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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.
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T E M P E R A T U R E D E P E N D E N T OPTICAL
P R O P E R T I E S O F INDIVIDUAL VASCULAR
WALL C O M P O N E N T S , MEASURED BY
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, Tnci Cilesiz,
Martin J.C. van Gemert, Ton G. van Leeuwen
pt AND n OF INDIVIDUAL VASCULAR COM PON ENTS
O
ptical properties of tissues and tissue components are important parameters in biomedical optics. We report measurements of tissue refractive index n, and the attenuation coefficient p.^ using optical coherence tomography of individual vascular wall layers and plaque components. Moreover, since the temperature dependence of optical properties is widely known, we compared measurements at room and body temperatures. A decrease of n and/^t was observed in all samples, with most profoundeffect on samples with high lipid content.
The sample temperature is of influence on the quantitative measurements within O C T images. For extrapolation of ex vivo experimental results, especially for structures with high lipid content, this effect should be taken into account.
I N T R O D U C T I O N
Since the beginning of biomedical optics, the optical properties of tissues and tissue components have been the subject of numerous studies. They arc vital parameters for the description of light transport in tissue, which in turn is important for light dosimetry and interpretation of optical imaging techniques.1 Using experimental techniques and their
accompanying theories, the optical properties as absorption coefficient, (reduced) scattering coefficient and anisotropy factor, have been determined for various tissues.2 Key factor in
these measurements is the path length that the light has traveled through the tissue under investigation. With optical coherence tomography (OCT), the amplitude of the (back) scattered light is measured as a function of the optical path length. Consequently, if the index of refraction of the tissue is known, the physical path length that the light has traveled can be determined.
CHAPTER 5
In OCT,3'"1 t h e o p t i c a l e q u i v a l e n t of B - m o d e u l t r a s o u n d i m a g i n g , t h e c o m b i n a t i o n o f t h e s o called c o h e r e n c e g a t i n g w i t h confocal gating, drastically r e d u c e s the c o n t r i b u t i o n of m u l t i p l e s c a t t e r e d light. C o n s e q u e n t l y , with O C T , high r e s o l u t i o n i m a g e s c a n be m a d e u p ro 5-10 s c a t t e r i n g d e p t h s in tissue." The a t t e n u a t i o n of t h e O C T signal, and t h u s t h e c o n t r a s t of t h e i m a g e s , d e p e n d s heavily o n the a t t e n u a t i o n coefficient (ju) o f t h e s a m p l e . By analysis o f t h e O C T signal in depth, //, of t h e s a m p l e c a n be d e t e r m i n e d . " R e c e n t s t u d i e s d e m o n s t r a t e d t h a t this intrinsic optical p r o p e r t y can c r e a t e a q u a n t i t a t i v e basis for O C T i m a g e i n t e r p r e t a t i o n . ' ' "'
Finally, T e a r n e y et a/, i n t r o d u c e d a suitable f o c u s - t r a c k i n g m e t h o d t h a t u s e d O C T t o t r a c k t h e focal-length shift that results from translating the focus o f a n o b j e c t i v e a l o n g the o p t i c a l axis w i t h i n a m e d i u m /1 T h e y used it t o d e t e r m i n e t h e r e f r a c t i v e index of skin t i s s u e in vivo a n d it h a s b e e n f u r t h e r d e v e l o p e d by o t h e r s for a p p l i c a t i o n to tissue.1 2' V a r i a t i o n s in refractive index n serve as a primary source o f t h e scattering in tissue and thus t h e c o n t r a s t in O C T imaging. Consequently, knowledge o f n is i m p o r t a n t for u n d e r s t a n d i n g a n d i n t e r p r e t a t i o n o f O C T i m a g e s and for i m p r o v e m e n t o f t h e O C T i m a g i n g p r o t o c o l . R e d u c i n g refractive i n d e x m i s m a t c h e s has b e e n r e p o r t e d t< > increase ( ) C T i m a g i n g d e p t h in h i g h l v s c a t t e r i n g t i s s u e s s u c h as skin,1"'1 a n d b l o o d1 8.
I n a p r e v i o u s study, w e r e p o r t e d m e a s u r e m e n t s o f /;, w i t h O C T o f a t h e r o s c l e r o t i c p l a q u e c o n s t i t u e n t s w i t h i n t h e vascular wall.1"1'' A l t h o u g h b o t h calcifications a n d lipid-rich r e g i o n s a p p e a r as d a r k , w e r e p o r t e d high//, values for the former and low values for the latter. Based o n the a p p e a r a n c e o f t h e i r borders, these vascular s t r u c t u r e s in an O C T image a r e m o r p h o l o g i c a l l y d i f f e r e n t i a t e d : i.e. sharply d e l i n e a t e d b o r d e r s a r e c h a r a c t e r i s t i c for a calcification a n d fuzzy b o r d e r s for lipid-rich regions.2" T o f u r t h e r investigate the n a t u r e of t h e s e differences w e i m a g e d isolated arterial wall and atherosclerotic p l a q u e c o m p o n e n t s by h i g h r e s o l u t i o n O C T in t h i s study. M e a s u r e m e n t s o f the optical p a t h l e n g t h w e r e used t o d e t e r m i n e the values o f n. T h e /? w a s then u s e d for a c c u r a t e m e a s u r e m e n t of/< with focus t r a c k i n g O C T . F u r t h e r m o r e , t h e effect of t e m p e r a t u r e c h a n g e s f r o m r o o m t e m p e r a t u r e ( 1 8 ° C ) t o b o d y t e m p e r a t u r e ( 3 7 ° C ) o n //and /z] w e r e a s s e s s e d .
M A T E R I A L S AND M E T H O D S
Tissues, phantom, and sample handling
H u m a n c a r o t i d arterial s a m p l e s w e r e collected from 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 M e d i c a l C e n t e r o f A m s t e r d a m (n = 8). T h e s a m p l e s w e r e o b t a i n e d within 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 were snap frozen in liquid n i t r o g e n a n d s t o r e d at 8 0 ° C . T o o b t a i n t h e i n d i v i d u a l i n t i m a l and medial layers, t h e adventitial layer was r e m o v e d f r o m t h e arterial s e g m e n t s (n = 4), including t h e external elastic l a m i n a . S u b s e q u e n t l y , t h e i n t i m a w a s carefully s e p a r a t e d f r o m t h e media by b l u n t p r e p a r a t i o n . Calcific n o d u l e s w e r e h a r v e s t e d from a s e c o n d set o f c a r o t i d s a m p l e s (n = 4). Arterial s e g m e n t s , in w h i c h lipid p o o l s w e r e c o n f i r m e d by m a c r o s c o p i c observation, were g e n e r o u s l y supplied by the U t r e c h t
//, AND II OF INDIVIDUAL VASCULAR COMPONENTS
Athero-Express biobank (n = 4). A mono layered tissue phantom with a biological rele-vant fatty acid composition was constructed from the triglyceride fraction of ordinary dairy butter. The lipids were either imaged between two glass plates (attenuation measurements) or as a droplet on a heating plate (refractive index measurement).
All experiments were done under controlled temperature, being either room temperature (18°C) or body temperature (37°C). The lipid phantom was studied stepwise from 18°C to 37°C. The temperature was monitored during the experiments, using a thermocouple placed next to the sample.
OCT imaging
The principle and physics of OCT have been extensively described.3'2" 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 ,um; dynamic range was 110 dB. The light was delivered via a single mode fiber with a mode field diameter of 5.3 ,um. The lateral resolution, determined by the spot size of the sample arm beam, was approximately 7 ,um. T h e measured depth of focus of the sample arm optics was 126 ± 6 ,um in air. In depth scanning (A-scan) was performed by a translating mirror in the reference arm, with or without synchronized (and corrected for the index of refraction) movement of the object lens in the sample arm (focus tracking mode or static mode, respectively). The amplitude and phase of the demodulated signal were digitized and stored in a computer. Each A-scan consisted of 8,192 data points. B-A-scan images were obtained by moving the tissue sample with respect to the fixed sample arm beam while performing A-scans.
Measurement of index of refraction
To determine the index of refraction of (isolated) tissue constituents, O C T imaging of the sample was performed in the static focus mode. During imaging, the sample was positioned on a reflective substrate. If the underlying surface was visible in the O C T image, the (group) refractive index n could be determined by measurement of the virtual optical path length outside the sample (d{) and the optical path length in the sample (d7)
as depicted in figure 5-1C. If the index of refraction of the surrounding medium (n ) is known, the index of refraction of the sample («,) can be determined via
«, = df », (5-1)
CHAPTKR 5
Attenuation measurement
In a previous study, we demonstrated that the attenuation coefficient ut could be
determined accurately using focus tracking during each A-scan.5 With the determined index
of refraction (see above), the O C T system was optimized for focus tracking imaging of the sample. For the given range of /J. 's and thicknesses, the O C T signal is dominated by single scattered light.5 Then, the decay of the O C T signal (/) with depth (d) simply follows
the Lambert-Beer law:
/'(y)ocexp(- fi,d) (5-2)
To obtain /j., an average of 50-100 adjacent A-scans was taken from a region of interest (ROT) in each O C T B-scan. With a Levenberg-Marquardt curve fitting algorithm, equation 5-2 (with added offset and amplitude to facilitate scaling) was fitted to the O C T signal within the ROl, with//, as the fitting parameter. During the fitting procedure, the offset was fixed at the average noise level of the recorded data and the amplitude was free running.
Statistical analysis
Single fit data arc given with the 95% confidence interval. All mean data are presented with standard deviation. The statistical significance of the measurements was tested with a paired Student's T-test.
R E S U L T S
T h e different components of the vessel wall were successfully isolated and imaged using O C T (figures 5-1). The high-resolution O C T images were used to verify that the different tissue components were indeed well separated (figure 5-1A and 5-1B). When the underlying surface was visible, the index of refraction of the arterial wall and plaque components could be determined using formula 5-1 (figure 5-1C). The results of the refractive index measurements are presented in table 5-1, along with reference values for better interpretation. The indexes of intima and media are in line with measurements of comparable tissues (1.352 ± 0.002 and 1.382 ± 0.008 resp.). Calcifications have a much higher index of refraction, 1.63 ± 0.05. The higher standard deviation could be attributed to the inhomogeneous structure of the calcifications. The lipid pool and lipid phantom have indexes in the same range (1.42 ± 0.04, and 1.417 ± 0.009, resp.). Increasing the temperature from 18°C to 37°C resulted in a decrease in n in all samples, with the most remarkable temperature dependent decrease in the lipid phantom.
Using the known index of refraction and the focus tracking mode, t h e ^t of the isolated
arterial and plaque segments could be determined (figure 5-2), except for the lipid pool. Due to the consistency and behaviour of the isolated lipid material, no reliable measurement
/ / , AND n OF INDIVIDUAL VASCULAR COMPONENTS
Figure 5-1 (A) An OCT image of a prepared medial layer. Part of the intima was carefully removed. Fragments of the internal elastic lamina (iel), the boundary between intima and media are visible. (B) An OCT image of the intimal fragment that was removed from the sample shown in panel A. (C) An OCT image of an arterial calcification. The optical path length outside the sample (d ) as well as within the sample (d,) is indicated. The bars represent 0.5 mm.
of /j.i could be done. The values of// at 37 °C are consistent with data reported in earlier
studies. '"''' In all studied samples a decrease in// is observed when increasing the sample temperature. This temperature dependent decrease in// was statistically significant in intima, media, and lipid phantom. To follow this trend more precisely, extensive measurements were performed on the lipid phantom. As can be observed in figure 5-3, the gradual decrease in// with increasing temperature coincides with a decrease in n.
D I S C U S S I O N
In this study, we investigated the nature of the differences in back-scattered and attenuation of O C T signals in arterial wall components. Using the almost histology like capabilities of a high-resolution O C T system, we were able to determine the n and // of individual blood vessel components. Furthermore, we observed decreases in both n and // when the sample temperature was raised from room to body temperature.
As shown in table 5-1, the measured values of n are in good agreement with previously reported values. For calcific nodules in the vascular wall, no values of n are available in
CHAPTER 5
OPI. measurement (800 nm Reference value
18°C 37°C Tissue (A.) Intima Media Calcification 1 .ipici pool 1 .ipici phantom 1.345 ± 0.002 1.391 ±0.007 1.64 ±0.02 1.52 r 0.02 1.494 ± 0.009 1.352 ±0.002 1.382±0.0021 1.63 ±0.05 L42 ± 0.04* 1.417 ±0.009* 1.350-1.367 1.382 1.651 1.467 Cells ;i Ventricular Muscle (1300 nm) " Apatite mineral (631 nm) 2I Adipose tissue (1300 nm) "
T a b l e 5-1 T h e refractive index of individual layers of the vascular wall, atherosclerotic plaque constituents and the lipid phantom. Measurements were done at 18°C and 37 °C. For better interpretation, also previously estimated or measured values of comparable tissue structures are given. ': p < 0.05, compared
to 18°C.
D18°C 137°C L in Situ
r * i
intima media calcif lipid
F i g u r e 5-2 T h e results of the fx measurements at 18°C (gray bars) and 37°C (black bars). T h e values of in situ measurements from ref.10 are also depicted (white bars). For lipid, /u values measured in the lipid phantom are presented next to the lipid pool measurements from ret. 10. F.rror bars represent the standard deviation. *: p<0.05
fl{ WDn OF INDIVIDUAL VASCULAR COMPONENTS
18 22
1,380
26 30 34 38
T(°C)
Figure 5-3 Measurements of n (black line, left y-axis) and // (dotted line, right y-axis) of the lipid phantom from 18°C to 37°C.
literature. The // of hydroxyapatite, the mineral in calcifications, is reported to be 1.651, in close agreement with our results of 1.63-1.64.2' The great variance in the values of n
presented in this paper can be attributed to the biological variability in hydroxyapatite. For osteones it has been reported that a variance in hydroxyapatite content from 50 to 100% results in refractive indexes of 1.564 to 1.604.22
The values for JJ, reported in this study are slightly higher, albeit without statistical significance, than prior in situ measurements. In the in situ studies10'19 O C T imaging was
done assuming a general refractive index of 1.33, thus underestimating the attenuation in structures with a higher n. In those previous studies, theLI was obtained in samples which consisted of multiple layers, like the intima on top of the media in the samples. Because of similar refractive indices of the individual components, the effect of these multiple layers on the measured attenuation likely is negligible. Furthermore, with the use of an average n of approximately 1.38 in cardiovascular O C T imaging, the axial dimensions of the calcified lesions will be overestimated. Finally, the large difference in n for calcified lesions compared to the surrounding medial and intimal tissues is expected to induce large back-scattered signals from those boundaries, which explains the characteristic sharp demarcations of calcified lesions.
Temperature dependence
Previous studies demonstrated that both the scattering2'1 and the absorption2"1'-3
coefficients can be temperature dependent. Scattering changes have been cleverly utilized to monitor temperature changes in laser welding26 2S. Moreover, O C T has been used to
mo-nitor biological tissue freezing during crvosurgery, based on changes in // with temperature.2''
In all samples studied in this study, we observed a persistent decrease in // when tissue temperature was raised from room temperature to 37°C. We hvpothesize that the decrease
CHAPTER 5
in // is the explanation for the decrease in,u,. In our study, the effect of temperature was largest for the lipid structures. In the range from room to body temperature, lipid structures have a transition from the rigid liquid-crystalline phase to the more fluid gel phase, which may explain the observed decrease in light scattering and index of retraction.'" Finally, these results clearly indicate that optical property measurements of fatty tissues should be performed at 37 °C.
CONCLUSION
We report accurate values of the refractive index of individual layers of the vascular wall and individual atherosclerotic plaque constituents, all important actors in vascular O C T imaging. The sample temperature is of influence on the quantitative measurements within O C T images. For extrapolation of ex vivo experimental results, especially tor structures with high lipid content, this effect should be taken into account.
ACKNOWLEDGEMENTS
T h e authors wish to thank Dr. G. Pasterkamp and the Utrecht Athero-Express biobank for the donation of atherosclerotic samples. This research is sponsored by the Netherlands Meart Foundation (grant 99.199) and is also part of the research program of the "Stichting voor Fundamenteel Onderzoek der Materie (FOM)', which is financially supported by the 'Nederlandse Organisatie voorwetenschappelijk Onderzoek (NWO)'. We acknowledge the Interuniversity Cardiology Institute of the Netherlands ( I O N ) for financial support.
U AND n OF INDIVIDUAL VASCULAR COMPONENTS
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CHAPTER 5
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