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Vascular applications of quantitative optical coherence tomography
van der Meer, F.J.
Publication date
2005
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Citation for published version (APA):
van der Meer, F. J. (2005). Vascular applications of quantitative optical coherence
tomography.
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TOMOGRAPHY OF ARTERIAL WALL
C O M P O N E N T S
Freek J. van der Meer, Dirk J. Faber, Jop Perrée, Gerard Pasterkamp,
David Baraznji Sassoon, Ton G. van Leeuwen
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 ) c a n v i s u a l i z e t h e a r t e r i a l w a l l a n d a t h e r o s c l e r o t i c p l a q u e s w i t h high r e s o l u t i o n . I n this study, we verified t h e q u a n t i t a t i v e analysis o f O C T for t h e d i m e n s i o n s o f p l a q u e s t r u c t u r e a n d t h e optical attenuation coefficient of the c o m p o n e n t s . We assessed the effect o f balloon dilation o n t h e O C T signal f r o m t h e m e d i a l layer o f p o r c i n e c a r o t i d a r t e r i e s ex vivo. I m a g i n g o f h u m a n a u t o p s y s a m p l e s w a s p e r f o r m e d f r o m t h e l u m i n a l side w i t h a h i g h (3.5 ^ m axial a n d 7 jum lateral) r e s o l u t i o n O C T s y s t e m ( a r o u n d 800 n m ) o r a regular (15-20 jum axial a n d 2 0 /itn lateral) r e s o l u t i o n O C T s y s t e m ( a r o u n d 1300 n m ) . F o r e a c h s a m p l e , d i m e n s i o n s w e r e m e a s u r e d by h i s t o m o r p h o m e t r y a n d O C T and t h e optical a t t e n u a t i o n w a s m e a s u r e d . I n a tissue culture setup, p o r c i n e carotid arteries were dilated and the a t t e n u a t i o n coefficients o f t h e dilated s e g m e n t s w e r e c o m p a r e d t o a c o n t r o l s e g m e n t f o r 4 h o u r s . Q u a n t i t a t i v e analysis showed a s t r o n g and significant correlation between O C T and histology cap thickness m e a s u r e m e n t s for b o t h O C T s y s t e m s . F o r b o t h s y s t e m s , t h e m e a s u r e d a t t e n u a t i o n coefficients of diffuse intimal t h i c k e n i n g and lipid-rich r e g i o n s differed significantly f r o m m e d i a a n d calcifications. B a l l o o n d i l a t i o n i n d u c e d a t i m e d e p e n d e n t i n c r e a s e in t h e a t t e n u a t i o n coefficient, w h i c h m a y b e a t t r i b u t e d t o t h e i n d u c t i o n o f a p o p t o s i s . B o t h t h e high and regular r e s o l u t i o n O C T s y s t e m s can precisely image t h e a t h e r o s c l e r o t i c p l a q u e s . Q u a n t i t a t i v e analysis o f t h e O C T signals allowed in situ d e t e r m i n a t i o n o f the i n t r i n s i c o p t i c a l a t t e n u a t i o n coefficient o f a t h e r o s c l e r o t i c 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 interest, w h i c h can f u r t h e r h e l p t o d i s c r i m i n a t e t h e p l a q u e a n d arterial wall c o m p o n e n t s .INTRODUCTION
Optical coherence tomography (OCT) is a modality for minimally invasive imaging of tissue, which, due to its high resolution (1-20 urn), has proven to be a powerful diagnostic tool in various medical disciplines.1 It is an optical analogue of ultrasound imaging; in an
A-scan the amplitude of the back-scattcrecl light from the tissue structures is plotted as a function of depth.2 Sequential A-scans form a cross-sectional image (B-scan), in which the
contrast is based on differences in optical scattering properties of tissue sample constituents. In cardiology, Brezinsky and coworkers demonstrated the ability of OCT to image vascular pathology,' and, although still in an experimental phase, it has already been shown that intravascular O C T is capable of imaging the arterial wall in vivo.A
Based on the observed morphology and differences in gray values in the OCT images, different types of atherosclerotic plaques can be distinguished. Due to its high resolution, O C T is the only imaging technique that is capable to identify the (fibrous) cap overlying lipid cores. Consequently, intravascular O C T is capable of in vivo imaging, and thus recognizing, the so called vulnerable plaque. Based on data from ex vivo morphological studies, these plaques are defined as diose in which a thin fibrous cap (<65-150>um)
overlies a substantial lipid core (>40%). Although the use of O C T in cardiovascular dia-gnosis has great potential, the ability of O C T to do quantitative measurements, and thus the full potential of O C T , still has not yet been fully investigated. Next to the straightforward quantitative analysis of e.g. lipid core and cap dimensions and back-scatter amplitude, the O C T data can comprise more localized information ot the (optical) properties of the tissue under investigation. For instance, from the reduction ot the OCT signal with depth, localized optical properties as the attenuation coefficient can be determined. T h e groups of de Boer and Chen have shown that from the change in polarization of the O C T light, the birefringence can be estimated."' Furthermore, by analysis of the variance in the O C T signal, the macrophage content of specific regions of the atherosclerotic plaque could be determined.
In a recent study, we demonstrated from a simple algorithm and a high resolution (3.5 ^ m axial and 7 ,um lateral) O C T system based on a Ti-Sapphire laser around 800 nm,s;' that
the attenuation of the O C T signal within the plaque volume can be measured in indicated regions of interest and used as additional information for discrimination of plaque components.'"•'' However, in the clinical application of intravascular O C T regular resolution (10 ,um axial and 20 /xm lateral) systems, based on light sources around 1300 nm, arc-utilized. We hypothesize that, although their image qualities may differ, both O C T systems are capable of accurately measure the clinically relevant dimensions. Also, we expect that the quantitative spectroscopic analysis for the discrimination of arterial wall (and plaque) c o m p o n e n t s is also feasible for a regular resolution O C T systems at 1300 nm.
In this study we investigate the possibility to use OCT for quantitative analysis of the dimensions of plaque structure (e.g. cap thickness) for both systems and compare the measured attenuation coefficients of plaque constituents for the regular resolution,
1300 n m s y s t e m w i t h o u r p r e v i o u s o b t a i n e d results at 800 n m . F u r t h e r m o r e , to m i m i c t h e effect o f o u r s p e c t r o s c o p i c m e a s u r e m e n t s d u r i n g i n t e r v e n t i o n a l p r o c e d u r e s , we assess t h e effect o f b a l l o o n d i l a t i o n o n t h e O C T signal from t h e m e d i a o f p o r c i n e c a r o t i d arteries in a tissue c u l t u r e s e t u p .
MATERIALS AND METHODS
OCT ImagingT h e h i g h r e s o l u t i o n (3.5 ,um axial a n d 7 ,um lateral) O C T s e t u p c o m p r i s e d o f a T k S a p p h i r e laser s o u r c e w i t h a c e n t e r w a v e l e n g t h (A,) o f 8 0 0 n m a n d a b a n d w i d t h (AX) o f 120 n m . I n d e p t h s c a n n i n g (A-scan) w a s p e r f o r m e d by a r a p i d s c a n n i n g o p t i c a l delay line.1 -. T h e a m p l i t u d e a n d p h a s e o f t h e d e m o d u l a t e d signal w e r e digitized a n d s t o r e d in a c o m p u t e r . E a c h A - s c a n c o n s i s t e d o f 4 0 9 6 data p o i n t s . B - s c a n i m a g e s w e r e o b t a i n e d b y m o v i n g t h e tissue s a m p l e w i t h r e s p e c t t o t h e fixed s a m p l e a r m b e a m while p e r f o r m i n g A scans. I n e a c h B s c a n , 100 Ascans p e r m m w e r e a c q u i r e d , and p e r lesion 5 c o n s e c u t i v e B -scans w e r e o b t a i n e d , s p a c e d at 0.5-1.0 m m from each o t h e r . L a r g e r lesions w e r e imaged b v adjacent B - s c a n s , w h i c h w e r e r e c o m b i n e d to o n e i m a g e .
T h e regular n o r m a l r e s o l u t i o n s e t u p ( 1 5 / f m axial a n d 20 ^ m lateral) c o m p r i s e d b r o a d b a n d light s o u r c e s w i t h 1 3 0 0 n m as c e n t r a l w a v e l e n g t h ( S L D , 1 mW, A.= 1 3 0 0 n m , AA,=36 n m a n d A F C L a s e r , 10 mW, A.= 1300 n m , A A , = 4 5 n m ) . D e p t h s c a n n i n g (2-3 m m typically) w a s o b t a i n e d by m o v i n g t h e r e f e r e n c e m i r r o r ( r e t r o reflector) o n a n a r t i c u l a t e d a r m m o u n t e d o n a g a l v a n o m e t e r . After r e c o m b i n a t i o n o f t h e r e f l e c t i o n s f r o m t h e s a m p l e and reference a r m s , t h e A C c o m p o n e n t o f the i n t e r f e r e n c e light intensity w a s m e a s u r e d b y a p h o t o d i o d e ( N e w F o c u s ) . T h e signal w a s d e m o d u l a t e d a n d digitized in the c o m p u t e r (2048 s a m p l e p o i n t s p e r A - s c a n ) . L a t e r a l s c a n n i n g w a s p e r f o r m e d by m o v i n g t h e tissue s a m p l e o v e r 4 - 1 0 m m ( 2 0 0 - 5 0 0 lateral A - s c a n s , r e s p e c t i v e l y ) . V o l u m e s c a n n i n g ( t h e collection o f m u l t i p l e parallel B-scans) w a s p e r f o r m e d at a n i n t e r - s c a n d i s t a n c e o f 200 (u m
o v e r 2-4 m m .
Tissue samples and tissue handling
H u m a n arterial s a m p l e s w e r e o b t a i n e d from t h e C o o p e r a t i v e H u m a n T i s s u e N e t w o r k ( C H T N ) b r a n c h at t h e U n i v e r s i t y H o s p i t a l s o f C l e v e l a n d ( n = 1 4 ) a n d f r o m t h e d e p a r t -m e n t o f p a t h o l o g y o f t h e A c a d e -m i c Medical C e n t e r o f A -m s t e r d a -m ( n = 1 6 ) . T h e s a -m p l e s 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 frozen in liquid n i t r o g e n and s t o r e d at - 8 0 ° C . A n i m a l s a m p l e s w e r e h a r v e s t e d at t h e time o f sacrifice. T o facilitate O C T i m a g i n g , vessel s e g m e n t s w e r e c u t l o n g i t u d i n a l l y t o e x p o s e t h e l u m i n a l surface. D u r i n g t h e O C T i m a g i n g , w i t h e i t h e r the h i g h o r r e g u l a r r e s o l u t i o n s y s t e m , t h e s a m p l e s w e r e i m m e r s e d in saline.
T h e i m a g e d areas w e r e m a r k e d u s i n g I n d i a n ink. After i m a g i n g , t h e s a m p l e s w e r e fixed in 4 % f o r m a l i n for m o r e t h a n 4 8 h o u r s . If n e c e s s a r y , t h e s a m p l e s w e r e decalcified in
E D T A ( p H = 7.0) for o n e w e e k . After e m b e d d i n g in paraffin, s e c t i o n s w i t h a t h i c k n e s s of 5 fim w e r e c u t at t h e m a r k e d sites, and stained w i t h e i t h e r h a e m a t o x y l i n a n d e o s i n , elastin V o n G i e s s o n , o r p i c r o Sirius r e d . Based o n the ink m a r k e r s a n d a n a t o m i c a l l a n d m a r k s , O C T a n d h i s t o l o g y w e r e m a t c h e d , a n d m o r p h o l o g i c features (e.g. intimal t h i c k e n i n g , lipid rich r e g i o n s a n d calcifications) w e r e identified in t h e O C T images.
Tissue culture and balloon dilation
P o r c i n e carotid arteries ( n = 3 ) were excised directly after euthanasia and transferred t o an ice-cold i s o t o n i c buffer. T h e vessels were stripped o f excess adventitial tissue a n d m o u n t e d in a t i s s u e c u l t u r e s e t u p c a p a b l e o f keeping arterial s e g m e n t s alive a n d f u n c t i o n a l up to 7 d a y s .1 3 T h e v e s s e l wall w a s d i l a t e d using a s t a n d a r d P T C A c a t h e t e r .1 4 T h e b a l l o o n was
d i l a t e d , 3 t i m e s for 1 m i n u t e at 6 atm. T h e d i a m e t e r r a t i o o f b a l l o o n o v e r l u m e n was a p p r o x i m a t e l y 1.4. O C T i m a g e s w i t h the high r e s o l u t i o n O C T s y s t e m w e r e o b t a i n e d p r i o r , d u r i n g a n d u p t o 4 h o u r s after balloon d i l a t i o n . A n a d j a c e n t vessel s e g m e n t was u s e d as a c o n t r o l .
Quantitative analysis
F i r s t , t h e m o r p h o l o g i c f e a t u r e s such a s i n t i m a , m e d i a , lipid p o o l s , f i b r o u s caps a n d calcific n o d u l e s w e r e identified in histology and O C T images. T w o i n d e p e n d e n t o b s e r v e r s m e a s u r e d t h e d i m e n s i o n s o f t h e s e features and o f n o r m a l arterial s e g m e n t s in e i t h e r h i s t o l o g y o r O C T . T o m i n i m i z e t h e effect o f the difficult m a t c h i n g o f t h e t w o types o f i m a g e s , t h e m e a s u r e m e n t s w e r e p e r f o r m e d o n and a v e r a g e d o v e r 3-4 a d j a c e n t i m a g e s .
N e x t , 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 (JI) o f the identified r e g i o n s o f i n t e r e s t ( R O I ' s ) in s e l e c t e d arterial s e g m e n t s w e r e o b t a i n e d in a p r o c e d u r e as d e s c r i b e d p r e v i o u s l y . " In s h o r t , t h e d e p t h d e p e n d e n c e o f t h e a m p l i t u d e of t h e O C T signal can b e d e s c r i b e d as t h e p r o d u c t o f t h e axial p o i n t s p r e a d f u n c t i o n (PSF) o f the o p t i c s u s e d a n d t h e a t t e n u a t i o n o f the l i g h t by t h e tissue structures.9 , 1 5 , 1 6 I n the R O T , t h e a v e r a g e signal o f 5 0 - 1 0 0 adjacent
A-s c a n A-s aA-s a f u n c t i o n o f d e p t h w a A-s fitted to the m o d e l with ,u| as t h e fitting p a r a m e t e r . U s i n g
this s i m p l e analysis a l g o r i t h m , w h i c h i n c o r p o r a t e s t h e p o s i t i o n o f t h e f o c u s in t h e tissue a n d t h e d e p t h o f f o c u s , t h e s c a t t e r i n g c o e f f i c i e n t ^ for t h e R O I ' s was d e t e r m i n e d in situ.
Statistical analysis
All d a t a o f t h e m e a s u r e d d i m e n s i o n s a r e p r e s e n t e d as t h e i r m e a n v a l u e and t h e i r s t a n d a r d d e v i a t i o n . T h e statistical significance o f the m e a s u r e m e n t s w a s tested u s i n g A N O V A . P o s t - h o c c o m p a r i s o n s w e r e done u s i n g t-tests w i t h B o n f e r r o n i c o r r e c t i o n . T h e fitting p r o c e d u r e , w i t h 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 , r e s u l t e d in values o f /J. a n d t h e i r 9 5 % c o n f i d e n c e intervals.
F i g u r e 4-1: Examples of O C T images and corresponding histology. A and B: High resolution O C T image of a rat aorta with the corresponding histology (EvG stain) showing the media (m) and adventitia (a). T h e elastin fibrils in the media have an increased scattering.
C and D: Regular resolution O C T image of a healthy human renal artery and its
corresponding histology (EvG stain). Again, note the increased scattering of the elastic laminae between the media (m) and adventitia (a) and herween media and lumen.
E to G: Regular resolution O C T image or a h u m a n thoracic aorta and its corresponding histology (F: H E stain, G: PicroSirius stain under polarized light). N o t e the coincidence of the thin highly back-scattering layer in the O C T image and the thin collagenous structures in the histologic image as marked by the arrow (I: ostium of an intercostal artery, L: plaque). All bars represent 0.5 mm.
RESULTS
Qualitative analysis
W i t h b o t h O C T s y s t e m s , d i f f e r e n t features of the a t h e r o s c l e r o t i c p l a q u e w e r e clearly identifiable. Using ink. m a r k e r s and anatomical l a n d m a r k s , the O C T images were successfully m a t c h e d to histology. As a s t a r t i n g point, figures 4 - 1 A and B s h o w a high r e s o l u t i o n O C T i m a g e of a rat a o r t a s a m p l e a n d its c o r r e s p o n d i n g histological i m a g e , respectively. T h e i m a g e s d e m o n s t r a t e d t h e s u p e r b r e s o l u t i o n o f O C T imaging. T h e m e d i a can clearly b e d i s t i n g u i s h e d from t h e a d v e n t i t i a . F u r t h e r m o r e , n o t e t h e h i g h e r reflectivity o f t h e elastin fibrils visible in the media, as can be confirmed in t h e histological section. Regular resolution O C T i m a g i n g w a s n o t able t o distinguish t h e small fibers in t h e m e d i a . H o w e v e r , as c a n be o b s e r v e d in t h e O C T i m a g e o f a h e a l t h y h u m a n r e n a l a r t e r y in s a n d w i c h a n d its c o r r e s p o n d i n g histology (figures 4 - 1 C and D ) , also for 1300 n m light, m e d i a a n d adventitia
E 3 600 500 400 300 -200 100 0
*
• - # s ^ ' 200 h i s t o l o g y ( p m ) 300 400B
1400 1200 -1000 600 400 200 -1300)
n m,_
• :
r
Ts
-V-T
- 1 200 400 - • i 600 h i s t o l o g y (f/m) y = 1.15x Rr = 0.96 800 10F i g u r e 4-2: T h e results of thickness measurements in high resolution (A) and regular resolution (B) OCT. T h e thick grey line represents the linear fit. T h e dotted line represents the function y=x.
30 -i 25 -_ 20 • E, 15
io ^
5 0 D800nm D1300nm I 10 T 16 13 T 3 T 6intima media lipid-rich calcif.
Figure 4-3: Attenuation coefficient (/it) of plaque components as measured in high (white bars) and regular (grey bars) resolution OCT. The numbers represent the measurement counts.
can be distinguished and elastin fibers induce a large back-scattered O C T signal. T h e appearance of the plaque constituents matched the previously described features.4 In figure
4-1E and F, the regular resolution O C T image of a human thoracic aorta (500 scans) and its corresponding histology, respectively, are displayed as an example. The image was obtained at the level of an intercostal artery with on both sides atherosclerotic lesions. T h e thin scatter-lucent layer overlying the plaque clearly corresponds with the collagenous layer in the histology image. In other images, lipid-rich areas and calcifications observed in the histologv could be matched to regions with a lower back-scatter signal in the O C T images. The calcification could be differentiated from the lipid-rich region by their demarcation, which was sharp for calcification and less defined in case of lipid-rich regions.
Quantitative measurements
O n a one to one basis, the measurements of the dimensions of the different arterial wall and plaque components correlated moderately with the histological measurements (R2=0.57 and R2=0.34 for high and regular resolution O C T images, respectively). After
averaging over the lesion, the intra lesion variation of the dimensions was reduced, resulting in a better correlation between O C T obtained dimensions and histological measurements for the high and regular resolution OCT images (R2=0.93 and 0.96, respectively). As depicted
by the fitted slope (figure 4-2), for both systems, the OCT overestimated the dimensions as measured by histology 15 -25%.
Using our model,// for all ROI's were obtained with high correlations (R2 > 0.7) and
confidence intervals better than 1 mm"1. Compared to the 800 nm light of the high
6 n 0.5 •
H-Kf-<
B
0.4 •0.3-1
0 50 100 150 200 250 300 16 i 14 12 10>
I++4—f
0 50 100 150 200 250 300 F i g u r e 4 - 4 : Ex vivo m e a s u r e m e n t s of the medial layer thickness (A) and attenuation coefficient fx (B) of dilated (grey dashed line with squares) and non-dilated (black line with dots) porcine carotid artery segments (n=3). T h e balloon dilation (at t=0) resulted in a temporary increase in u,, probably due to compression or the media. After a return to base level,// of the media in the dilated segment showed an increase and a subsequent return to base level after 4 hours.for the regular resolution system, as depicted in figure 4-3, was lower but followed the trend. For the 1300 nm O C T light, the lipid-rich regions had the lowest attenuation of O C T light (2.3 ± 0.5 mm '). The diffuse intimal and the medial tissue showed intermediate attenuations (3.2 ± 1.2 mm ' and 6.7 ± 1.1 mm ', respectively). The attenuation coefficient of calcified tissue was largest: 26 ± 3.2 mm ' and differed significantly from diffuse intimal tissue and lipid-rich tissue (p<0.01).
In the ex-vivo culture setup, we were able to visualize the top part of the arteries with the high resolution O C T system, from the adventitia through the whole media. Analysis of the O C T images of the control segments revealed that the thickness and attenuation coefficient of the media was constant (0.49 ± 0.01 m m and 7.8 ± 0.3 mm ', respectively) over a period of more than 4 hours (figure 4-4). In contrast, the media of balloon dilated segments demonstrated a marked time dependant change in thickness and attenuation. During dilation (t=0), the thickness decreased approximately 35% (figure 4-4A), while the u increased with 7 5 % (figure 4-4B). Directly after dilation, the thickness and attenuation
returned to their baseline values. Whereas the thickness of the dilated segment remained constant and similar to its control segment (0.50 ± 0.01 mm), the attenuation coefficient showed a marked increase (41%) between 45 to 90 minutes after dilation. Although all three dilated segments showed an increase in/z[? there was a biological variability in the
onset of this increase. This results in a higher standard deviation in the rising edge of figure 4-4B compared to the standard deviation in the 'plateau phase' 120 to 240 minutes after dilation. Furthermore, the attenuation tended to return to the control values at approximately 4 hours after dilation for two of the three arteries.
D I S C U S S I O N
OCT has shown that its high resolution can result in very detailed images of the vessel wall. In this study, we confirmed the appearance of the different vascular and plaque components for both O C T systems. The fatty plaque and calcified lesions showed as darker regions than the normal intimal and medial arterial wall layers. We demonstrated that even elastin fibers that are not visual with any other current imaging technique, are clearly identified by O C T as bright but thin lines in the arterial wall. These differences in gray values in the O C T clearly help to distinguish the different constituents qualitatively. To investigate the quantitative aspects of the O C T images, we compared dimensions measured in the O C T images with those obtained in histology. Surprisingly, the correlation between OCT and histological measurements was low for both O C T systems. We attribute these low correlations to mismatches in the position of the images taken. Both O C T and histology resultin across sectional image of the sample of a mere 5-lO^m. It is impossible to obtain a histology section of 5 jum at exactly the same location as where the tomographic image of similar proportions was made. This, in combination with the local variations in dimensions, as depicted by the error bars in figure 4-2, explains the poor correlation. Taking the average dimensions of a lesion resulted in good correlations for both O C T systems. The slope of fitted lines, however, was larger than one, which can be attributed to dehydration of the sample during the histological procedure which will result in shrinkage of the histological samples. This finding may influence the threshold of cap thickness for classification of plaque as being vulnerable. For OCT, this threshold should be 15-25% more than the 65 micron as obtained from histological classifications.
In a previous study, we demonstrated that quantitative analysis of the attenuation coefficient of the different constituents was feasible with the high resolution O C T system."'" In this study, we verified that the differences in attenuation coefficients for these constituents were measurable for the regular resolution O C T system (figure 4-3). Furthermore, the differences between attenuation coefficients for the arterial wall and plaque constituents followed the same trend as for the high resolution (800 nm) O C T system, except for the calcified lesions. The data clearly demonstrate the lower attenuation for the 1300 nm O C T light, which explains the deeper imaging with this system. This finding is in agreement with the work of Schmitt et a/., who demonstrated that the
a t t e n u a t i o n c o e f f i c i e n t s at 1300 n m were a p p r o x i m a t e l y 20" '<> l o w e r than at 800 n m for rat a o r t i c t i s s u e . " ' T h e r e a s o n for the increased a t t e n u a t i o n for calcified R O I for 1300 n m O C T c o m p a r e d to 8 0 0 n m O C T light remains u n c l e a r . F u r t h e r s t u d i e s are n e e d e d t o clarify w h e t h e r the l o w b a c k - s c a t t e r i n g w i t h i n the calcification c o m b i n a t i o n w i t h t h e large index o f r e f r a c t i o n m i s m a t c h b e t w e e n t h e calcified tissue a n d its s u r r o u n d i n g may lead t o a n o v e r - e s t i m a t i o n o f t h e a t t e n u a t i o n coefficient as d e t e r m i n e d by o u r m o d e l .
T h e ex vino b a l l o o n dilation o f a pig carotid artery d e m o n s t r a t e d an u n e x p e c t e d increase in t h e a t t e n u a t i o n coefficient o f t h e media, w h i c h could n o t b e explained by a c h a n g e in the t h i c k n e s s (e.g. d u e t o e d e m a f o r m a t i o n ) . T h e t r e n d t o a n i n c r e a s e in the a t t e n u a t i o n c o e f f i c i e n t w i t h t i m e s u g g e s t s a n a p o p t o t i c r e s p o n s e o f t h e m e d i a . In a study by Perlman
et ai, if w a s s h o w n t h a t b a l l o o n dilatation induces a time d e p e n d a n t increase and s u b s e q u e n t
d e c r e a s e in t h e n u m b e r o f a p o p t o t i c cells.'4 We h y p o t h e s i z e t h a t the c h a n g e in s c a t t e r i n g ,
t h e b a s i s o f o u r O C T m e a s u r e m e n t s , can b e e x p l a i n e d by this a p o p t o t i c p r o c e s s , w h i c h i n v o l v e s a series o f m o r p h o l o g i c changes, in w h i c h m a n y p o t e n t i a l scatterers are involved. T h e i n i t i a l i n c r e a s e in s c a t t e r i n g could b e d u e t o c e l l u l a r s h r i n k a g e , t o c h r o m a t i n c o n d e n s a t i o n , n u c l e a r f r a g m e n t a t i o n o r cell b l e b b i n g . A p o p t o s i s is a n i m p o r t a n t phvsiological p a r a m e t e r , w h i c h currently can only be assessed by histological and biochemical a s s a v s . Recentlv, h i g h f r e q u e n c y u l t r a s o u n d was s h o w n t o d e t e c t a p o p t o s i s . - F u r t h e r e x p e r i m e n t s are n e e d e d t o v a l i d a t e t h e d e t e c t i o n o f O C T b a s e d o n s c a t t e r i n g c h a n g e s . P r e l i m i n a r y e x p e r i m e n t s o n isolated cells c o n f i r m o u r h y p o t h e s i s : an initial i n c r e a s e and a s u b s e q u e n t d e c r e a s e in s c a t t e r i n g after induction o f a p o p t o s i s h a s b e e n o b s e r v e d .
Clinical implications and limitations
U n s t a b l e atherosclerotic plaques are morphologically characterized by a lipid core covered by a t h i n fibrous cap.1 8 T h e s e u n s t a b l e plaques are very p r o n e t o r u p t u r e / f i s s u r e , especially
in t h e s h o u l d e r s o f t h e fibrotic c a p , with s u b s e q u e n t e x p o s u r e o f t h e t h r o m b o g e n i c lipid c o r e t o t h e f l o w i n g b l o o d , r e s u l t i n g in t h r o m b o s i s . U n f o r t u n a t e l y , u n s t a b l e p l a q u e s a r e d i f f i c u l t t o d e t e c t w i t h c u r r e n t l y e m p l o y e d i m a g i n g t e c h n i q u e s . T h e y a r e o f t e n h e m o d y n a m i c a l l y i n s i g n i f i c a n t a n d t h e r e f o r e difficult t o d e t e c t w i t h a n g i o g r a p h y .1' '
A n g i o s c o p y c a n , in s o m e c a s e s , d e t e c t the p o s i t i o n o f a n u n s t a b l e p l a q u e . H o w e v e r , a n g i o s c o p v only p r o v i d e s i n f o r m a t i o n on m o r p h o l o g y o f t h e e n d o - l u m i n a l surface a n d is t h e r e f o r e , like a n g i o g r a p h y , u n a b l e t o identify the e x t e n t o f an a t h e r o s c l e r o t i c p l a q u e i n t o t h e v e s s e l wall. F u r t h e r m o r e , intravascular u l t r a s o u n d i m a g i n g , a l t h o u g h b e i n g able t o i m a g e t h e vascular wall, is limited in the specific identification o f lipid-rich p l a q u e s . O t h e r t e c h n i q u e s like m a g n e t i c r e s o n a n c e angiography ( M R A ) a n d n u c l e a r m a g n e t i c r e s o n a n c e i m a g i n g ( M R I ) h a v e b e e n r e p o r t e d t o b e able t o identify u n s t a b l e p l a q u e s in t h e a o r t a b u t t h e i r r e s o l u t i o n is p r o b a b l y t o o limited to detect t h e specific m o r p h o l o g i c a l characteristics o f u n s t a b l e p l a q u e s in c o r o n a r y arteries.2" T h e r e f o r e , the need for a high resolution imaging
t e c h n i q u e that c a n d e t e c t u n s t a b l e coronary a t h e r o s c l e r o t i c p l a q u e s is p a r a m o u n t . 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 ) may b e able t o fulfill that n e e d .
c o n s t i t u e n t s . In gray scale i m a g e s , as u s e d in this p a p e r , i n t i m a l tissue s h o w s u p 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 and calcification m a n i f e s t as a d a r k e r a r e a c o m p a r e d t o t h e i n t i m a l a n d medial 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 b e m a d e o n t h e gray levels a l o n e , a n d c o n s e q u e n t l y has t o be 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 d a r k e r areas: a s h a r p l y 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 o f 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 .4 T h e m e a n O C T signal (i.e. gray level in t h e image) f r o m a c e r t a i n tissue r e g i o n o f
interest is d e t e r m i n e d by the specific local o p d c a l properties. Quantification o f these intrinsic optical p r o p e r t i e s c o u l d p r o v i d e a n e x t r a , o b j e c t i v e , classification p a r a m e t e r . T h e a p p l i e d simple q u a n t i t a t i v e analysis o f t h e O C T signals allows spatial d e t e r m i n a t i o n o f the intrinsic o p t i c a l a t t e n u a t i o n coefficient o f a t h e r o s c l e r o t i c 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 interest. O u r m o d e l only takes the c o n t r i b u t i o n of single scattered light in a c c o u n t . Recently, L e v i t z et al. m e a s u r e d t h e a t t e n u a t i o n coefficients for 1 3 0 0 n m O C T light o f p l a q u e c o n s t i t u e n t in a o r t i c tissue w i t h a m o d e l t h a t i n c o r p o r a t e s t h e i n c r e a s e d c o n t r i b u t i o n o f multiple scattered light w i t h i m a g i n g d e p t h .2' O n a v e r a g e t h e i r m e a s u r e d / t are larger t h a n
o u r s , b u t t r e n d s b e t w e e n t h e d i f f e r e n t a t t e n u a t i o n c o e f f i c i e n t s for t h e d i f f e r e n t tissue types are similar for o u r m e a s u r e m e n t s at 1300 n m . I n t h e i r m o d e l , t h e selected r e g i o n s o f i n t e r e s t n e e d e d u n i f o r m s c a t t e r i n g p r o p e r t i e s , w h i c h actually limit t h e a p p l i c a t i o n of t h a t m o d e l . B e c a u s e of t h e c o m p l e x i t y o f t h a t m o d e l , w h i c h d o e s n o t allow for s t r a i g h t f o r w a r d e x t r a c d o n o f t h e a t t e n u a t i o n coefficient o f layered tissue, w e state t h a t o u r d e p l o y e d single s c a t t e r i n g m o d e is a p p r o p r i a t e for clinical a p p l i c a t i o n o f t h e l o c a l i z e d a t t e n u a t i o n m e a s u r e m e n t s . 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 bv O C T with t h e o b s e r v e d differences in optical a t t e n u a t i o n coefficients o f t h e v a r i o u s r e g i o n s may e n h a n c e differentiation b e t -w e e n v a r i o u s p l a q u e types -w i t h i n t h e vessel -wall. T h i s may c o n t r i b u t e t o 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 .
In c o n c l u s i o n , b o t h t h e h i g h a n d regular r e s o l u t i o n O C T s y s t e m s c a n precisely i m a g e t h e a t h e r o s c l e r o t i c p l a q u e s . T h e d i m e n s i o n s o b t a i n e d from t h e O C T images correlate well w i t h t h e h i s t o l o g i c a l d a t a . Q u a n t i t a t i v e analysis o f t h e O C T signals a l l o w e d in situ d e t e r m i n a t i o n o f t h e i n t r i n s i c 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 o f a t h e r o s c l e r o t i c tissue c o m p o n e n t s w i t h i n R O I ' s . T h e a t t e n u a t i o n coefficient o f R O I ' s in t h e p l a q u e c a n f u r t h e r h e l p to d i s c r i m i n a t e t h e p l a q u e a n d arterial wall c o m p o n e n t s , c a n follow the effect o f i n t e r v e n t i o n o n the arterial wall a n d can e n h a n c e t h e d e t e c t i o n o f t h e v u l n e r a b l e p l a q u e s .
ACKNOWLEDGEMENTS
T h i s r e s e a r c h is s p o n s o r e d by t h e N e t h e r l a n d s H e a r t F o u n d a t i o n (grant 99.199) and is also p a r t of t h e r e s e a r c h p r o g r a m o f t h e " S t i c h t i n g v o o r F u n d a m e n t e e l O n d e r z o e k deiMaterie ( F O M ) ' , w h i c h is financially s u p p o r t e d by the ' N e d e r l a n d s e O r g a n i s a t i e v o o r w e -t e n s c h a p p e l i j k O n d e r z o e k ( N W O ) ' . W e a c k n o w l e d g e -t h e I n -t e r u n i v e r s i -t y C a r d i o l o g y I n s t i t u t e o f t h e N e t h e r l a n d s ( I O N ) for financial s u p p o r t .
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