UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Vascular applications of quantitative optical coherence tomography
van der Meer, F.J.
Publication date
2005
Link to publication
Citation for published version (APA):
van der Meer, F. J. (2005). Vascular applications of quantitative optical coherence
tomography.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)
and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open
content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please
let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material
inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter
to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You
will be contacted as soon as possible.
L O C A L I Z E D M E A S U R E M E N T O F OPTICAL
A T T E N U A T I O N C O E F F I C I E N T S O F
A T H E R O S C L E R O T I C PLAQUE
C O N S T I T U E N T S BY QUANTITATIVE OPTICAL
C O H E R E N C E T O M O G R A P H Y
Freek J. van der Meer, Dirk J. Faber,
David Baraznji Sassoon, Maurice C. Aalders,
Gerard Pasterkamp, Ton G. van Leeuwen
LOCALIZED/t MEASUREMENTS OF PLAQUE COMPONENTS
O
p t i c a l c o h e r e n c e t o m o g r a p h y ( O C T ) is a n o v e l , h i g h - r e s o l u t i o n diagnostic tool that is capable of imaging the arterial wall and plaques. The differentiation between different types of atherosclerotic plaque is based o n qualitative differences in gray levels and structural appearance. We hypothesize that a quantitative data analysis of the O C T signal allows measurement of light attenuation by the local tissue components, which can facilitate quantitative spatial discrimination bet-ween plaque constituents.High-resolution O C T images (at 800 nm) of human atherosclerotic arterial segments obtained at autopsy were histologically validated. Using a new, simple analysis algorithm, which incorporates the confocal properties of the O C T system, the light attenuation coefficients for these constituents were determined: for diffuse intimal thickening (5.5 ± 1.2 mm'1) and lipid-rich regions (3.2 ± 1 . 1 mm"1), the attenuation differed
significantly from media (9.9 ± 1.8 mm"1), calcifications (11.1 ± 4.9 mm'1) and thrombi
(11.2 ± 2.3 m m ' ) (p<0.01).
These proof of principle studies show that simple quantitative analysis of the O C T signals allows spatial determination of the intrinsic optical attenuation coefficient of atherosclerotic tissue components within regions of interest. Combining morphological imaging by O C T with the observed differences in optical attenuation coefficients of the various regions may enhance discrimination between various plaque types.
I N T R O D U C T I O N
Optical coherence tomography (OCT) is an imaging modality for minimally invasive imaging of tissue, which, due to its high resolution (l-20yam), has proven to be a powerful
CHAPTER 3
i m a g i n g ; in an A - s c a n t h e a m p l i t u d e o f the b a c k - s c a t t e r e d light from t h e tissue s t r u c t u r e s is p l o t t e d as a f u n c t i o n o f d e p t h .2 S e q u e n t i a l Ascans form a c r o s s s e c t i o n a l i m a g e ( B
-s c a n ) , in w h i c h t h e c o n t r a -s t i-s b a -s e d o n difference-s in optical -scattering p r o p e r t i e -s o f ti-s-sue s a m p l e c o n s t i t u e n t s . In c a r d i o l o g y , B r e z i n s k y and c o w o r k e r s d e m o n s t r a t e d t h e ability o f O C T t o i m a g e v a s c u l a r p a t h o l o g y ,1 a n d , a l t h o u g h still in an e x p e r i m e n t a l p h a s e , it h a s a l r e a d y b e e n s h o w n t h a t in vivo vascular O C T is c a p a b l e o f i m a g i n g t h e arterial wall, d i f f e r e n t i a t i n g b e t w e e n v a r i o u s t y p e s of a t h e r o s c l e r o t i c p l a q u e s .4' ' D u e to t h e high
r e s o l u t i o n it is t h e only i m a g i n g t e c h n i q u e capable o f precisely m e a s u r i n g the c a p t h i c k n e s s o f v u l n e r a b l e p l a q u e s ( < 6 5 - 1 5 0 jum), which overlies a s u b s t a n t i a l lipid c o r e ( > 4 0 % ) . ~
W i t h a g o o d sensitivity a n d specificity, O C T c a n identify t h e arterial wall a n d p l a q u e c o n s t i t u e n t s . I n gray scale i m a g e s , as used in this p a p e r , intimal tissue s h o w s up as a b r i g h t layer o n t o p o f a d a r k e r m e d i a . B o t h lipid a n d calcification m a n i f e s t as a d a r k e r area c o m p a r e d to t h e intimal a n d m e d i a l arterial wall layers. F u r t h e r differentiation b e t w e e n t h e lipid a n d calcified tissue c a n n o t be m a d e on t h e gray leyels a l o n e , and c o n s e q u e n t l y has to b e m a d e o n t h e d e m a r c a t i o n o f t h e s e darker areas: a sharply d e m a r c a t e d b o r d e r i n d i c a t e s t h e p r e s e n c e of c a l c i f i c a t i o n s , w h e r e a s a diffuse b o r d e r i n d i c a t e s t h e p r e s e n c e o f a lipid p o o l . " T h e m e a n O C T signal (i.e. g r a y level i n the i m a g e ) from a c e r t a i n tissue region of interest (ROI) is d e t e r m i n e d by t h e specific local optical properties in the ROT. Quantification o f t h e s e i n t r i n s i c o p t i c a l p r o p e r t i e s could p r o v i d e an e x t r a , o b j e c t i v e , classification p a r a m e t e r .
R e c e n t l y , w e d e m o n s t r a t e d t h a t analysis o f the d e c r e a s e o f t h e O C T signal allowed d e t e r m i n a t i o n o f t h e o p t i c a l a t t e n u a t i o n coefficient (jx) o f a h o m o g e n e o u s s c a t t e r i n g s u s p e n s i o n .8 We h y p o t h e s i z e t h a t t h e high spatial r e s o l u t i o n o f O C T c o m b i n e d with a
s i m i l a r q u a n t i t a t i v e d a t a analysis o f the O C T signal from arterial tissue allows highly l o c a l i z e d m e a s u r e m e n t of/zt, a n d t h a t spatial differences 'm/u , w h i c h is a n intrinsic optical tissue p r o p e r t y , allows f u r t h e r differentiation b e t w e e n plaque c o n s t i t u e n t s o n a quantitative basis. T o test o u r h y p o t h e s i s , we subjected h u m a n carotid artery s p e c i m e n s t o O C T imaging a n d s u b s e q u e n t histological analysis, f r o m t h e O C T data, the local a t t e n u a t i o n coefficients o f a r t e r i a l wall a n d p l a q u e c o n s t i t u e n t s were d e t e r m i n e d .
MATERIALS AND METHODS
Tissue Samples and Tissue Handling
H u m a n c a r o t i d a r t e r y s a m p l e s (N = 13) w e r e o b t a i n e d w i t h i n 12 h o u r s o f p o s t m o r -t e m e x a m i n a -t i o n , a n d w e r e s n a p f r o z e n in liquid ni-trogen a n d s -t o r e d a-t -80°C." T o facili-ta-te
OCT i m a g i n g , v e s s e l s e g m e n t s w e r e cut l o n g i t u d i n a l l y to e x p o s e t h e l u m i n a l s u r f a c e .
D u r i n g t h e O C T i m a g i n g p r o c e d u r e , the s a m p l e s w e r e i m m e r s e d in saline a n d k e p t at 3 7 ° C . T h e imaged areas ( N = 20) w e r e marked using I n d i a n ink. After i m a g i n g , the s a m p l e s w e r e fixed in 4 % formalin for m o r e than 48 h o u r s . If necessary, the samples w e r e decalcified
J.( >( AI.I/.I Du MEASt IREMENTS 1 >!• PLAQI E C( >\I!>( >NENTS
in E D T A (pH=7.0) for one week. After embedding in paraffin, sections with a thickness of 5 fxm were cut at the marked sites, and stained with haematoxylin and eosin, elastin Von Giesson, or picro Sirius red. Based on the ink markers and anatomical landmarks, O C T and histology were matched, and regions of interest (e.g. intimal thickening, lipid rich regions and thrombus) were identified in the O C T images.
OCT Imaging
The principle and physics of O C T have been extensively described.2-3 In our setup,
imaging was performed with a high-resolution O C T system using a femtosecond TkSapphire laser (Femtosource) with a center wavelength of 800 nm and a bandwidth of 120 nm. Axial resolution was 3.5 /j.m; dynamic range was 110 dB. The light was delivered via a single mode fiber with a mode field diameter of 5.3 urn. The lateral resolution, determined by the spot size of the sample arm beam, was approximately 7 ^ m . T h e depth of focus of the sample arm optics was 100 ^ m in air. In depth scanning (A-scan) was performed by a rapid scanning optical delay line.'" The amplitude and phase of the demodulated signal were digitized and stored in a computer. Each A-scan consisted of 4096 data points. B-scan images were obtained by moving the tissue sample with respect to the fixed sample arm beam while performing A-scans. In each B-scan, 100 A-scans per mm were acquired, and for larger lesions overlapping B-scans were combined to one image. Per lesion, at least 5 consecutive image data sets were acquired with a step size of 0.25 to 0.5 mm, measured perpendicular to the B-scan.
Data Analysis
During O C T imaging, the O C T signal is measured as a function of depth d in the tissue. When O C T is assumed to detect only light that has been scattered once, the decay of the O C T signal with depth simply follows the I.ambert-Beer law, i.e. an exponential decay in which the decay constant is the attenuation coefficient (p.). In addition, analogous to confocal microscopy," the O C T signal is influenced by the confocal properties of the sample arm optics, e.g. the position of the focus in the tissue and the depth of focus (figure 3-1).
Consequently, the depth dependence of the amplitude of the OCT signal, i (d), can be described as the product of the axial point spread function (PSF) of the optics used, and the attenuation of the light by the tissue structures.12-13 According to previous work8:
exp(-M)
CHAPTF.R3
x
° d-xji
Figure 3-1. The ( ) C T signal is measured as a function of depth cl. To incorporate the effect of the confocal properties of the sample arm optics, given by the position 'AT and depth of focus is 2Z of lens /. . the < >CT signal is e n e n as a function of the d-X
In t h e d e n o m i n a t o r o f this r e l a t i o n , the axial PSF d e s c r i b e s the effect of the d i s t a n c e
d-x) [mm] o f t h e o b j e c t ai d e p t h d [mm] to the focus p o s i t i o n x() [ m m ] . T h e PSF is
c h a r a c t e r i z e d b y t h e Rayleigh length z0 (half the d e p t h o f focus: w h i c h , for o u r setup, was
calculated to be 0.133 m m in s c a t t e r i n g media. T h e n u m e r a t o r «>i the e q u a t i o n describes the a t t e n u a t i o n o f t h e light in the tissue, using L a m b e r i Beer's law and a s s u m i n g only single s c a t t e r e d light is d e t e c t e d . I [ere, u | m m |. is the tissue p a r a m e t e r to be o b t a i n e d .8
T o o b t a i n JX for the different tissue c o n s t i t u e n t s , the average o f 50 100 adjacent \ -s c a n -s wa-s taken from e a c h R O I in each O C T B--scan. C o m b i n i n g the k n o w n p o -s i t i o n oi the f o c u s in t h e t i s s u e s a m p l e with a L e v e n b e r g - M a r q u a r d t c u r v e fitting a l g o r i t h m , e q u a t i o n 3-1 (with a d d e d offset and amplitude to facilitate scaling) was fitted to the ( >< I signal w i t h i n the R( )I, w i t h / / as t h e fitting p a r a m e t e r . D u r i n g the fitting p r o c e d u r e , the offset was fixed at the a v e r a g e n o i s e level o f the r e d trded data and the a m p l i t u d e was tree-r u n n i n g . P tree-r o v i d e d single s c a t t e tree-r i n g applies, o u tree-r m o d e l is also able to d e t e tree-r m i n e the a t t e n u a t i o n coefficient in a layered m e d i u m . O u r p h a n t o m c o n s i s t e d o f a p l a s t i c scattering layer with a t t e n u a t i o n coefficient o f 1 0 m m u n d e r n e a t h 0.5 m m intralipid s o l u t i o n s with different c o n c e n t r a t i o n s (0-2" i») and thus different a t t e n u a t i o n coefficients (0 6 m m i. We tested t h e capability for d e t e r m i n i n g the a t t e n u a t i o n of the d e e p e r layer as a function of
the a t t e n u a t i o n coefficient o f t h e u p p e r intralipid layer, by a p p l y i n g the same data analysis
p r o c e d u r e as t o r the tissue s a m p l e s .
Statistical Analysis
Single fit data are g i v e n with its estimated s t a n d a r d error. All m e a n data are p r e s e n t e d w i t h s t a n d a r d d e v i a t i o n . T h e statistical significance of the m e a s u r e m e n t s was tested by analysis of v a r i a n c e \X< )VA . a statistical tool for the c o m p a r i s o n o f several g r o u p s of o b s e r v a t i o n s . Post h o c c o m p a r i s o n s were clone using t-tests with B o n f e r r o n i c o r r e c t i o n . All m e t h o d s used are d e s c r i b e d in ret. 1-1.
LOCALIZED// MI \SI REMENTSOI PLAQUI COMPONENTS
RESULTS
l sing ink m a r k e r s and a n a t o m i c a l l a n d m a r k s , the images that w e r e o b t a i n e d u s i n g () C T w e r e successfully m a t c h e d to histology for 20 lesions in 13 arterial s e g m e n t s . U s i n g t h e histology, different regions of interest w e r e identified in the O C T images, e.g. diffuse intimal t h i c k e n i n g in c o n j u n c t i o n with d e v e l o p i n g lipid-rich are.:- \ 6), and calcifications
N = 4 ) figure 3-2) Ba^ed on differences in gray values in the ( )CT images, different features
o f the a t h e r o s c l e r o t i c lesion were clearly distinguishable. Diffuse intimal t h i c k e n i n g s h o w s as t h e u p p e r m o s t reflective layer, b o u n d by a high reflective internal elastic lamina. T h e media is present as a u n i f o r m m e d i o c r e b a c k - s c a t t e r i n g layer u n d e r n e a t h the internal elastic lamina, a n d is e n c o m p a s s e d by the external clastic lamina, which a p p e a r s as a s t r o n g b a c k -s c a t t e r i n g layer 'figure 3-2A). Lipid rich areas a n d calcifications o b s e r v e d in the h i s t o l o g y c o u l d be m a t c h e d to r e g i o n s with a l o w e r b a c k - s c a t t e r signal in the ( ) C T i m a g e s (figures 5-2B and 32C). T h e calcification could be differentiated from the lipid-rich region bv t h e i r d e m a r c a t i o n , which was s h a r p for calcification and less defined in case of lipid-rich regions. O C T i m a g i n g of the p h a n t o m visualized the t w o layers as t w o different r e g i o n s o f
c
F i g u r e 3-2. O C T images (A C) with corresponding histology (D-F) of different
lesions: (A and D) early intimal thickening (i), with media (m) and the underlying external clastic lamina (eel) clearly visible, (B .\u<.\ I. : calcified lesions (c) in the
intima and media; (C and 1 ): lipid-rich region (1) in the intima. Bars indicate
0.5 m m . T h e r e c t a n g l e s i n d i c a t e t h e r e g i o n f r o m w h i c h t h e d a t a d e p i c t e d in figures 3 4 are t a k e n .
CHAPTER 3
b a c k - s c a t t e r e d O C T light. I n t h e a v e r a g e A - s c a n s o f t h e R O I , the O C T signal o f t h e s e c o n d l a y e r fitted very well t o t h e m o d e l (eq. 3-1) for t h e w h o l e r a n g e of/^ o f t h e u p p e r layer o f i n t r a l i p i d (figure 3-3A t o E ) . F u r t h e r m o r e , t h e d e t e r m i n e djut for t h e d e e p e r layer
s e c o n d layer c o r r e s p o n d e d very well t o the e x p e c t e d value o f 10 m m ', as d e t e r m i n e d in a s e p a r a t e m e a s u r e m e n t u s i n g f o c u s tracking (figure 3 - 3 F ) . F o r t h e v a s c u l a r tissue similar r e s u l t s w e r e o b t a i n e d . U s i n g t h e fitting a l g o r i t h m (eq. 3-1), it w a s p o s s i b l e t o f i t ^ for all r e g i o n s o f i n t e r e s t . In figure 3-4A, t h e average A - s c a n (thin line) o f t h e ROT m a r k e d in figure 3 - 2 A is p l o t t e d . T h e r e s u l t i n g fits for t h e i n t i m a l and m e d i a l layers (fig 3 - 4 A , t h i c k line) s h o w a very g o o d c o r r e l a t i o n (R2 = 0.99) with the p l o t t e d A-scan (fig 3-4A, t h i n line).
F o r calcified a n d lipid-rich tissue (fig 3-4B a n d 3-4C, respectively), similar results w i t h high c o r r e l a t i o n b e t w e e n fitted a n d m e a s u r e d signals w e r e o b t a i n e d (0.7 < R2 < 0.98). T h e data
o f g r a p h 3-4J3 is d e r i v e d from t h e calcified tissue s a m p l e s h o w n in figure 3-213, a n d t h e d a t a o f g r a p h 3 - 4 C w a s d e r i v e d f r o m the l i p i d - r i c h tissue s a m p l e s h o w n in figure 3 - 2 C . T h e 9 5 % c o n f i d e n c e i n t e r v a l o f t h e fitted 4u_ was a p p r o x i m a t e l y 0.1 m m ' in o u r data set,
i n d i c a t i n g a c c u r a t e fitting p r o c e d u r e s . T h e m e a n a t t e n u a t i o n coefficients for the different p l a q u e c o m p o n e n t s were d e t e r m i n e d and are s h o w n in figure 3-5. T h e attenuation coefficient o f t h e O C T light is h i g h e s t in t h e t h r o m b u s (11.2 ± 2.3 m m ' ) , w h e r e a s lipid-rich r e g i o n s ^ W depth ( 0 0 5 1 depth (mm)
B
\
0 5 1 depth (mm) 2% mtralipiO\
V
) 0 5 depth (mm) '- )**«» CF
Iioj
s 8 Z 6 a. 4 0.5 1 depth (mm) fitting result (*/,) 2 4 6 f . upo« byer <m m >F i g u r e 3-3 Average O C T A-scan data obtained from the intralipid/plastic phantom (thin black line) with the fitted signal, using equation 3-1, of the plastic lower layer (thick grey line).The intralipid (upper layer) varies in c o n c e n t r a t i o n (A: water, B: 0.2 %, C: 0.5%, D: 1%, E: 2%). In panel F the measured ,u is shown as a function of the ,u of the intralipid. The focus was placed at 0.61 m m in depth.
1000: m 1 0 0 , O O 10 L O C A L I Z E D JX MEASI R E M E N T S O F PLAQUE C O M P O N E N T S Intima: /J, = 6.1 ± 0.1 m m ' ^ / M e d i a : / J , = 7.6 ± 0 . 1 mm 0.0 0.2 0.4 0.6 0.8 1.0 1.2 depth (mm)
B
Intima: /v, = 5.3 ± 0.1 m m ' Calcification: /;, = 12.8 ± 0.1 mm V K y Fibrous: \i, = 7.6 ± 0.1 mm 0.2 I.4 0.6 0.8 1.0 1.2 1.4 1.6 depth (mm) 100- 10- 1-/ c a p : 1-/j, = 5.5 ± 0.1 mm V /Lipid-rich:/j, =J V
0.0 0.2 0.4 0.6 O.i depth (mm) 1.0 1.2F i g u r e 3-4 Average O C T A-scan data (thin grey line) of the regions depicted by the rectangles in the O C T figures 3-2A-C, respectively, and the fitted signal using equation 3-1 (thick dark line) with the calculated a t t e n u a t i o n coefficient /J. (± the 9 5 % confidence interval) depicted for the several ROI's.
CHAPTI R 3
h a v e t h e lowest a t t e n u a t i o n o f O C T light (3.2 ± 1.1 m m1) . T h e diffuse intimal tissue s h o w s an i n t e r m e d i a t e a t t e n u a t i o n [5.5 + 1.2 m m ). T h e a t t e n u a t i o n coefficient o f calcified tissue w a s 11.1 ± 4.9 m m and for medial tissue m e d i a 9.9 ± 1.8 m m . T h e a t t e n u a t i o n c o e f f i c i e n t s o f b o t h diffuse i n t i m a l tissue and lipid-rich tissue were f o u n d to differ significantly from o t h e r p l a q u e c o m p o n e n t s . ( p < 0 . 0 1 .
In o u r m e a s u r e m e n t ^ t h e / / measured t o r t h r o m b u s are ejuite high. Macroscopically, this t h r o m b u s was assessed as red t h r o m b u s , c o n s i s t i n g of mainly red b l o o d cells (RBC's) a n d fibrin. RBC's are k n o w n t o be highly s c a t t e r i n g , which explains the high value of //..
The m o r e a d v a n c e d w h i l e t h r o m b u s (platelet-rich), or a d v a n c e d i n t r a m u r a l t h r o m b i , will c o n t a i n less R B C ' s , w h i c h p r o b a b l y will result in a lower a t t e n u a t i o n coefficient.
T h e p o s i t i o n o f the focal p l a n e within the tissue is of influence on the a m o u n i ot d e t e c t e d light, and s u b s e q u e n t l y o n the O C T signal, i.e. u affects the a p p e a r a n c e o t a c e r t a i n feature. In figure 3 6, < ) C T images of the s a m e s a m p l e were taken with the focus p o s i t i o n at t h e l u m e n - i n t i m a b o u n d a r y figure Ï-6A . ami a s e c o n d image figure 3-6B with t h e focus shifted a p p r o x i m a t e l y 0.1 m m d o w n w a r d s c o m p a r e d to figure 3-6A. N o t e the difference in b a c k - s c a t t e r e d signal of the calcification, m a r k e d ' c \ and its s u r r o u n d i n g s . W h e r e a s the p o s i t i o n of the focus in the tissue s a m p l e clearly influences the b a c k - s c a t t e r e d --ignal in R( )I, the m e a s u r e m e n t o f t h e a t t e n u a t i o n coefficient s h o u l d not be affected d u e t o t h e c o r r e c t i o n w i t h i n the d e n o m i n a t o r o f t h e fit f u n c t i o n (eq. 3-1). I n d e e d , t h e a t t e n u a t i o n of the light in ibis R( )I did not differ and m e a s u r e d to be 1'L4 i 0." m m and
11.1 ± 0.6 m m . respectively. T h i s o b s e r v a t i o n was typical for all o t h e r lesion types,
i m a g e d w i t h different focus p o s i t i o n s . £ E a. 18 16 14 12 10 8 6 4 2 0
intima media lipid-rich
Figure 3-5. Average values for the attenuation coefficient u of intimal,
lipid-rich, calcific, and medial tissue and thrombus. Error h.\v< depict the standard deviation. ': p < 0 . 0 1 .
L o t VI l/l D/J, MEASI KI Ml NTS OF PLAQI I COMPON1 S'TS
= 0.5 mm
Figure 3-6 The effect of focus position on grej values is depicted in the two (>CT images of a calcification (c) in the intimal layer (i). The arrows indicate the position of the focus in each image. The vascular lumen is marked (I.). The rectangles indicate the ROI with the calculated u.
B
Figure 3-7 Examples ol < >CT images, previously shown in figure-- 2A and 2B, combined with a false color overlay oi the attenuation coefficient^ . To accentuate calcified \ l e s i o n s , area'- w i t h ti larger t h a n 12 m m ! are p l o t t e d , to a c c e n t u a t e
lipid l e s i o n s H areas w i t h fi s m a l l e r t h a n 4 m m ' are p l o t t e d . Bars i n d i c a t e 0.5 m m . c o l o r b a r s d e p i c t s v a l u e s of u .
CHAPTER 3
DISCUSSION
In this study, we demonstrated the proof of principle that quantitative analysis of the O C T signal allows the determination of the attenuation coefficient (ju) at 800 nm O C T light in a layered phantom as well as in layered arterial tissues. By fitting the O C T data of selected regions to a model describing the decay of the O C T signal with tissue depth, the
jj. could be determined for several components of the arterial wall including human
atherosclerotic plaque. Most remarkably, even in this limited data set, the clinically interesting lipid-rich regions were distinguishable from calcifications, which is not possible based on the mean O C T signal (the gray level) alone. In the future, this quantitative analysis of intrinsic and thus characteristic (optical) properties of the different tissue types can increase sensitivity and specificity of O C T in plaque detection and differentiation and, thus, its diagnostic potential. Figure 3-7 demonstrates the value of this additional information: Using a color overlay, the ju data of selected ROl's are added to the morphologic O C T image, facilitating the identification of calcification (figure 3-7A) or lipid-rich regions (figure 3-7B) at a glance.
As shown in figure 3-4, incorporation of the depth and the position of the focus of our O C T system in the fitting model of eq. 3-1, resulted in fitted curves with good to excellent correlation with the measured signals, f r o m these fits, the// of the ROI could be obtained within 10% uncertainty. The mean O C T signal in the ROI not only depends on the tissue back-scattering intensity, but also on the optical properties of the overlying tissue and on the strength of reflection at the boundary with the overlying region. Since optical properties and strength of reflection are not strictly related, due to the absorption of proximal tissue and backscattering influences of distal tissues, it is possible to have various combinations of mean O C T signals and attenuation coefficients. Support for the robustness of our fitting algorithm that corrects tor out of focus positions comes from the fact that the same value of the (u was found for two different focal positions
(figure 3-6). These results demonstrate that additional information from the raw O C T data can be obtained, enabling differentiation of plaque constituents based on their differences in(u . This additional information depends on the u of the tissue and not on
the experimental conditions.
Comparison with Other Models
The single scattering model used in this manuscript is widely used to describe the OCT signal, and its range of validity has been subject of many investigations. In a previous study we found that for a fixed focus geometry the single scattering regime extends to approximately 4 mean free paths'^ (mfp = /id) in accordance with findings by Bi/hcva, Wax and Pan."' IS In general, the range of validity of the single scattering model will
depend on the optical properties of the tissue, and the O C T setup (confocal parameters, signal to noise ratio). In 1994, Schmitr et a/, were among the first to apply the single scattering model for measuring the optical properties of rat aorta by OCT.13 They calculated
LOCALIZED/z MEASUREMENTS OF PLAQUE COMPONENTS
interfaces o f t h e s a m p l e h o l d e r with their 830 n m O C T s e t u p t o b e 14.9 ± 2.3 mm"1, w h i c h
is h i g h e r c o m p a r e d t o o u r r e s u l t s o f 9.9 + 1.9 mm"1. We a t t r i b u t e this difference t o the
h i g h c o n t e n t o f s t r o n g b a c k - s c a t t e r i n g elastin in a o r t i c s e g m e n t s c o m p a r e d t o o u r , less elastic, c a r o t i d s a m p l e s , and differences in s e t u p .
S c h m i t t etal. r e c o g n i z e d t h e effect o f m u l t i p l e s c a t t e r i n g o n t h e signal a t t e n u a t i o n for O C T and p r o p o s e d a m o r e c o m p r e h e n s i v e m o d e l , based o n t h e E x t e n d e d H u y g e n s F r e s n e l ( E H F ) f o r m a l i s m t h a t i n c l u d e d t h e d e g r a d a t i o n o f t h e m u t u a l c o h e r e n c e o f the s a m p l e b e a m .1 3 T h i s a p p r o a c h w a s f u r t h e r e x p l o r e d by T h r a n e et a/.. T h e i r m o d e l is t h e m o s t
c o m p r e h e n s i v e m o d e l o f the O C T signal to date.1 9 N e x t t o the so-called r o o t m e a n s q u a r e
s c a t t e r i n g angle, a m e a s u r e of the s a m p l e ' s s c a t t e r i n g a n i s o t r o p v , it p r o v i d e s e x t r a c t i o n o f t h e scattering coefficient (ja) o f arterial tissue, a n optical p r o p e r t y similar to t h e / / e x t r a c t e d in this study, p a r t i c u l a r l y l o r n o n - o r weakly a b s o r b i n g tissues.
Recently, L e v i t z et al. u s e d t h e E H F m o d e l t o d e t e r m i n e a o f 14 a o r t a s e g m e n t s a t 1300 n m O C T light.2" T h e t r e n d s b e t w e e n t h e m e a s u r e d / / o f the d i f f e r e n t tissue t y p e s s h o u l d be and are similar to o u r results, except for calcified lesions. H o w e v e r , their findings, d e s c r i b e d in ranges rather than average values, gave larger values than o u r s . A p p r o x i m a t e l y 9 5 % o f t h e i r n o r m a l arterial s a m p l e s h a d / / b e t w e e n 15 a n d 39 mm"1, w h i l e // w a s l o w e r
t h a n 15 m m ' in a b o u t 6 0 % o f lipid-rich a n d fibro-calcific p l a q u e s . F u r t h e r m o r e , f i b r o u s l e s i o n s d e m o n s t r a t e d c o n s i d e r a b l e v a r i a t i o n i n / * . T h e s e a b s o l u t e d i f f e r e n c e s c a n n o t b e a t t r i b u t e d t o t h e difference in w a v e l e n g t h u s e d , since a t t e n u a t i o n coefficients at 1300 n m a r e a p p r o x i m a t e l y 2 0 % l o w e r t h a n at 800 n m . " Alternatively, an e x p l a n a t i o n c o u l d b e f o u n d in t h e e f f e c t o f t h e t e m p e r a t u r e o f t h e i r s a m p l e s d u r i n g i m a g i n g . I n o u r m e a s u r e m e n t s , care w a s taken to keep the s a m p l e s at 37 ° C b e c a u s e especially t h e / / o f fatty tissue d e p e n d s o n the t e m p e r a t u r e . Preliminary results from o u r lab indicate that i n c r e a s i n g the e x p e r i m e n t a l e n v i r o n m e n t t r o m 2()°C t o 3 7 ° C may result in a 5-fold decrease i n / t (data n o t s h o w n ) . A s e c o n d e x p l a n a t i o n for the differences in the results o f Levitz c o m p a r e d to o u r s c o u l d be ascribed t o the different and m u c h m o r e c o m p l i c a t e d m o d e l o f O C T signals in s c a t t e r i n g t i s s u e s . B o t h m o d e l s a r e n o t perfectly a p p l i c a b l e t o t h e O C T m e a s u r e m e n t g e o m e t r y . A d i s c u s s i o n o f t h e l i m i t a t i o n s o f t h e E H F m o d e l is b e y o n d t h e s c o p e o f t h i s p a p e r , t h e r e a d e r is r e f e r r e d t o ref. 21 and ref. 2 2 . I n s u m m a r y , w h e n b e y o n d their r a n g e of applicability, o u r m o d e l will u n d e r e s t i m a t e // ; t h e E F I F m o d e l will o v e r e s t i m a t e fx . Finally, w e did n o t c o n s i d e r t h e differences in b a n d w i d t h o f t h e light s o u r c e u s e d in b o t h s t u d i e s . F o r u s , t h e b a n d w i d t h w a s a p p r o x i m a t e l y twice t h a t o f t h e s o u r c e u s e d in t h e s t u d y of L e v i t z . F u r t h e r analysis, like t h e w o r k r e c e n t l y p u b l i s h e d by X u et al. in w h i c h spectrally d e p e n d e d c h a n g e s in s c a t t e r i n g a n d a b s o r p t i o n w i t h i n t h e O C T s o u r c e s p e c t r u m w e r e d e t e r m i n e d , m i g h t p r o v i d e m o r e i n f o r m a t i o n .2 3
In a r e c e n t study, we d e m o n s t r a t e d t h a t t h e m o d e l d e p l o y e d in this p a p e r is equally a p p r o p r i a t e as the E H F m o d e l for d e s c r i b i n g t h e O C T signal for weakly scattering media,1"
w h i c h is n o t s u r p r i s i n g since t h e E H F m o d e l d e s c r i b e s t h e single and m u l t i p l e s c a t t e r i n g r e g i m e s s i m u l t a n e o u s l y . " We h a v e f o u n d t h a t t h e r e is n o significant i m p r o v e m e n t o f fit statistics c o m p a r e d t o the single s c a t t e r i n g m o d e l for a t t e n u a t i o n coefficients o f u p to
CHAPTER 3
5 mm"1.1 5 2 4 U p u n t i l 17 m m ' t h e single s c a t t e r i n g m o d e l still yields reliable r e s u l t s for a
m o n o layered s a m p l e . I n this study, we d e m o n s t r a t e d t h a t o u r single s c a t t e r i n g m o d e l c a n also b e a p p l i e d t o layered arterial samples. W h e t h e r this a s s u m p t i o n is also valid for larger s c a t t e r i n g d e p t h s a n d t h a t , i n s t e a d of m e a s u r i n g t h e tissue p r o p e r t y the O C T signal a t t e n u a t i o n is u s e d for d i f f e r e n t i a t i o n of t h e p l a q u e c o n s t i t u e n t s , still h a s t o b e e x p l o r e d . T h e E H F m o d e l has p r o v e n to e x t r a c t the optical p r o p e r t i e s of layered tissue correctly in a n u m e r i c a l p h a n t o m c o n s t r u c t e d w i t h M o n t e C a r l o simulations.2"1
T h e a p p r o a c h o f u s i n g q u a n t i t a t i v e data from t h e m e a s u r e d O C T signals is in line w i t h t h e a p p r o a c h f r o m o t h e r g r o u p s a n d o t h e r i n t r a v a s c u l a r i m a g i n g t e c h n i q u e s . R e c e n t l y T e a r n e y et a/, d e m o n s t r a t e d t h a t by analysis o f the v a r i a n c e in t h e O C T signal, t h e m a c r o p h a g e c o n t e n t of specific regions of t h e atherosclerotic plaque could be determined.2'1
T h e s e r e s u l t s s u g g e s t t h a t O C T n o t only c a n d e t e c t t h e lipid p o o l a n d precisely m e a s u r e t h e c a p thickness, b u t also can detect inflammation, the third characteristic o f the vulnerable p l a q u e . O t h e r optical t e c h n i q u e s that arc c a p a b l e o f q u a n t i t a t i v e d e t e r m i n a t i o n of p l a q u e c o m p o s i t i o n are R a m a n , n e a r - i n f r a r e d s p e c t r o s c o p y , and speckle analysis.2 2' T e a r n e y a n d B o u m a d e m o n s t r a t e d t h a t t h i n c a p p l a q u e , thick c a p p l a q u e and n o r m a l a o r t a c o u l d b e d i f f e r e n t i a t e d by analysis o f s p e c k l e pattern f l u c t u a t i o n s , based o n e x p e c t e d differences in B r o w n i a n motion.2'1 B e c a u s e t h e s e are en face t e c h n i q u e s , they will yield i n f o r m a t i o n o f t h e
w h o l e u n d e r l y i n g tissue as o n e p a r a m e t e r , i m p l y i n g that critical features as the p o s i t i o n o f t h e c o m p o n e n t s c a n n o t b e r e v e a l e d . In c o n t r a s t , O C T h a s t h e a d v a n t a g e o f c o m b i n i n g m o r p h o l o g i c i m a g i n g w i t h localized functional i m a g i n g , r e s u l t i n g in a c o m b i n a t i o n o f t i s s u e i d e n t i f i c a t i o n and p o s i t i o n .
Finally, i n t r a y a s c u l a r u l t r a s o u n d (IVUS) also allows q u a n t i t a t i v e m e a s u r e m e n t s , in situ, o f p l a q u e c o n s t i t u e n t s . B o t h e l a s t o g r a p h y a n d r a d i o f r e q u e n c y d a t a analysis c a n identify d i f f e r e n t p l a q u e features. H o w e v e r , these t e c h n i q u e s have limited spatial r e s o l u t i o n , w h i c h is a b o u t t e n f o l d l o w e r t h a n with OCT,3 0 , 3 1 which m a k e s it i m p o s s i b l e to precisely m e a s u r e t h e c a p t h i c k n e s s .
Clin if a 11 i/ip tic a tio n s
T h e ability t o d e t e c t a n d m o n i t o r t h e y u l n e r a b l e p l a q u e is keenly s o u g h t t o d e f i n e its n a t u r a l h i s t o r y a n d s u p p o r t the s t u d i e s t o eyaluate p r o g r e s s i o n and r e g r e s s i o n . C u r r e n t l y , O C T is t h e o n l y i n t r a v a s c u l a r i m a g i n g technicjue c a p a b l e o f i m a g i n g the m o r p h o l o g y o f t h e v a s c u l a r wall w i t h a r e s o l u t i o n t h a t allows v i s u a l i z a t i o n o f small a n a t o m i c a l s t r u c t u r e s . I t s clinical applicability is c u r r e n t l y u n d e r i n v e s t i g a t i o n , and already has s h o w n g r e a t p o t e n t i a l . ' ' In this study, we d e m o n s t r a t e that a d d i t i o n a l i n f o r m a t i o n is available in t h e b a c k - s c a t t e r e d signal, w h i c h c a n b e u s e d t o also o b t a i n q u a n t i t a t i v e i n f o r m a t i o n of imaged tissue s t r u c t u r e s . U s i n g a false c o l o r overlay indicating regions with a low o r high a t t e n u a t i o n c o e f f i c i e n t (figure 3-7) o n t h e o r i g i n a l image m a y h e l p t o faster identify p l a q u e t y p e s . A l t h o u g h t h e s e l e c t i o n of t h e R O l for these o v e r l a y s w a s d o n e manually, p r e l i m i n a r y a n a l y s i s i n d i c a t e t h a t g e n e r a t i n g a u t o m a t e d c o l o r o v e r l a y by m e a n s o f a sliding w i n d o w will r e s u l t in r o u g h l y t h e s a m e i m a g e s as p r e s e n t e d in t h e m a n u s c r i p t . F u r t h e r r e s e a r c h o n
LOCALIZED/i MEASUREMENTS OF PLAQUE COMW >\r\ I S
t h e o p t i m i z a t i o n o f t h e a l g o r i t h m t o p r e s e n t the tissue p r o p e r t i e s h a s to b e p e r f o r m e d . F o r e x a m p l e , t h e effect o f s u d d e n i n c r e a s e s in t h e O C T signal w i t h d e p t h (as c a n be s e e n in figure 3-4) h a s t o b e e x p l o r e d . F u r t h e r m o r e , t h e calcification a p p e a r s less t h i c k in t h e a t t e n u a t i o n coefficient p l o t , c o m p a r e d t o t h e O C T a n d the h i s t o l o g i c a l i m a g e s . T h i s is p r o b a b l y d u e to t h e i n h e r e n t loss o f signal w i t h d e p t h . T h e r e s u l t i n g p o o r s i g n a l - t o - n o i s e r a t i o in d e e p e r r e g i o n s o f t h e i m a g e will r e s u l t in u n d e r e s t i m a t i o n o f t h e local a t t e n u a t i o n coefficient. A similar e x p l a n a t i o n c a n b e given for t h e i n d i c a t i o n o f lipid in t h e l o w e r r i g h t c o r n e r in figure 3-7B. Additionally, t h e use o f t h e a t t e n u a t i o n coefficient d e t e r m i n e d f r o m t h e O C T signals m a y also h e l p t o identify p a t h o l o g i c a l tissues for o t h e r clinical specialties. T h e d e t e c t i o n o f t u m o r s , e.g. in t h e e s o p h a g u s , is h a m p e r e d by t h e loss o f a n a t o m i c a l s t r u c t u r e c o m p a r e d t o n o r m a l tissue. A d d i t i o n a l i n f o r m a t i o n b a s e d o n a n i n t r i n s i c t i s s u e p r o p e r t y m i g h t h e l p t o b e t t e r identify a n d d i s c r i m i n a t e t h e t u m o r s .
Study Limitations
In t h i s p i l o t study, c a r o t i d a r t e r i e s with relatively small a n d b e g i n n i n g l e s i o n s w e r e u s e d r a t h e r than v u l n e r a b l e lesions in c o r o n a r y arteries. Still, t h e c o m b i n a t i o n o f g r a y scale O C T i m a g e s w i t h tissue specific a t t e n u a t i o n coefficients was feasible. We d o n o t claim t o d i f f e r e n t i a t e l e s i o n s b a s e d o n , u m e a s u r e m e n t s a l o n e , b u t state t h a t q u a n t i t a t i v e a n a l y s i s m i g h t e n h a n c e t h e d i a g n o s t i c capabilities o f O C T . T h e r e f o r e , t h e use o f c a r o t i d a r t e r i e s w i t h i n c i p i e n t a t h e r o s c l e r o t i c l e s i o n s a n d t h e lack o f g e n u i n e a t h e r o m a s h o u l d n o t b e c o n s i d e r e d to be a p r o b l e m . E v e n m o r e , the d e t e c t e d differences iniut o f t h e i n c i p i e n t fat
d e p o s i t s c o m p a r e d t o t h e o t h e r tissue c o m p o n e n t s i n d i c a t e a high sensitivity for lipid d e t e c t i o n , u s i n g o u r a l g o r i t h m .
T h e light s o u r c e o f a n O C T s e t u p d e t e r m i n e s t h e s y s t e m r e s o l u t i o n a n d t h e i m a g i n g d e p t h . In t h e s e e x p e r i m e n t s , t h e O C T s y s t e m light s o u r c e had a c e n t e r w a v e l e n g t h o f 800 n m . T h e O C T systems t h a t are c u r r e n t l y available for clinical uses o p e r a t e w i t h a light s o u r c e at 1300 n m . F o r this w a v e l e n g t h r e g i o n t h e a t t e n u a t i o n o f light by tissue a n d b l o o d is l o w e r , w h i c h i n c r e a s e s t h e i m a g i n g d e p t h . F u r t h e r m o r e , b o t h t h e axial a n d lateral r e s o l u t i o n o f o u r system w a s a p p r o x i m a t e l y t h r e e fold b e t t e r t h e n t h e clinical u s e d O C T s y s t e m . F u r t h e r s t u d i e s a r e n e e d e d t o d e t e r m i n e t h e effects o f t h e s e d i f f e r e n c e s o n t h e m e a s u r e d a t t e n u a t i o n coefficients. T h e o r y and p r e l i m i n a r y e x p e r i m e n t s b o t h i n d i c a t e t h a t o u r a p p r o a c h and results still will be valid for l o w e r r e s o l u t i o n s a n d t h e w a v e l e n g t h r a n g e a r o u n d 1300 n m .
CONCLUSION
Simple q u a n t i t a t i v e analysis o f t h e O C T signals allows in situ d e t e r m i n a t i o n o f t h e intrinsic optical a t t e n u a t i o n coefficient o f atherosclerotic tissue c o m p o n e n t s w i t h i n r e g i o n s o f i n t e r e s t . C o m b i n i n g m o r p h o l o g i c a l i m a g i n g by O C T w i t h t h e o b s e r v e d d i f f e r e n c e s i n o p t i c a l a t t e n u a t i o n c o e f f i c i e n t s o f t h e v a r i o u s r e g i o n s m a y e n h a n c e d i f f e r e n t i a t i o n b e t w e e n various plaque types within the vessel wall. T h i s may c o n t r i b u t e to a b e t t e r d e t e c t i o n a n d m a n a g e m e n t o f t h e v u l n e r a b l e p l a q u e .
CHAPTER 3
REFERENCES
1. Fujimoto, J. G , Brezinski, M. E., Tearney, G. J., Boppart, S. A., Bouma, B., Hee, M. R.. Southern, |. F., and Swanson, E. A., "Optical biopsy and imaging using optical coherence tomography," Nat Med, 1, 970-972 (1995).
2. Huang, D., Swanson, E. A., Lin, C. R, Schuman, j . S., Stinson, W. G., Chang, W., Ilee, M. R., H o t t e , T., Gregory, K., Puliafito, C. A., Fujimoto, |. G , and et al, "Optical coherence tomography," Science, 254, 1178-1181 (1991).
3. Brezinski, M. E., Tearney, G J., Bouma, B. E., Izatt, J. A., Hee, M. R., Swanson, E. A., Southern, J. E , and Fujimoto, J. G , "Optical coherence tomography for optical biopsy -Properties and demonstration of vascular pathology," Circulation, 93, 1206-1213, (1996). 4. Fujimoto, J. G , Boppart, S. A., Tearney, G. ]., Bouma, B. E., Pitris, C , and Brezinski, M. I.., "High resolution in vivo intra-arterial imaging with optical coherence tomography," Heart, 82. 128-133, (1999).
5. Jang, I. K., Bouma, B. E., Kang, D. II.. Park, S. J., Park, S. W., Seung, K. B.. Choi, K. B., Shishkov, M., Schlendorf, K., Pomerantsev, E., ïlouser, S. I.., Aretz, H. T , and Tearnev, G. J., "Visualization of coronary atherosclerotic plaques in patients using optical coherence t o m o g r a p h y : C o m p a r i s o n with intravascular ultrasound," j.Aw.('.oil.Cardiol. 39, 604-609 (2002)'.
6. Yabushita, H., Bouma, B. E., Houser, S. L., Aretz, II. T , Jang, I. K., Schlendorf, K. II., K a u f f m a n , C. R., Shishkov, M., Kang, D. 11., H a l p e r n , E. E , and Tearney, G. [., "Characterization of human atherosclerosis by optical coherence tomography," Circulation,
106, 1640-1645 (2002).
7. Kolodgie, F. D., Burke, A. P., Farb, A., Gold, II. K., Yuan, J. V., Narula, [., Finn, A. V., and Virmani, R., " T h e thin-cap flbroatheroma: a type of vulnerable plaque - T h e major precursor lesion to acute coronary syndromes," Curr.Opinion Cardiol. 16, 285-292 (2001). 8. Van Leeuwen, T. G., Faber, D. )., and Aaldcrs, M. C , "Measurement of the axial point spread function in scattering media using single-mode fiber-based optical coherence tomography," IEEE J.Sel.Top.Quantum Electronics 9, 227-233 (2003).
9. Cilesiz, I. F. and Welch, A. ]., "Optical properries of human aorta: are they affected bv cryopreservation?," Lasers Surg.Med.X4, 396-402 (1994).
10. Rollins, A. M., Kulkarni, M. D., Yazdanfar, S., L'ng-arunyawee, R., and Izatt, J. A.. "In vivo video rate optical coherence tomography," Opt.Express, 3, 219-229, (1998). 1 1 . Sheppard, C. ]. and Wilson, T , "The theory of the direct-view con focal microscope,"
J.Microsc. 124', 107-117 (1981).
12. Izatt, J. A., Hee, M. R., Owen, G. M., Swanson, E. A., and Fujimoto, |. G., "Optical coherence microscopy in scattering media," Opt.Lett. 19, 590-592 (1994).
1 3 . Schmitt, J. M., Knuttel, A., Yadlowsky, M., and Eckhaus, M. A., "Optical-coherence tomography of a dense tissue: statistics of attenuation and backscattering," Phys.Med.Bio/. 39, 1705-1720 (1994).
14. Altman, D. G , "Comparing groups - continuous data," in Altman, D. G (ed.) Practical statistics for medical research London: Chapman and Hall, 1997, pp. 205-217.
15. Faber, D. J., van der Meer, F. J., and Aaldcrs, M. C. G , "Quantitative measurement of attenuation coefficients of weakly scattering media using optical coherence tomography." Opt.Express, 12, 4353-4365 (20(14).
16. Bizheva, K. K., Siegel, A. M., and Boas, D. A., "Parh-Iength-resolved dynamic light s c a t t e r i n g in highly s c a t t e r i n g r a n d o m media: T h e t r a n s i t i o n to diffusing wave spectroscopy," Phys.Kev.E, 58, 7664-7667 (1998).
17. Pan, Y, Birngrubcr, R., and Engelhardt, R., "Contrast limits of coherence-gated imaging in scattered media," Appl.Opt. 36,2979-2983 (1997).
18. Wax, A., Yang, C , Dasari, R. R., and Feld, M. S., "Path-length-resolved dynamic light scattering: modeling the transition from single to diffusive scattering," Appl.Opt. 40, 4222-4227 (Aug.2001).
1 9. Thrane, L., Yura, II. T , and Andersen, P. E., "Analysis of optical coherence tomography systems based o n the extended Huygens-Fresnel principle," J.Opt.Soc.Aw. A, 17, 484-490 ( 2 0 0 0 ) .
2 0 . Levitz, D., T h r a n e , I.., Frosz, M. II., Andersen, P. E., Andersen, C. B., Valanciunaite, J., Swarding, J., Andersson-Engels, S., and Hansen, P. R., "Determination of optical scattering
LOC U J Z E D / i MEASUREMENTS < )F PLAQUE COMPONENTS
p r o p e r t i e s of highly-scattering media in optical c o h e r e n c e t o m o g r a p h y i m a g e s , "
0pt.Express,\2, 249-259 (2004).
2 1 . Karamata, B., Lambelet, P., Laubscher, M., Leutenegger, M., Bourquin, S., and Lasser, T., "Multiple scattering in optical coherence tomography. Part I: Investigation and modeling.," ].Opt.Soc.Am.A, 22, 1369-1379 (2005).
22. Karamata, B., Lambelet, P., Leutenegger, M., Laubscher, M., Bourquin, S., and Lasser, T., "Multiple scattering in optical coherence tomography. Part II: Experimental and theoretical investigation of cross-talk in wide-field optical coherence tomography.,"
j.Opt.Soc.Am. A, 22, 1380-1388 (2005).
2 3 . Xu, C. Y., Marks, D. L., Do, M. N., and Boppart, S. A., "Separation of absorption and scattering profiles in spectroscopic optical coherence tomography using a least-squares algorithm," Opt. Express, 12, 4790-4803 (2004).
2 4 . Faber, D. |., Aalders, M. C. G., and Van Leeuwen, T. G., "Curve fitting for quantitative measurement of attenuation coefficients," Proc.SPlE, vol. 5690 in press (2005). 2 5 . Thrane, 1.., Frosz, M. H., Jorgensen, T. M., Tycho, A., Yura, II. T., and Andersen, P. E.,
"Extraction of optical scattering parameters and attenuation compensation in optical coherence tomography images of multilavered tissue structures.," Opt. Lett., 29, 1641-1643 (2004).
26. Tearney, G. J., Yabushita, H., Houser, S. L., Aretz, H. T., Jang, I. K., Schlendorf, K. H., Kauffman, C. R., Shishkov, M., Halpern, E. E , and Bouma, B. E., "Quantification o f macrophage c o n t e n t in atherosclerotic plaques by optical c o h e r e n c e tomography,"
Circulation, 107, 113-119 (2003).
27. Brennan, J. E , I I I , Romer, T. J., Lees, R. S., Tercyak, A. M., Kramer, J. R., Jr., and Feld, M. S., "Determination of human coronary artery composition by Raman spectroscopy,"
Circulation, 96, 99-105 (1997).
2 8 . Moreno, P. R., Lodder, R. A., Purushothaman, K. R., Charash, W. E., O'Connor, W. N . , and Muller, J. E., "Detection of lipid pool, thin fibrous cap, and inflammatory cells in human aortic atherosclerotic plaques by near-infrared spectroscopy," Circulation, 105, 923-927 (2002).
29. Tearney, G.J. and Bouma, B. E., "Atherosclerotic plaque characterization by spatial and temporal speckle pattern analysis," Opt.Lett. 27, 533-535 (2002).
30. de Korte, C. 1.., van der Steen, A. F., Cepcdes, E. I., Pasterkamp, G., Carlier, S. G., Mastik, F., Schoneveld, A. FL, Serruys, P. W., and Bom, N., "Characterization of plaque components and vulnerability with intravascular ultrasound clastographv," Pbys.Med.Bio/. 45, 1465-1475 (2000).
3 1 . Komiyama, N., Berry, G. J., Kolz, M. L., Oshima, A., Metz, J. A., Preuss, P., Brisken, A . E , Paulina, M. XL, Yock, P. G , and Fitzgerald, P. J., " T i s s u e characterization o f atherosclerotic plaques by intravascular ultrasound radiofrequency signal analysis: an ///
