomography (OCT)
and Micro-OCT
Optical Coherence Tomography (OCT) [26] is a high-speed cross-sectional optical
imaging technique that can relatively easily be incorporated into miniaturized probes
and is therefore uniquely well suited for in-vivo imaging of tissue microstructure or
"optical biopsy" [27] without the use of contrast agents or ionizing radiation. Today,
31
OCT is in wide-spread clinical use in opthalmology and is gaining popularity in the
evaluation of coronary artery disease, gastrointestinal pathologies, and maladies of
the respiratory tree [27].
OCT can be considered the optical analogue of ultrasound, as it measures the
echo time delays of backscattered light at changes in refractive index within the
tissue. The consequence of using light instead of sound is that OCT can provide
significantly better spatial resolution (on the order of 10
[tm)
and imaging speed
(> 100 fps) compared to ultrasound at the cost of lower penetration depth (< 2
mm in scattering tissue).
Because the frequency of light (> 200 THz for visible
and near-infrared light) is beyond the limit of electronic detection, OCT employs an
interferometric technique to detect the echo time delay between the various reflecting
and backscattering structures within the sample [27]. OCT acquires a 1-dimensional
image (A-line) along the entire imaging depth with each analysis of the interference
signal and mapping of the intensity of backscattered light from the different reflecting
and backscattering structures along the axis of illumination onto a grey- or colorscale to create the image. In order to obtain a two- or three-dimensional image, the
OCT beam needs to be scanned in the lateral directions - for example in a raster
scan pattern along perpendicular lateral dimensions or in a spiral pattern within
a luminal organ. Different OCT technologies can be further specified according to
employed strategies for detecting and processing the interference signal. In spectraldomain OCT as used in this thesis, a broadband light source illuminates the sample
and the entire spectral interference signal is acquired simultaneously, as a function of
wavelength using a spectrometer. Rescaling the spectrometer output from wavelength
to wave number and then Fourier transforming the interference signal provides the
depth location of the scattering events [27, 28].
In order to visualize ciliary microstructure, the spatial resolution of typical OCT
systems (on the order of 10 pm) is, however, insufficient.
In these systems, the
transverse resolution is intentionally kept low to provide sufficient depth of focus and
enable fast, cross-sectional imaging. Similarly, the axial resolution may be kept low
in favor of imaging speed and/or cost and availability of detectors and light sources.
32
Other optical reflectance techniques, such as confocal microscopy, which could provide
finer spatial resolution (often on the order of 2 pm for in-vivo imaging systems) are
not suitable either, in particular for in-vivo imaging. Their lateral resolution and
optical depth sectioning capabilities are coupled via the numerical aperture of the
lens and thus, they typically require point scanning in the depth direction, which is
technically challenging, comparably unstable, and time-consuming.
Micro-OCT combines the advantages of OCT (speed, cross-sectional imaging, decoupling of lateral and axial resolution) with an order of magnitude higher spatial
resolution than conventional OCT systems. Micro-OCT differs from most conventional OCT systems primarily by 1) using a particularly large bandwidth source for
higher axial resolution, 2) using a high numerical aperture (NA) objective lens for
higher lateral resolution, and 3) shaping of the imaging beam and aperture to an
annular profile that provides a usable, extended depth of focus in addition to the
high lateral resolution. The following paragraphs describe these differences in more
detail and Table 2.2 summarizes qualitatively the different factors governing axial
and transverse resolution as well as depth of focus in confocal microscopy, OCT, and
Micro-OCT.
2.4.2
Axial resolution
The axial resolution in OCT is primarily governed by coherence gating and is proportional to the ratio of the center wavelength A, to the bandwidth A A of the sourcedetector combination, A
(equation 2.1 [29], derived for a Gaussian source spectrum).
Thus, to improve axial resolution, one can image at shorter wavelengths and increase
the bandwidth of the system. The current implementation of our Micro-OCT system
operates within the visible to near-infrared spectrum with particularly large bandwidth, namely in the approximate wavelength ranges from 650 to 950 nm (FWHM
of Gaussian spectral envelope), such that A, = 800 nm and AA = 300 nm. The theoretical diffraction-limited axial resolution in air (n=1) according to equation 2.1 [29]
33
is therefore 0.9 pm. 1
6z =
2.4.3
21n2 A 2
7r
C
nAA
(2.1)
Tranverse resolution versus depth of focus in OCT
The transverse resolution in OCT is, similar to confocal microscopy, primarily governed by the numerical aperture (NA) of the imaging lens and is proportional to
the ratio n.
A large numerical aperture allows focusing of the beam to a smaller
spot and collecting backscattered light from smaller structures (increased spatial frequency) and therefore improves resolution. Depth of focus, however, decreases simultaneously with increasing diameter of the numerical aperture and is proportional to
the ratio N.
Since typical OCT systems acquire the one-dimensional depth image
without scanning in depth, they require the depth of focus to be at least the desired
imaging depth.2 Therefore, even if a desired lateral resolution can be obtained, the
resulting depth of focus may be insufficient.
For example, in order to achieve a theoretical, diffraction limited 2 pm lateral
resolution at A, = 800 nm using a fiber-based illumination/collection OCT probe, a
NA on the order of 0.18 would be required according to equation 2.2 [31, 28]. The
resulting depth of focus at this wavelength and NA, however, would only be on the
order of 16 pm according to equation 2.3 [32, 28]. Although on the order of the
combined height of the ciliary (7 pm) and mucus layer (10 pm), such a depth of focus
would be insufficient: the epithelial cell bodies could not appear in focus at the same
time, the short axial range would be a major challenge for any probe design, and there
would be no margin to accommodate tissue irregularities or the presence of cardiac,
respiratory, and operator motion.
1Note that in addition to the coherence effect, the use of a high numerical aperture (NA) lens
can improve axial resolution further in systems with higher center wavelength and lower bandwidth
[30, 28]. This effect is not considered here as it small in most commonly used OCT systems and
negligible in the considered Micro-OCT system.
2
In reality, the depth of focus is often maximized to not only cover the imaging depth but also
potential surface variations in the tissue and operator/scanning motion.
34
6x = 0.44
=
(2.2)
NA
2 A~
A
7r N A2
(2.3)
Table 2.2: Comparison of governing parameters of spatial resolution and depth of focus in
confocal microscopy, OCT, and Micro-OCT. Ac: Center wavelength, AA: Bandwidth, n:
Refractive index, NA: Numerical aperture
Confocal
Axial Resolution/Depth Sectioning
A2
NA
A
cLNA
Lateral Resolution
DethofFocus
Dept ofNA
2.4.4
oc
OC
2
Ax
OCT
Micro-OCT
oc
L
cxnAA
_C_
nA
c -A
NA
0c NA
A
cxNAA2
0
cxNA' 2
Annular sample beam and aperture improve both lateral resolution and depth of focus in Micro-OCT
Micro-OCT uses an annular sample beam and collection aperture, also referred to as
"donut beam", to provide extended depth of focus in addition to fine lateral resolution
and inspite of using a relatively high NA.
W. T. Welford first showed in 1960 the benefit of obstructing a significant central
fraction of a circular aperture in order to increase depth of focus [33]. For the case
of photography, he gives equations 2.4 with 2.5 and 2.6 with 2.7, which describe
the intensity distribution on axis away from the focus and in the radial direction at
the focus, respectively. Figures 2-1 and 2-2 show these intensity distributions in a
graphical way, for a qualitative understanding of the effect of the fractional central
obstruction c: 1) In order to increase the depth of focus significantly, a large portion
(greater 0.6) of the aperture radius should be blocked. 2) In doing so, the Airy pattern
35
in the radial direction becomes narrower as well, however at the cost of higher order
Airy rings with increased intensity. Thus, lateral resolution is slightly improved as
the depth of focus is extended, but the image may be prone to more artifacts from
the increased side lobes, and "ringing" around point objects may be observed. In the
case of Micro-OCT, not only the collecting aperture is annular, but the sample beam
is of annular profile and focused onto the tissue of interest.
a4
I(z, 0) = A 2 R 2
{
sin[Ip(1 - E2)]
2
(2.4)
1
is the intensity along the axis, where
p = 7ra2z/AR 2
(2.5)
and a is the diameter of the aperture, A the wavelength of light, R the focal distance
of the lens, e the fractional central obstruction, and z the distance from the focus
along the axis.
-epsion=0.3
0.6
-0.9
-
0.9 -
o
0.8
-
L
E
-
2 0.7
U-
z 0.5
0.3
--
E0.2
--
-
0.6 -
0
0
0.02
0.04
0.12
0.14 0.16
0.06 0.8
0.1
Axial Distance From Focus [mm]
0.18
0.2
Figure 2-1: Qualitative comparison of axial intensities from the focus for a circular and
annular apertures of different inner diameters. Factor epsilon is the fractional radius of the
obstructed center portion as described in [33]. Computed from equation 2.4 with 2.5 using:
a=0.672mm, A=0.08mm, R=2.8mm
36
I(0, p) =
a42J
A2 R 2
V
v
E)2(2.6)
is the radial intensity at the focus, where
(2.7)
v = 27rap/AR
and a is the diameter of the aperture, A the wavelength of light, R the focal distance
of the lens, E the fractional central obstruction, and p the radial distance from the
axis at the focus.
-epsilon=0
-0.3
0.9
-0.6
.
-0.9
.
C 0.6
4)
-
A-_
<0.7
-o
~0
04
0.1
01
0
1
2
4
3
5
Radial Distance From Focus [mm]
6
7
x103
Figure 2-2: Qualitative comparison of radial intensities at the focus for a circular and
annular apertures of different inner diameters. Factor epsilon is the fractional radius of the
obstructed center portion as described in [33]. Computed from equation 2.6 with 2.7 using:
a=0.672mm, A=0.08mm, R=2.8mm
2.4.5
Design requirements for a miniaturized Micro-OCT probe
to visualize MCC in vivo
The main goal of this thesis was to provide visualization of MCC in vivo enabled by
a miniaturized Micro-OCT probe. A swine model was chosen for the resemblance of
its pulmonary anatomy to the human lower respiratory system and for its relevance
with respect to the existing genetic CF pig model [34, 35, 36] for future studies of CF
37
pathophysiology. Further, the probe should be adaptable to imaging the human nasal
cavity and smaller airways (down to about 7 generations) in the future. A commonpath probe design was chosen that combines the beam shaping and focusing function
of the probe with integrated splitting and recombination of sample and reference
arm beam to minimize dispersion. It can be seen from the overview of governing
parameters of axial and lateral resolution as well as depth of focus for Micro-OCT
in table 2.2 that the axial resolution requirement is mainly provided by the choice of
the source-detector combination and thus need not be considered in the probe design.
In addition, both the lateral resolution and depth of focus depend on the numerical
aperture of the focus, which is created within the probe. Other design requirements
to consider for the probe include its dimensions (diameter and length) to reach the
tissue of interest, scanning speed to allow quantification of CBF, motion stability,
and sufficient working distance to avoid touching the mucus, as well as a general noncontact design with respect to the region of interest, such that the natural, unaltered
mucus flow can be visualized.
38
Table 2.3: Design requirements for a miniaturized Micro-OCT probe to visualize mucociliary
function in living swine trachea. ET: endotracheal. CBF: ciliary beat frequency
Requirement
Comment
Lateral Resolution
< 2.5 pm
half the ciliary length in trachea
Depth of Focus
> 200 pm
covering working distance, epithelial
Parameter
thickness, surface variations, and perpendicular motion during scanning
Diameter
allowing
< 4 mm
side-by-side
bronchoscopic
guidance within ET tube
Length
> 40 cm
to access respiratory epithelium beyond
ET tube
Frame rate
> 34 Hz
twice the CBF within swine trachea
and as fast as possible to limit motion
artifacts
mechanical and/or software algorithms
to limit/correct for motion artifacts
Motion stability
Working distance
20 pm from
outer most sur-
minimum required to observe unaltered
ASL and mucus transport, the larger
entire
the better (within the limits of the
depth of focus)
>
face of
probe
39
40
Chapter 3
Miniaturized Micro-OCT probe for
real-time visualization of
mucociliary function in vivo
3.1
Motivation
Our lab has recently introduced a novel imaging technology for advanced study of
microscopic mucociliary anatomy and function, termed Micro-OCT [11]. Micro-OCT
was demonstrated to provide several key parameters of mucociliary clearance, simultaneously, in real-time, without the use of exogenous contrast. The quantitative assessment of airway surface liquid depth (ASL), periciliary liquid depth (PCL), ciliary
beat frequency (CBF), and mucociliary transport rate (MCT) was validated against
standard techniques - none of which can provide all four parameters at once. Further,
the high resolution of Micro-OCT was capable of visualizing the ciliary forward and
recovery stroke as well as glandular secretion of mucus.
Like current techniques, however, these parameters were studied in tissue cultures
and excised tissue requiring an invasive procedure and precise control of experimental
conditions. Further, it remains unclear how the observed ciliary behavior compares
to their natural behavior in vivo and the exact effect of therapeutic interventions or
41
insults cannot be studied in their full complexity within the living host.
We have therefore developed a miniaturized probe (4 mm outer diameter) for
Micro-OCT imaging and demonstrate its capability to visualize real-time mucociliary
function of tracheal epithelium in a living swine model.
3.2
3.2.1
Methods
Common-path optical probe design with annular sample beam profile
The principles behind Micro-OCT have been described in section 2.4 of this thesis
and prior publications [10, 11]. The Micro-OCT probe developed in this thesis integrates both the sample and reference arm of our previous bench-top system within
a common-path configuration to minimize dispersion mismatch between the sample
and reference arms (Figure reffig:ProbeASchematic). Compared to most conventional
OCT probes, the common-path design is particularly critical in Micro-OCT, as both
the lower wavelength (A, = 800nm versus 1300 nm) and order of magnitude higher
spatial resolution (1 pm versus 10 jtm) make Micro-OCT more sensitive to dispersion
artifacts. Beam splitting into sample and reference arm within the probe is achieved
through use of two serially assembled prisms (Figure reffig:ProbeASchematic). The
first is coated with gold on its long side except for a circular opening in the center,
thus reflecting the sample arm light at 90 degrees and letting the reference arm light
through. Reflection of the reference arm light is achieved through a reflective surface
mounted to match the sample arm length at its approximate focus. Reflection of the
sample arm light is provided by the tissue structures. In addition to beam splitting,
the two-prism configuration provides a change of the sample beam profile from an
approximate Gaussian to an annular shape. Because the opening within the gold
coating is circular on the long side of the prism and the beam is focused towards the
surface at 90 degrees, the obstructed center of the beam is oval shaped.
Figure 3-la illustrates the optical probe components in more detail. A single mode
42
fiber is centered within a stainless steel housing (driveshaft, 2 mm OD) with help of a
ferrule (1 mm OD, BK7). The light exiting the fiber is then allowed to diverge within
a glass spacer of center length 2.9
0.05 mm. The exit surface of the light from the
fiber as well as the spacer surface are polished to 0.3 pm roughness and at 8 degrees
to minimize Fresnel back reflections at this first interface between the driveshaft and
spacer. The diverging light is then focused by a 5-mm long gradient index (GRIN)
lens (Grintech, 2 mm OD, pitch 0.23) towards two right triangular prisms. The first
rectangular prism face, which is oriented at 45 degrees to the beam is coated with
a gold reflective surface excluding a circular opening in its center of about 250 pm
diameter. The outer portion of the beam is thus reflected at 90 degrees to focus on
the tissue in a side-viewing configuration and with a beam profile resembling an oval
shaped donut. The center portion of the beam is allowed to continue along the axis of
the probe through the uncoated, second right triangular prism. This center portion
will then subsequently be reflected by a reference reflector along the axis of the probe
and serve as the reference arm. Returning light within both arms is recombined and
the interference signal is transported to the Micro-OCT console.
All serial components of the optical probe are attached using UV-curing adhesive
(NOA 65, Norland Products, NJ) with nearly perfect transmission in the visible and
near-infrared wavelength ranges.
3.2.2
Probe scanning
In order to create a two-dimensional image over time, the optical probe subsystem
is scanned over a 400 pm distance at 40 frames per second (fps) using a rigid steel
driveshaft connected to a Piezo stack actuator.
3.2.3
Outer housing design characteristics
The stationary outer housing of the endoscopic probe is comprised of a cylindrical steel
hypotube (4 mm OD) incorporating a thin imaging window and an end-cap to provide
distance rails and a seal against body fluids. The window was realized as a cut-out
43
within the hypotube overlaid by polyolefin heat shrink. The end cap was custom
designed and 3D printed and features distance rails to facilitate placing the probe's
focus with respect to the tissue surface. The working distance of the probe from the
center of the optics is on the order of 2.8 mm, or about 0.8 mm from the imaging
window. A hand piece (custom design, 3D printed) stabilizes the outer housing as
well as the piezo scanner and facilitates manipulation of the endoscopic probe. Figure
3-1b through e illustrates the assembly and appearance of the components of the outer
housing.
3.2.4
Fabrication of prototype
During fabrication, it is critical to assure minimal back-reflections from the surfaces
between two serially assembled components. Such back-reflections would be lossy and
could act as additional undesired reference reflectors. Therefore, all optical components and adhesives should be index matched. For the same reason, the fiber tip and
subsequent spacer surface were angle polished at 8 degrees. Further, all serial optical
components should be aligned precisely to minimize the diameter of the optical probe
subsystem and to avoid clipping of the beam at any surface. Precise positioning of the
gold-coated prism is important, such that the hole within the coated surface obstructs
the center portion of the beam and leaves a rotationally symmetric outer beam profile. Lastly, the reference reflector should be adjusted for maximized, non-saturating
reference reflector power.
Figure 3-2 illustrates the assembly steps of the optical probe subsystem. First, the
shuttle tube should be threaded over the driveshaft as placing it later poses a risk of
damaging the assembled optics. A single mode fiber (SMF) is threaded through a side
opening towards the proximal end of the probe and advanced through the inside of
the drive shaft until it exits the drive shaft. The exiting end is then stripped to reveal
several mms of bare fiber, coated with epoxy, and a 1-mm outer diameter (OD) ferrule
is threaded over it. The epoxy is allowed to cure at room temperature over night. By
pulling on the fiber, the ferrule is then carefully placed and epoxied within the drive
shaft in a similar manner. The drive shaft assembly is polished at an 8 degree angle
44
and to a 0.3-micron surface quality. The spacer is polished at an 8 degree angle and
to length (2.9 mm center length). The spacer and subsequently the GRIN lens (5 mm
length) are then aligned to the subassembly with help of a custom made holder and
attached using UV-curing Norland Optical Adhesive (NOA) 65. The custom made
holder piece allows the components surfaces to contact above a cut-out to prevent
attaching the assembly to the holder. The subassembly is then oriented vertically and
connected to a handheld diode laser for alignment. The coated, first prism is placed
on top of the subassembly on a dot of NOA and its position is optimized by centering
the diffraction pattern within the reflected light spot before being UV-cured. The
uncoated, second prism is then attached and the desired donut beam can be observed.
The shuttle tube, which is tightly fit on a nylon bearing, is put in place and the nylon
bearing is attached to the driveshaft with epoxy. A polished, polycarbonate (PC)
set screw is positioned within the shuttle tube to provide optimized reference path
length compared to a sample at the focus. The optical probe subsystem is now fully
assembled and connected to the scanner. A stationary outer housing is placed over
the optical probe subsystem for protection.
3.2.5
Experimental design of animal study
All animal experiments were approved and carried out in accordance with the regulations set forth by the Massachusetts General Hospital Institutional Animal Care and
Use Committee (IACUC). Three healthy female Yorkshire swine (40 to 50 kg) were
intubated with an 8.5 endotracheal (ET) tube and breathing spontaneously throughout the study while anesthesia was maintained on 2% isoflurane. The ET tube had
been precut to approximately 23 cm length and a 7-fr Arndt Bronchial Blocker (Cook
Medical) accompanied the ET tube on its outside during intubation. The endoscopic
probe was then inserted through the straight section of a Y-adapter, with a pediatric
size bronchoscope (Olympus, 2.7 mm OD) inserted through the angled section to
guide the probe and provide visual feedback for probe placement. Once a suitable
imaging location within the trachea was identified, the bronchial blocker balloon was
positioned behind the probe and inflated for several seconds during image acquisition
45
behind the probe's imaging window to stabilize the outer housing of the probe with
respect to the tissue region of interest. Figure 3-3 shows an example bronchoscopic
image during balloon inflation. Following the first in-vivo imaging experiment, the
trachea was further freshly excised and images of beating cilia were obtained with the
probe in the ex-vivo tissue.
3.2.6
Data processing
Each resulting three-dimensional image (cross-sectional image over time) was motion
corrected and analyzed using ImageJ [371.
Computation of CBF: The image stack was re-sliced to yield a stack of en-face
images, then the en-face images were visually evaluated to identify periodic patterns
of ciliary beat. Once such a pattern was identified, a line profile over the pattern
was computed and CBF was calculated as the number of peaks in 100 frames, corresponding to 2.5 seconds.
ASL measurement: The cross-sectional images were displayed and ASL was measured manually using the line tool.
MCT visualization: The transport of particles on the mucus surface was visualized
using a time-lapsed color image. 13 frames corresponding to approximately 0.32 s of
transport were displayed in a single two-dimensional image, where the maximum
intensity in each of the 13 images was represented by a color on a continuous color
scale from blue (first image) to white (last image). The motion of a bright particle
on the mucus surface can therefore be seen as its color changes from blue to red to
white.
3.3
3.3.1
Results
Miniaturized rigid probe
A miniaturized, 4 mm OD, 50 cm driveshaft length, common-path Micro-OCT probe
was built where beam apodization and beam splitting was provided by serial ar46
rangement of a gold-coated and uncoated prism, where the coated surface contained
a circular uncoated region to provide passage of the reference arm light. The length
of the probe was chosen long enough to reach tracheal epithelium unaffected during
intubation and as short as possible for convenience during fabrication and handling.
Figure 3-1 shows the full probe assembly schematically. Figure 3-4 shows the resulting
donut beam. The probe performance was evaluated qualitatively only and validated
as providing similar quality and detail to the previously used bench-top system.
3.3.2
Lessons from the animal study
Three in-vivo swine studies were performed, and only the third study provided the
desired in-vivo image as shown in figure 3-6.
Initially challenging was the probe
placement within the trachea, which was blind in the first two studies. Thus, if
no tissue was within the ranging depth of the Micro-OCT image, it could not be
identified whether the probe was too close or too far from the tissue and in what way
the position should be altered. The addition of bronchoscopic guidance of the probe
in the third study then allowed visually guided adjustment, thus easier and faster
trouble shooting, finally yielding the desired image.
A second challenge was the encountered respiratory and cardiac motion. In the
initial study, the tissue could not be kept consistently enough within the ranging depth
of the Micro-OCT image. This issue was solved through addition of an inflatable
balloon that pushed the probe towards the tissue as well as distance rails on the
outer housing of the probe that assured a minimum distance between tissue and
probe optics.
Further, the design of the imaging window, which was a polyolefin heat shrink
placed over the window cut-out of the outer housing, increased experimental time
significantly. It was easily removed by normal handling during the experiment such
as insertion and retraction of the probe and had to be replaced a few times.
Overall, finding a good position within the trachea with visible ciliary motion
remains time-consuming and challenging, such that only a few percent of the obtained
images from each experiment provide visual ciliary motion and mucociliary transport.
47
3.3.3
Images of MCC in swine trachea
The image quality provided by our miniaturized probe design allowed visualization
of beating cilia and mucociliary transport within tracheal tissue. Figure 3-5 demonstrates the periodic ciliary beating pattern, which allows computation of CBF, observed using the probe within freshly excised tracheal swine tissue. CBF was calculated to be 8 Hz, which appears low for swine tracheal epithelium, but remains in the
expected range.
Figure 3-6 shows an in-vivo image of swine tracheal respiratory epithelium that
clearly visualizes ASL. ASL was measured to be 18 pm, which is within the expected
range. Also, a color-coded time-lapsed image is shown that is averaged over about
1/3rd of a second (13 frames at 40 fps) or approximately one to three ciliary strokes
and qualitatively visualizes MCT.
3.4
Discussion
We have demonstrated a first miniaturized micro-OCT endoscopic probe and visualization of mucociliary micro-anatomy and function within swine trachea in vivo.
Current limitations and future developments include:
e Further miniaturization:
The current endoscope is 4 mm in outer diameter and thus within the range of
the larger, current pediatric bronchoscopes and rhinoscopes. This size is therefore suitable for imaging applications in swine as well as potential future clinical
applications. However, simultaneous navigation using a standard bronchoscope
is difficult since the Micro-OCT probe is too large to be guided through the
accessory channel of the scope and inserting both alongside through the endotracheal (ET) tube significantly narrows the ET tube diameter and increases
resistance and work of breathing, which can be limiting in a fragile or diseased
population, such as children. Blind placement of the micro-OCT probe is feasible, but undesirable even in a swine study, as it prolongs the search for a
48
suitable imaging location, increases uncertainty about the exact placement and
limits probe placement to locations that do not require navigation in further
airway generations. Further miniaturization of the probe is therefore merited to
extend the application space of the novel micro-OCT technology and probe and
a readily realizable solution could be to preserve the 4-mm OD at the very tip
of the probe to provide sufficient space for the bearing and shuttle tube, but to
reduce the more proximal diameter of the outer housing. However, to provide
navigation of the probe within the accessory channel of a bronchoscope in the
future, the design of the optical probe subsystem should also be reviewed and
miniaturized further.
o (Semi-) flexible probe design:
Secondly, a rigid endoscope design was chosen for this first generation probe for
ease of scanning the imaging beam, where the rigid driveshaft could precisely
transmit a translating motion provided by a piezo scanner. Rigid endoscopes
are clinically used and can be preferable in certain cases, for example because
they allow one-handed navigation.
However, the rigidity of the design also
prevents navigation of the probe within the accessory channel of a common
bronchoscope, which is required to enable precise navigation, access to smaller
airways, and future translation into a clinical bronchoscopy setting. Moreover,
even in this study, we project that a more flexible probe design would have
facilitated stabilization of the probe within the airway for better image quality
and easier extraction of the parameters of interest, because in the employed
stabilization method the balloon was found to act more by pulling the tissue
towards the probe than by fixing the probe position within the airway. The
development of a flexible probe is thus a further goal of future research.
o Stable reference reflector position:
A clear practical limitation of the current probe is the placement of the reference
reflector by means of a set screw. In this design, the reference reflector position
was not stable enough and as it moved during the experiment, image quality is
49
significantly degraded or the image was lost. Readjusting the reference reflector
was difficult, as the optical probe subsystem (OPS) was protected by the outer
housing during the experiment.
Readjusting therefore required extraction of
the probe and disassembly and re-assembly of the outer housing from the OPS.
A novel probe design should therefore provide a reference reflector position that
is set once without further readjustments.
" Shortening distance between sample beam exit and probe tip:
Further, the distance between the exit of the imaging beam and the tip of the
probe was prone to limiting imaging locations in this study. For example, when
the tip was in good contact with a bent portion of the tissue, the region of
interest could be outside of the working range of the probe. We therefore aim
to shorten this distance in a subsequent probe design.
" Permanent imaging window:
The current imaging window covering the cut-out section in the outer housing
was comprised of polyolefin heat shrink that was applied before the experiment
and would provide a suitably thin, non-birefringent, transparent seal. However,
this window design was fragile and did not withstand multiple extraction and
repositioning of the probe within one experimental setting. It therefore had to
be regularly re-placed in a given setting, which was inconvenient and time consuming. A more permanent imaging window is desirable for a second-generation
probe.
" Finer navigation control:
Lastly, the hand piece should be redesigned to provide finer navigation control
with one hand. For example, it should be able to be held and navigated similarly
to a pen and common standard rigid bronchoscopes and rhinoscopes.
50
3.5
Conclusion
In this chapter, we demonstrated a first Micro-OCT rigid endoscopic probe, which incorporates both the sample and reference arm of the previous bench-top probe within
a miniaturized, 4 mm outer diameter, common-path design. Using this probe in a
bronchoscopically guided swine imaging procedure, we demonstrated the first visualization of mucociliary micro-anatomy and function in vivo. Further improvements of
the probe design are merited to increase the research application space and facilitate
translation into the clinical setting.
51
a
Fiber Ferrule
Shuttle
Bearing
i
Uncoated Prism
I
PC
Driveshaft Spacer
GRIN Lens Coated Prism Set Screw
b
/
Endcap
Outer housing
C
Polyolefin window
Distance Rails
Handpiece
stotionary
Piezo Scanner
Electrical
Connection
d
translating
Optical
Connection
e
Endcap
Handpiece
Piezo
,Scanner
Figure 3-1: Schematic and pictures of common-path configuration and components of first
Micro-OCT probe. a. Optical probe subsystem (OPS). The sample beam is focused on the
tissue via a GRIN lens and a two-prism assembly, the first of which is coated with a thin
layer of gold on its long side, with the exception of a central circular opening. A shuttle tube
is placed over the optics to hold a reference reflector in place at the focus of the reference
beam. b. OPS within the outer housing. The outer housing remains stationary with respect
to the sample and at a pre-defined distance corresponding to the height of the distance rails
on the endcap. A polyolefin heat shrink is placed over the outer housing and provides the
imaging window. c. The OPS is translated for scanning of the beam via a piezo scanner.
The outer housing and piezo scanner are manipulated by the user and held stationary with
respect to the tissue with a handpiece. d. and e. Pictures of the fully assembled probe.
52
a
Drive Shaft,
7
F--
-
Shuttle Tube
L--I=--
'SMF
b
Ferrule
cI
Epoxy
d
4-
4
f
UV-Light
e
GRIN
NOA Spacer
h
g
PC
Set Screw
Coated Prism
_HC
Prism
rl hEi
o
- - -U7
Figure 3-2: Schematic illustration of main assembly steps of Micro-OCT optical probe
subassembly. a. The shuttle tube was threaded over the driveshaft before the assembly of
any optics. A single mode fiber (SMF) was threaded through the lumen of the driveshaft.
b. A 1-mm outer diameter (OD) ferrule was threaded over the bare fiber and held in place
with epoxy. c. By pulling on the fiber, the ferrule was carefully placed and epoxied within
the drive shaft. d. The drive shaft assembly was polished at an 8 degree angle. The spacer
was polished at an 8 degree angle and to length (2.9mm). The spacer and GRIN lens (5mm
length) were then carefully aligned to the subassembly and attached using a custom-made
holder and Norland Optical Adhesive (NOA) 65. e. NOR was cured with UV-light. f. The
subassembly was oriented vertically and connected to a handheld diode laser for alignment.
The coated prism was placed on top of the subassembly and its position was optimized by
centering the diffraction pattern within the reflected light spot. g. The uncoated prism was
placed and the desired donut beam could be observed. h. A polished, polycarbonate (PC)
set screw was positioned within the shuttle tube to provide optimized reference path length
compared to a sample at the focus. The optical probe subsystem was then connected to
the scanner and was protected by a stationary outer housing.
53
Tracheal epithelium
Blocker Balloon
Probe Outer Housing
Figure 3-3: Picture of a bronchoscopic image during balloon inflation for stabilization of
the Micro-OCT probe during image acquisition with respect to the tissue region of interest.
Figure 3-4: Picture of the optical probe subsystem, which is the scanning portion of the
probe, and the donut-shaped beam profile.
54
Yt
x
x
4-,
z
Gray Value
Figure 3-5: Image of mucociliary clearance in freshly excised swine trachea obtained with
first-generation Micro-OCT probe. The square in the cross-sectional (x-z) image indicates
the position of the time-lapsed image (x-t), in which the periodic pattern of ciliary beat can
be observed and measured from the line profile. CBF was calculated here as 8 Hz (single
cilium).
X t
x
x
_A
k
z
z
P
Figure 3-6: Image of mucociliary clearance in vivo obtained with first-generation MicroOCT probe in swine trachea. Left: One cross-sectional frame showing airway surface liquid
depth (ASL), measured to be 18 pm. Right: Time-lapsed color image showing 13 frames
corresponding to about 1/3rd of a second of observed mucociliary transport. The transport
of particles on the mucus was visualized as the particle's color changes from blue (first
frame) to white (last frame). MCT could be quantified by measuring the traveled distance
over the observed time.
55
56
Chapter 4
Novel common-path Micro-OCT
probe designs
4.1
Motivation
In the previous chapter, we demonstrated the first generation Micro-OCT probe which
enabled unprecedented images of mucociliary micro- structure and function in vivo.
However, the ease of use was limited due to various factors, such as an unstable
reference reflector position, long distance between the imaging beam and probe tip,
suboptimal window and hand piece design, rigid driveshaft, and limited potential
for further miniaturization. This chapter describes our second generation probe design addressing the above mentioned limitations for improved stability and usability
and with the potential to enable the assessment of mucociliary clearance in smaller
airways.
The major change compared to the previous design is the shift from the two prism
approach and a reference reflector held in position by an additional shuttle tube to an
integrated reference reflector design. This new design eliminates the need for a second prism and additional shuttle tube for improved stability and ease of use and is a
promising approach and important step towards further miniaturization of the probe.
Further, a semi-flexible driveshaft is provided by switching from the stainless steel to
copper driveshaft. The outer housing is improved through a more permanent poly57
carbonate imaging window and new hand piece design allowing manipulation that is
similar to manipulating a pen and clinically available bronchoscopes and rhinoscopes.
4.2
4.2.1
Methods
Integrated reference reflector design
Two major limitations of the first generation Micro-OCT probes were an unstable
reference reflector position requiring time-consuming readjustment during the experiments and potential loss of position of the probe at a desired imaging location, and
a relatively long distance between the imaging beam and tip of the probe limiting
potential locations that could be assessed. Further, a shuttle tube was required in
addition to the drive shaft and outer housing with the sole purpose of holding a
reference reflector in place, increasing the diameter of the probe.
This second generation probe design is based on the idea that the reference reflection could be provided by the back-surface of a glass spacer, which is an integral part
of the probe optics. This design element eliminates the need for and instability of a
separate reference reflector. Compared to the first generation probe design, the second prism here is a spacer polished at 45 degree and still provides an on-axis passage
of the reference reflector light, but its length (1.5 mm center length) is adjusted such
that the glass-air interface at its back surface provides the reference reflection at equal
optical path length compared to the sample beam. Thus, the position of the reference
reflector is fixed and stable, the shuttle tube can be eliminated with the potential to
decrease the probe diameter, and the distance from the sample beam to the tip of
optical probe subsystem (OPS) (and thus to the probe tip itself) is minimized as the
reference reflection is located to the very last surface of the OPS.
Fabrication: The glass spacer was initially polished on the 45 degree angled side
to a surface quality of 0.3 pm and then to length on the opposite side. Once attached
to the probe optics, the flat surface of the spacer was repolished to be approximately
perpendicular to the axis of the reference arm light.
58
Figure 4-1, parts a to d illustrate the new design of the OPS: The sample beam is
focused on the tissue of interest via a GRIN lens and a prism-spacer assembly. The
prism is coated with a thin layer of gold on its long side, with the exception of a
central circular opening. The reference arm light passes through this opening and is
reflected from the back-surface of the spacer, the first surface of which is polished at
45 degrees to fit the prism and the back-surface of which is polished perpendicular to
the axis of the OPS.
4.2.2
Semi-flexible probe design
As a first step towards a more flexible probe design, the steel driveshaft was replaced
by a semi-flexible copper driveshaft. The copper driveshaft remains rigid enough to
transmit linear scanning motion initiated by a piezo scanner, but has the potential
for better stabilization by the bronchial blocker balloon and can be inserted into the
accessory channel of a bronchoscope as long as the bronchoscope is navigated using
small angles (<30 degrees).
4.2.3
Improvements to the outer housing
Two major improvements were made to the outer housing:
1. The imaging window was provided by a 125 pm-thick polycarbonate sheet that
was epoxied to the steel outer housing at minimum distance from the OPS. This
design is permanent, withstands the forces and manipulations during an experiment and subsequent disinfection of the device for reuse. This design therefore
reduces overall preparation and experimental time and increases the success
rate of a given procedure. The improvement is therefore convenient for current
animal studies and critical in view of potential future clinical applications.
2. An ergonomically shaped hand piece was designed and 3D-printed that allows
the imaging probe to be held and navigated similarly to a pen and to clinically
used rigid rhinoscopes.
Navigation and placement of the probe is therefore
59
facilitated, refined, and approaches a clinical standard of care in view of future
clinical testing of the Micro-OCT probe. Figure 4-1, parts c and e show the
hand piece.
4.2.4
Animal study
A single in-vivo swine study was performed according to the same procedures as described in the previous chapter, namely under bronchoscopic guidance of the secondgeneration Micro-OCT probe and stabilization with an intermittently inflated balloon
for stabilization.
4.3
4.3.1
Results
Second-generation Micro-OCT probe
Figure 4-1d and e show pictures of the second-generation Micro-OCT probe with improved reference reflector stability, semi-flexible driveshaft, permanent imaging window and novel hand-piece design.
Figure 4-2 shows a performed "Knife Edge Test" on an image of a glass slide
suspended in air to test the lateral resolution provided by the probe.
The mean
gray value of pixels representing air was calculated to be 40, the mean gray value of
maximum intensity (reflection from the glass slide) 142. The transition from low to
high intensity characterized at the 10% to 90% points (gray values of 50 and 132,
respectively) was 5.5 pm which would correspond to a 1/e 2 spot size radius of a
Gaussian beam of 4.3 pm according to equation 4.1 [38].
WO = XIO(4.1)
1.28
60
4.3.2
First image of tracheal epithelium in living swine model
Figure 4-3 shows an example image from a preliminary swine study. The study set
up was equivalent to imaging MCC with the first generation Micro-OCT probe as
described in the previous chapter.
Layers of the respiratory epithelium could be
visualized, but no ciliary motion could be observed.
It can be seen that, in this
first prototype, the reference power provided by the reflective surface was insufficient
compared to the reflective power of the mucus surface. In other words, the autocorrelation signal was stronger than the desired cross-correlation signal, the signal
from surface of interest saturated the detector and the features of interest could not
be visualized.
4.4
Limitations and future work
During fabrication of the device, a major limiting, time-consuming, step was found
to be the repolishing of the reflective spacer surface after assembly to assure perpendicularity to the reference beam axis and maximize optical path distance match
with respect to the sample beam. The assembly could only be held at the height of
the driveshaft rather than as close as possible to the surface-to-polish, which did not
allow minimizing the forces exerted on the assembled spacer. Thus, polishing had to
be performed extremely carefully. Secondly, the exact length of the spacer had to be
tested in a reiterative fashion - polishing off a few tens of micrometers was alternated
with tests of the reference position using the Micro-OCT bench top system. A more
precise method of alignment of the spacer before assembly could eliminate the need
for repolishing and should be found.
Further, the reference power provided by the reflective surface was insufficient
in the first prototype. Potential options to increase reference reflector power could
include more precise alignment/repolishing to assure precise straight back-reflection
of the reference arm light and/or the addition of a reflective coating.
Lastly, the usefulness of the semi-flexible driveshaft remains to be demonstrated.
For the preliminary animal study, no copper outer housing was available and steel
61
was used as in the previous chapter.
An important advantage of the integrated reference reflector design is its potential
for significant miniaturization of the OPS. The 90 degree reflection of the sample
beam, which in the current first (chapter 3) and second-generation probe (Figure
4-1) designs is assured by a gold-coated prism surface, could be achieved without
any coating and solely through total internal reflection if the 45-degree spacer had
a diameter corresponding to the desired central obstruction of the sample beam. As
a side effect, the obstructed portion of the resulting donut beam would then also be
circular instead of oval further improving image quality. Figure 4-4 illustrates this
design idea (c) named third-generation Micro-OCT probe, in comparison to the two
previous generation probes.
4.5
Summary
We have shown a second generation common-path Micro-OCT probe with integrated
reference reflector, semi-flexible driveshaft, and improvements to the outer housing.
Advantages compared to the first generation are:
* Stable reference position, which is additionally advantageous for further miniaturization as well as flexible probe designs
* Minimized distance from sample beam to probe tip
" Elimination of the shuttle tube simplifying fabrication and adjustment of the
OPS and further facilitating future miniaturization
* Semi-flexible copper drive shaft for better stabilization with respect to the tissue
of interest
* Permanent polycarbonate window within the outer housing reducing preparation and experimental time
* Novel ergonomic hand piece design for finer probe navigation control
62
The major current limitations observed in the first prototype of the secondgeneration Micro-OCT probe include lower than expected lateral resolution inspite of
dispersion compensation and lower reference reflector power, which was insufficient
in a first imaging experiment. Future work should focus on facilitating angled placement and alignment of the spacer with respect to the prism and other options such
as surface coating such that reference reflector power can be significantly increased.
In the discussion, we further presented a third-generation probe design idea that
is a further development from our second-generation. It therefore is prone to similar
challenges to the second-generation design, but provides great potential for further
miniaturization of the Micro-OCT probe.
63
Fiber
Engineered
Surface
Bearing
Uncoated Spacer
Ferrule
- --
--
Brass
Driveshaft
Spacer
GRIN Lens
- --
Coated Prism
b
Outer housing
C
PC window
Distance Rails
Handpiece
stationary
Piezo Scanner
Electrical
Connection
d
translating
Optical
Connection
e
Handpiece
Piezo
Scanner
Electrical
Connection
Optical
Connection
Figure 4-1: Schematic and pictures of common-path configuration and components of
second-generation Micro-OCT probe. a. Optical probe subsystem (OPS). The sample
beam is focused on the tissue via a GRIN lens and a prism-spacer assembly. The prism is
coated with a thin layer of gold on its long side, with the exception of a central circular
opening. The reference arm light passes through this opening and is reflected from the
back-surface of the spacer. b. OPS within the outer housing. The outer housing remains
stationary with respect to the sample and at a pre-defined distance corresponding to the
height of the distance rails on the endcap. A 125-pm thick polycarbonate (PC) sheet is
epoxied on a cut-out in the outer housing and provides the imaging window. c. The OPS
is translated for scanning of the beam via a piezo scanner. The outer housing and piezo
scanner are manipulated by the user and held stationary with respect to the tissue with
a pen-like ergonomic handpiece. d. Picture of the distal probe optics. e. Picture of the
proximal probe.
64
...........
...........
---------------
.....................
x
x
a
C
b
140
90%
140
130
120
120
1.28wo
110
100
100
90
TO80
80
10%,
60
60
40
50
-
-
-
70
40
Figure 4-2: Schematic illustrating the knife edge test for evaluation of transverse resolution
achieved with second generation Micro-OCT probe. a. Micro-OCT image of cover glass
edge. b. Color-coded equivalent to image a demonstrates corresponding gray values. c.
Gray values profile plot corresponding to image b illustrates conversion from the 10% and
90% knife edge response of the Micro-OCT probe into a measure of spot size radius wO.
Here, a knife edge response over 5.5 pm corresponds to a spot size radius of 4.3 pm.
t
x
Auto-correlation image
Tissue surface
Cross-correlation image
Z
Figure 4-3: First in-vivo image obtained with second-generation Micro-OCT probe. The tissue surface provided a stronger reflection than the reference reflector resulting in a stronger
auto-correlation than cross-correlation image.
65
Figure 4-4: Schematic illustrating the the three generations of Micro-OCT probe designs.
a. First-generation Micro-OCT probe optics based on the two-prism design with separate
reference reflector. An example donut beam is shown at a significant distance from the focus to exaggerate misalignment and imperfections. b. Second-generation Micro-OCT probe
optics based on a prism-spacer design of integrated reference reflector. The star indicates
that the sample beam profile picture was taken before assembly of the spacer and thus
illustrates the diffraction pattern caused by centrally placed cut-out within the gold coating. c. Third-generation Micro-OCT probe optics with great potential for miniaturization.
Based on a prism-fiber design employing total internal reflection at the prism surface and
thus eliminating the need for gold coating. The resulting donut beam here is shown after
accidental misalignment of the fiber with respect to the beam center. However, the round
profile of the central obstruction can be observed in comparison to the oval-shaped central
obstruction of design a and b.
66
Chapter 5
Summary and Outlook
Mucociliary clearance (MCC) is an absolute critical defense mechanism of our respiratory system. Its importance is underlined by the fact that patients with cystic fibrosis
(CF), and thus impaired MCC, used to die in their childhood and only over the last
decades with more advanced and aggressive therapy their life span has extended to
an average of 30 or 40 years. In addition, the prevalence of mucociliary impairment
extends far beyond the classical rare genetic diseases such as CF and primary ciliary
dyskinesia (PCD). Recently, there has been an increasing understanding that patients
with chronic obstructive pulmonary disease (COPD) exhibit mucociliary impairment
and that MCC is altered in any lung infection and may be influenced by antibiotic
use [1, 2, 16, 17]. In spite of its significance and high prevalence, there has been no
tool available to study the complexity of MCC at the level of the cilia and mucus in
real time in vivo.
This thesis filled the gap as it demonstrated a first and unique endoscopic probe
for visualization of ciliary micro-function in vivo. The probe employs a novel optical
imaging technique termed Micro-OCT, which had previously been shown to be superior to commonly used techniques for studying MCC. This chapter summarizes the
achieved milestones as initially illustrated in Figure 1-2 and discusses next steps and
the projected impact.
67
5.1
Thesis summary
We demonstrated an innovative tool for visualization of mucociliary micro-anatomy
and -function in vivo based on a novel optical imaging technique termed Micro-OCT.
First images of MCC in living swine trachea were shown and novel probe designs are
introduced as the groundwork for future translation of the Micro-OCT probe into a
clinical endoscopic prototype.
A miniaturized rigid endoscopic Micro-OCT probe was shown that provides the
high spatial (< 2 pm) and temporal (> 34 Hz) resolution necessary for the evaluation of ciliary micro-function. The common-path probe design minimizes dispersion
artifacts and combines the functionality of both the sample and reference arm of the
previously used bench-top system within a 2-mm diameter optical assembly for a total probe diameter of 4 mm, which corresponds to the size of commonly used clinical
rhinoscopes and bronchoscopes.
This first-generation probe enabled unprecedented visualization of mucociliary
function in healthy swine trachea in vivo. The probe was guided alongside a pediatric
bronchoscope for visual identification of a region of interest. Motion artifacts were
minimized by an intermittently inflatable balloon stabilizing the probe with respect
to the tissue. Longitudinally placed distance rails on the tip of the probe positioned
the probe and focusing optics such that the tissue surface appeared in focus while
keeping the mucus flow undisturbed.
CBF was calculated from an ex-vivo image, ASL was measured and MCT visualized from the in-vivo image.
In order to lay the groundwork for research studies in further generations of the
lower airways and clinical translation, newer generation probe designs were tested with
improved reference reflector stability, semi-flexible driveshaft and a greater potential
for miniaturization among other improvements.
The presented results open new avenues for studying MCC in health and disease,
in pre-clinical and clinical research settings and have great potential to eventually
revolutionize diagnosis, phenotyping, and therapeutic management for all patients
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with respiratory disease.
5.2
Next steps
The results presented in this thesis open new avenues for studying MCC. Interesting
next steps include:
" Study of mucociliary modulators:
Novel therapies to improve mucociliary function are intensely searched and studied, and the application of recently developed CFTR potentiators is extended
from CF to COPD patients [39, 40, 41]. While the therapeutic efficacy can
be demonstrated in clinical studies, the exact mechanistic effect and its influence on ciliary function versus mucus properties remains incompletely understood and cannot be quantified in these studies. With the developed endoscopic
Micro-OCT probe, modulators of MCC can now be studied in a complex invivo system, the exact effect on cilia and mucus can be observed from onset to
weaning and can be quantified in real time. Different modulators with different
mechanism of action can be compared with respect to their effect on a common parameter, such as the resulting mucociliary transport (MCT). Similarly,
novel therapeutic compounds can now be tested for accelerated development
and translation into the clinic.
* Non-invasive evaluation of MCC in the human nasal cavity:
The developed rigid probe should be translated for non-invasive assessment of
MCC within the human nasal cavity. Size, materials and single-handed manipulation are close to existing rigid rhinoscopes and the respiratory epithelium of
the inferior turbinate within the human nasal cavity is commonly evaluated for
diagnosing PCD or studies of CF and easily accessible. Remaining challenges
for the adaption of the current probe into a clinical tool include assurance of
electrical safety and appropriate navigation to the region of interest. Such a
tool, which provides non-invasive evaluation of ciliary beating patterns in vivo
69
could then provide a novel and more sensitive way of diagnosing patients with
primary ciliary dyskinesia - a disease which is thought to be largely underdiagnosed with current methods.
Further, since the respiratory epithelium is continuous from the upper to lower
airways, it would be interesting to show that the pathophysiology observed in
the human nasal cavity is representative of the defects that would be observed
in the lower airways. If the nose could serve as a window to the lung, studies
of MCC and any therapeutic effects would be non-invasive and could be easily
performed on each individual patient and large patient populations with the
potential to transform the diagnosis, phenotyping and treatment management
of patients with respiratory disease.
* Evaluation of the early CF defect in CF piglets and children:
The demonstrated probe should be adapted into a fully flexible design and
be further miniaturized to allow navigation within the accessory channel of a
bronchoscope. Such a design would enable assessment of the smaller airways
in swine and humans and could significantly enhance our understanding of CF
disease pathogenesis. It could elucidate the unclear relationship between the
micro-anatomy and function of the respiratory epithelium and the mucus properties in CF piglets [34, 35, 36] and children before potentially confounding
secondary changes occur due to lung inflammation. Observing the natural history of disease in children could finally prove or revoke the hypothesis that CF
lung disease begins in the lower airways - consequently, this may allow more
targeted therapeutic management. Similarly, longitudinal studies in CF piglets
could lead us to a better understanding of the initial events in the onset of
disease and its underlying cause to guide the development of novel therapies.
70
5.3
Impact
The presented work is only the first step in the development and translation of a
completely novel technique for the assessment of ciliary function in general and mucociliary clearance in particular.
We expect our Micro-OCT probe to become a cornerstone technology for studying
MCC and novel therapies in the pre-clinical and clinical setting. The new capabilities
provided by our endoscopic Micro-OCT probe will greatly increase our understanding
of the pathogenesis of all diseases of MCC and to accelerate the development and
translation of therapeutics. In PCD, the technique could become the standard of
care for the diagnosis as ciliary beating patterns could be analyzed in vivo in a rapid,
non-invasive way.
In COPD, the technique could entirely replace spirometry for
phenotyping and therapeutic management. Patients could be categorized depending
on the extent of impairment of MCC or reversal thereof rather than non-specific
parameters such as impairment of FEVI or other spirometry results, which includes
patients with very different presentations and morbidity. Such improved phenotyping
would further greatly facilitate the development of novel therapeutics as their efficacy
can be more easily be established in a patient population of similar pathophysiology.
Further, the technique could be adapted to evaluate other organ systems with
ciliated respiratory epithelium, such as the fallopian tube, and potentially even to
elucidate the often insufficiently understood function of stereo cilia in organs such as
the inner ear or kidneys.
In summary, a clinical Micro-OCT endoscope has the potential to provide noninvasive improved and personalized diagnosis, phenotyping and therapeutic management in CF, PCD, COPD and all respiratory disease and could provide new avenues
for studying other organ systems with motile and stereo-cilia.
71
72
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