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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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A P O P T O S I S I N D U C E S T E M P O R A L I N C R E A S E
I N A T T E N U A T I O N AS 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, Maurice C. G. Aalders,
J op Perrée, Ton G. van Leeuwen
O C T i.\iAc;iN<; < >!• API > P K >SIS A M ) M:< R< >SI>
O
ptical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis was induced. We analyzed the O C T data including both the scattering properties of the medium and the axial point spread function of the O C T system. We measured that the optical attenuation coefficient in necrotic cells decreased from 2.2 ± 0.3 mm ' to 1.3 ± 0.6 mm"1,whereas with the apoptotic cells a clear increase (up to 6.4 ± 1 . 7 mm ') in scattering was observed. The results on cultured cells a presented in this study indicate the ability of O C T to detect and differentiate between viable, apoptotic and necrotic cells based on their backscatter properties. This functional supplement to high-resolution O C T imaging can be of great clinical benefit, enabling on line monitoring of tissues, e.g. for feedback in cancer treatment.
INTRODUCTION
Tn every multi cellular organism a delicate balance exists between cell division on one hand and cellular death on the other hand. Cellular death can be executed by two different pathways, necrosis and apoptosis.' Necrosis, the pathological pathway, is also known as accidental cell death which is triggered by external disturbances e.g. physical trauma, chemical stress or hypoxia. Its morphology is characterized by slight initial cellular swelling, followed by cell lysis. In vivo, the resulting cell debris triggers an inflammatory response.
The physiological pathway of cell death is called apoptosis, also known as programmed cell death. It is a general mechanism for the clearance of cells that have become superfluous or show an aberrant function, without causing inflammation. The apoptotic pathway is conducted by a series of tightly regulated biochemical processes in which a cell, once triggered, goes through consecutive phases of cell shrinkage, chromatin condensation and breakdown,
CHAPTER 6
n u c l e a r d i s i n t e g r a t i o n , cell b l e b b i n g and t h e f o r m a t i o n o f so-called a p o p t o t i c b o d i e s . T h e s e a p o p t o t i c b o d i e s c o n t a i n n u c l e a r fragments a n d cell o r g a n e l l e s (Figure 6-1). U n d e r n o r m a l p h y s i o l o g i c a l c o n d i t i o n s they are cleared by e i t h e r m a c r o p h a g e s o r n e i g h b o r i n g cells t h r o u g h p h a g o c y t o s i s .2
A s t h e p h y s i o l o g i c a l c o u n t e r p a r t o f cell g r o w t h , a p o p t o s i s plays an i m p o r t a n t role in t h e b a l a n c e of tissue d y n a m i c s . D i s t u r b a n c e s in this b a l a n c e results in d i s e a s e . If a b e r r a n t cells d o n o t u n d e r g o a p o p t o s i s , a t u m o r can develop. O n the o t h e r h a n d , excessive a p o p t o s i s c a n r e s u l t in d e g e n e r a t i v e s y n d r o m e s such as a t r o p h y a n d c a r d i o m y o p a t h y .3 In clinical
p r a c t i c e , t r e a t m e n t o f t h e s e s y m p t o m s and d i s e a s e s i n v o l v e s r e d u c t i o n o r s t i m u l a t i o n o f a p o p t o s i s , respectively. C o n s e q u e n t l y , there is a s t r o n g n e e d for m e t h o d s t h a t c a n d e t e c t a n d quantify a p o p t o s i s on a m i c r o s c o p i c level. Currently, the standard m e t h o d s o f a p o p t o s i s d e t e c t i o n in tissue a r e h i s t o l o g i c a l o r b i o c h e m i c a l , w h i c h are t i m e c o n s u m i n g and r e q u i r e b i o p s i e s . ' /// vivo d e t e c t i o n u s i n g r a d i o n u c l i d e i m a g i n g5 a n d m a g n e t i c r e s o n a n c e imaging''
a r e 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 .
F i g u r e 6-1. A schematic representation of the different stages of apoptosis. Once apoptosis is triggered in a normal cell (A), some shrinkage will occur (B) and the nucleus condensates (B, C). T h e loss of cvtoskeleton integrity induces blebbing of the cellular membrane (C). The condensated nucleus is fragmented (D) followed by disintegration of the whole cell into apoptotic bodies, containing remnants of the nucleus and other cell components (I7.). Apoptotic bodies are
cleared by phagocytosis by macrophages or neighbouring cells, or undergo secondary necrosis.
Recently, C z a r n o t a a n d c o w o r k e r s r e p o r t e d t h a t a p o p t o s i s c o u l d b e d e t e c t e d u s i n g h i g h - f r e q u e n c y u l t r a s o u n d . T h e y o b s e r v e d a n increase in b a c k s c a t t e r i n g o f t h e u l t r a s o u n d signal, w h i c h they a t t r i b u t e d t o t h e d i s i n t e g r a t i o n o f the n u c l e u s , a d i s t i n c t i v e feature o f a p o p t o s i s . 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 ) i m a g i n g , the optical a n d high r e s o l u t i o n e q u i v a l e n t of u l t r a s o u n d i m a g i n g , is based o n time o f flight d e p e n d a n t intensity differences o f b a c k s c a t t e r e d light.H I n a p r e v i o u s p u b l i c a t i o n , we u s e d O C T t o i m a g e p o r c i n e c a r o t i d
a r t e r i e s in an ex vivo tissue c u l t u r e setup.'' We r e p o r t e d an i n c r e a s e in back s c a t t e r i n g in the m e d i a l layer after b a l l o o n d i l a t i o n . Since t h e r e is a rapid o n s e t o f a p o p t o s i s after b a l l o o n a n g i o p l a s t y , ' " w e h y p o t h e s i z e d t h a t the c h a n g e s in O C T signal can b e a t t r i b u t e d to this c h a r a c t e r i s t i c t o r m o f cellular d e a t h . Functional O C T allows for q u a n t i t a t i v e m e a s u r e m e n t o f t i s s u e a b s o r p t i o n a n d s c a t t e r i n g p r o p e r t i e s , at t h e m i c r o s c o p i c level. B e c a u s e b o t h a p o p t o s i s a n d n e c r o s i s directly influence t h e cell's main scatterers (i.e. n u c l e u s , m e m b r a n e , m i t o c h o n d r i a , a n d o t h e r cellular organelles"""') w e h y p o t h e s i z e d t h a t the local a t t e n u a t i o n
O C T IMAGING OF APOPTOSIS AND \ H R< ISIS
coefficient (jx) could be used ro assess cellular death. Moreover, due to the differences in the morphology of the two pathways to cell death, we hypothesized that differences could be observed in attenuation of light by necrotic and apoptotic cells as compared to normal cells, and that those changes could be monitored in time, after induction of cell death. In this paper, we present O C T measurements of the attenuation coefficient of pelleted human fibroblasts, in which necrosis or apoptosis was induced.
M A T E R I A L S AND M E T H O D S
Cells and reagentia
Human fibroblasts were maintained in D M E M (Gibco/BRL) supplemented with 10% fetal calf serum, streptomycin at 100 tig/ ml, penicillin at 100 U/ml in a fully humidified atmosphere containing 5% C 0 2 at 37 °C. Prior to the experiment, subconvluent grown cells were trypsinized, and collected by centrifugation at 500g for 10 minutes. At t = 0 , the pelleted cells were resuspended in DMEM containing either 10% ethanol, to induce necrosis, 200/iM cytosine arabinoside (AraC, Sigma) to induce apoptosis, or 0.1 mg/ml colchicine (CX, Sigma) to induce a mitotic arrest. After 30 minutes incubation, the cells were pelleted and imaged with O C T for 6 hours, hi a second set of experiments, the dose-dependency of apoptosis induction by AraC was studied. Culture flasks with subconvluent grown cells were incubated with 50 LIM, 100 /JM or 200 LIM AraC for two hours. Cells were trypsinized, and collected by centrifugation at 500g for 10 minutes. Pelleted cells were immersed in medium, and imaged with O C T at 3, 6, 9,12 and 24 hours after the induction of apoptosis. Between and during measurements, the cells were kept at 37°C.
Verification of apoptosis
At the end of the O C T experiment, samples of pelleted cells were subjected to immunofluorescence analysis, using a commercially available apoptosis detection kit (Sigma). The double labeling assay with annexin-V-Cy3 (AnnV) and 6-carboxyfluorescein diacetate (CEDA) allows differentiation between apoptotic, necrotic and viable cells. The CFDA is processed in metabolic active cells into fluorescing 6-CF. The AnnV-Cy3 label binds to phosphatidylserine residues when they appear in the outer leaflet of the cellular membrane, which occurs in compromised cells, being apoptotic or necrotic. Therefore, cells, only labeled with 6-CF (green) are viable cells, cells only labeled with Ann-Cy3 (red) are necrotic cells, and cells staining with both are apoptotic. Labeling was performed con-form the instructions, as provided by the manufacturer.
Flow cytometry
To measure the necrotic and apoptotic fraction after induction of cellular death, cells were harvested at different time points, and washed in ice-cold FIEPES buffer (10 mM H E P E S , 150 mM KC1, 1 mM MgC12 and 1.3 mM CaC12, pH 7.4) supplemented with 1 mg/ml glucose and 0.5% (w/v) BSA. Cells were then incubated with FITC-labeled
CH \PTER6
Annexin V (diluted 1:200 in H E P E S buffer) for 15 min and washed again in H E P E S buffer. )ust before analysis of the samples by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA), PI was added (final concentration 5 ,t/g/ml) to distinguish necrotic cells (Annexin V - / P 1 + ) from early apoptotic cells (Annexin V + / P I - ) and late apoptotic cells (Annexin Y + / P I + ) . The samples were analyzed on a FACSCalibur (Becton Dickinson) equipped with CellQuest software. The cytometer was calibrated by eye with fluorochrome beads supplied by the manufacturer.
OCT imaging
O C T imaging of cells was performed with an high resolution O C T setup in which a TkSapphire laser (FemtoSource, Vienna, Austria), operating at a central wavelength of 800 nm with a bandwidth of 125 nm, was used as light source. Depth scanning, by changing the (optical) path-length in the reference arm, was performed using a so-called rapid-scanning-optical-delay (RSOD) line.1" This allowed precise and constant (speed and
intensity) depth ranging, and has the additional advantage to allow easy correction for dispersion mismatch between the reference arm and sample arm. In depth scanning was performed at 2 lines per second. For this system, we measured a dynamic range of 110 dB and a free space resolution of 5 am. For each cell pellet and each time point 3 to 5 b-scans were made.
Data analysis
In each b-scan, the attenuation coefficient (u) of the pelleted cells was obtained in a procedure as described previously.'s In short, the depth dependence of the amplitude of
the O C T signal 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.1''21 An average
Control 2 0 0 / j M A r a C 10% EtOH
Figure 6-2. Examples of OCT images of pelleted cells. Untreated control cells (A) remained unchanged during the entire experiment. Apoptotic AraC treated cells (B) showed an increase in scattering in the top layer, whereas necrosis (C) resulted in a decrease in signal.
O C T IMAGING OF APOPTOSIS AND NECROSIS
signal of 50-100 adjacent A-scans as a function of depth was fitted to the model w i t h ^ as the fitting parameter. Using this simple analysis algorithm, which incorporates the position of the focus in the tissue and the depth of focus, the scattering coefficient,ut for the ROI's
was determined in situ.
9 i I • 5 • 4 • 3 • 2 • 1 • 0 0 100 200 300 t i m e ( m i n ) 400 500
F i g u r e 6-3. The measured attenuation coefficient for 800 nm light in pelleted human fibroblasts, as a function of time. Time was measured in minutes from the point that the cells forced into necrosis (filled squares) or apoptosis (filled dors). Sham treated (control) cells show no change in scattering (circles).
RESULTS
In the experiments described in this study, cells were pelleted and were proven to be viable (data not shown), similar to other studies."-24 When compared to untreated cells
(figure 6-2A), the treatment of the cells with 200 / J M AraC resulted in a temporary increase in OCT signal and a decrease of imaging depth (figure 6-2B). O n the contrary, cells treated with 10% ethanol showed an increase in imaging depth (figure 6-2C). The measurements of the fxx of the samples in time are presented in figure 6-3. The AraC treated cells show an
initial increase in/^, followed by a decrease, ending up below the level of the control cells. Treatment with 10% E t O H results in an immediate decrease in backscatter, compared to untreated control cells.
As shown in the fluorescence images (figure 6-4), apoptosis was induced in the AraC-treated cells. Panels 6-4A and 6-4B show unAraC-treated control cells, and panels 6-4C and 6-4D show AraC treated cells. The green fluorescence (fig 6-4A and 6-4C) is a marker for metabolic active cells (i.e. either viable or apoptotic cells), whereas the red fluorescence marks the compromised cells (i.e. necrotic or apoptotic cells). Therefore, the double labeled cells can be discerned between viable (green), apoptotic (green and red), and necrotic (red) cells.
CHAPTER 6
Viable Apoptotic
i
Control
AraC
F i g u r e 6-4. Images of immunofluorescence labeling of control cells (A,B) and cells treated with 200 mM AraC (C,D) at 24 hours. Using the green fluorescing label C F D A (A,C), viable cells can b e identified, whereas the red fluorescing label AnnV (B,D) is specific for apoptotic cells. At 24 hours 5 % of the control cells has become apoptotic, while after treatment with 200 mM AraC 6 1 % of cells is apoptotic.
T 100
F i g u r e 6-5. After induction with 10% ethanol (t = 0), the number of viable-cells (gray line, o p e n dots) decrease, a n d the n u m b e r of necrotic viable-cells (black line, filled dots) increases. T h e total number of cells (gray dotted line) decreases, due to loss of necrotic cells. T h e decrease in,ur (black dotted line) coincides with the
increase in necrosis.
O C T [MAGING OF APOPTOSIS AND NECROSIS 1 1 -I 10 • g • 8 -7 • fi 5 4 3 --L
B
11 1 •o 9 8 -7 • 6 • 5 -3 •r.
9 12 time (hrs)c
"E E a! 1 1 -I 10 j 9 • R • 7 • 6 • 0 • 4 • 3 • 15 24I
\
\
\
\ N - - f 6 9 12 time (hrs)n
6 9 12 15 24 time (hrs)Figure 6-6. Dose-dependency curves of the increase in u after treatment with 50 ^M (A), 100 /iM (B), and 200 /zM (C) AraC. The higher the dose, the earlier is the onset of the ,ur increase. The black lines depict the untreated control cells,
and the dotted lines are AraC treated apoptotic cells.
Since the necrotic process rapidly results in clearance of cells bv rupture of the cell membranes, the E t O H - treated cells were not visualized using fluorescence microscopy. FACS analysis can detect necrotic cells based on the scattering of remnants. The result of the cell counting of is presented in figure 6-5. The decrease in average attenuation (dotted line) coincides with a decrease in viable cells (gray line), and a synchronous increase in necrotic cells. No apoptotic cells were detected, therefore the decreasing total cell count has to be contributed to the total clearance of necrotic cells.
The induction of apoptosis by AraC is known to be dependent on the concentration. In figure 6-6, the results are plotted of the attenuation measurement of cells treated with 50/*M (6-6A), ÏOO^M (6-6B), and 2 0 0 / M (6-6C). The higher the concentration of AraC, the earlier the rise in attenuation is observed.
To study the effect of nuclear condensation on the attenuation, cells were treated with colchicine (figure 6-7). The resulting increase in/i mimicked the apoptosis curves.
CHAPTER 6
D I S C U S S I O N
I n t h e e x p e r i m e n t s p r e s e n t e d in this p a p e r , a n i n c r e a s e in /n is o b s e r v e d in pelleted cells, a f t e r t r e a t m e n t w i t h A r a C . A r a C is k n o w n to i n d u c e a p o p t o s i s via i n c o r p o r a t i o n i n t o D N A d u r i n g r e p l i c a t i o n , a c t i n g as a chain t e r m i n a t o r .2 1 H o w e v e r , A r a C c a n a l s o i n d u c e
a p o p t o s i s directly t h r o u g h o x i d a t i v e stress, with a n i n c r e a s e in t h e g e n e r a t i o n o f reactive o x y g e n s p e c i e s a n d p 5 3 - d e p e n d e n t cytotoxicity.2 6" T h i s latter, r a p i d p a t h w a y h a s b e e n
s h o w n t o i n d u c e a p o p t o s i s in l y m p h o i d cells w i t h i n 3 h o u r s .2 8 A p a r t from t h e use in
l e u k e m i a t r e a t m e n t , is k n o w n t o i n d u c e a p o p t o s i s in n e u r o n a l cells2'' a n d f i b r o b l a s t s . " '
A p o p t o s i s was i n d e e d d e t e c t e d using a commercially available viability assay, c o m b i n i n g 6 - C D F A w i t h a n a n n e x i n - V - C y 3 label. A n n e x i n - V b i n d s t o t h e p h o s p h a t i d y l s e r i n e t h a t r e d i s t r i b u t e s f r o m t h e i n n e r t o t h e o u t e r leaflet o f t h e p l a s m a m e m b r a n e as an early e v e n t in t h e a p o p t o t i c p r o g r a m .1- B i n d i n g o f a n n e x i n V t o e x t e r n a l i z e d p h o s p h a t i d y l s e r i n e h a s
f o r m e d t h e b a s i s for w i d e l y u s e d optical m e t h o d s ( f l u o r e s c e n c e m i c r o s c o p y a n d flow c y t o m e t r y ) for d e t e c t i n g a p o p t o s i s . F u r t h e r m o r e , A r a C is k n o w n to i n d u c e a p o p t o s i s in a d o s e - d e p e n d e n t f a s h i o n . '2" A similar d e p e n d e n c y is o b s e r v e d in t h e c h a n g e s in/z in o u r
e x p e r i m e n t s .
T h e r e is a g r e a t variety in a p o p t o s i s i n d u c e r s ., 4 In an e x p e r i m e n t a l s e t u p similar t o the
o n e d e s c r i b e d h e r e , w e i n d u c e d a p o p t o s i s in m o u s e fibroblasts (AIF) with s t a u r o s p o r i n e ,v l
a n d s u b j e c t e d h u m a n l u n g c a r c i n o m a cell line (SW 1573) t o a l a - P D T .3 5 T h e p r e l i m i n a r y
r e s u l t s i n d i c a t e t h a t in b o t h cell lines, an increase i n / / is o b s e r v e d (data n o t s h o w n )
10 -I 9 8 7 6 5 4 -3 • 2 -1 • 0 0 * — c o n t r o l • c o l c h i c i n e A AraC 100 200 300
time (min)
400 500F i g u r e 6-7 Colchicine treated cells (black dotted line) mimic the /a -curve of AraC treated cells (gray line). However, the maximum values of fx are higher, and secondary necrosis is not significantly detected. Untreated conrrol cells are represented by the black line.
O C T IMAGING OF VPOPTOSIS VND NECROSIS
The origin of light scattering from cells is still subject of studies. Scattering occurs due to the mismatch in indices of refraction between these different cellular compartments and is also dependent on the size and shape of the scatterer.36 J8 In the case of necrosis, the cell
and its organelles disintegrate, resulting in an amorphous mass. The decrease in scattering from necrotic cells can therefore probably be explained by a removal of potential scatterers, resulting in less scattering events. Although, due to the cellular swelling prior to cell lysis, one could expect an initial increase in scattering, this was not observed in our experiments. Valenzeno et al. proposed a technique for measurement of the blood hematocrit based on the decrease of forward scattering during erythrocyte lysis. The loss of refractive index mismatches reduced the number of scattering events.'9 The degree of hemolysis is inversely
related to the intensity of small angle scattering. Furthermore, in experiments monitoring the optical density (OD) of cell suspensions, Kravtsov eta/, reported a clear decrease in O D after the induction of necrosis.4"
The apoptotic process involves a series of morphologic changes, in which many potential scatterers are involved. It has been reported that the initial increase in scattering could be due to cellular shrinkage,41 to chromatin condensation,"' or nuclear fragmentation42-4'.
Kravtsov and coworkers designed an assay in which they monitored the optical density (OD) of cells in suspension. When inducing apoptosis, they observed a temporary increase in O D , comparable to the increase in tu in this study. Morphological analysis of their
samples revealed that the increase in O D coincided with the blebbing of the apoptotic cell membrane. The initial O D increase is followed bv a decrease when the majority of the cells pass the blebbing-stage. Considering the similarities with the results presented here, especially the transient tendency of the ju increase, it is likely that membrane blebbing is responsible for the results of our study.
In flow cytometry, apoptotic cells are also identified based on changes in scattering. Whereas this study focuses on back-scattered light, cytometry registers forward and side (90° angle) scattering of single cells. Forward scatter is related to the cell size, and side scattering is affected bv the cells refractive and reflective properties and reveals optical inhomogeneity, such as resulting from condensation of cytoplasm or nucleus and granularity. During the apoptotic process, there is a slight initial decrease in forward scatter (cell shrinkage), followed bv an increase in side scatter (nuclear condensation and fragmentation). The formation of apoptotic bodies is characterized by a decrease in both forward and side scatter.44
Czarnota etal. used colchicine to image mitotic arrested cells, which would be normal cells with a condensated nucleus. Whereas the initial goal was to image cells with mere condensated nuclei, apparently also apoptosis was induced in the colchicine treated cells. Colchicine is described as an apoptosis inducer.45,46
Clinical implications and study limitations.
Monitoring of apoptosis could play an important role in the clinic. Apart from an apparent diagnostic value, online monitoring of apoptosis in response to treatment could
C.HAITI ;R 6
g r e a t i y i m p r o v e t h e r a p e u t i c efficacy in e.g. skin c a n c e r t r e a t m e n t .
A p o p t o s i s also plays a r o l e in c a r d i o v a s c u l a r d i s e a s e . A p o p t o s i s o f v a s c u l a r s m o o t h m u s c l e cells4 •4S a n d macrophages"1'' I0cali7.es in s o called ' v u l n e r a b l e ' l e s i o n s , i.e. t h o s e
l e s i o n s m o s t likely to r u p t u r e , a n d at sites o f a c t u a l r u p t u r e d p l a q u e s . I n in vivo s t u d i e s , i n d u c t i o n of a p o p t o s i s in e n d o t h e l i a l cells as well as s m o o t h m u s c l e cells h a s b e e n s h o w n t o r e s u l t in t h r o m b o s i s ^ " a n d p l a q u e r u p t u r e " ' respectively. H o w e v e r , b e f o r e O C T c a n b e u s e d t o d e t e c t v a s c u l a r a p o p t o s i s , t h e specificity and sensitivity o f t h e d e t e c t i o n o f cell d e a t h h a s t o b e i n v e s t i g a t e d .
A l t h o u g h t h e l o s s o f cell i n t e g r i t y d u r i n g n e c r o s i s c o u l d be d e t e r m i n e d u s i n g flow c y t o m e t r y , the identification o f a p o p t o s i s in fibroblast m e t with i n s u r m o u n t a b l e difficulties. T h e flow c y t o m e t r y t e c h n i q u e w a s d e v e l o p e d t o r the analysis o f single cells that g r o w in s u s p e n s i o n . A d h e r e n t cells, like t h e fibroblasts u s e d in this study, are very difficult to a n a l y z e w i t h c y t o m e t r y d u e t o c l u m p i n g o f t h e cells, a n d r u p t u r i n g o f cells w h e n r e s u s p e n d i n g . F u r t h e r m o r e , it s h o u l d be s t r e s s e d t h a t b e c a u s e t h e cells d e t a c h d u r i n g late s t a g e s o f a p o p t o s i s , m a n y a p o p t o t i c cells m a y b e selectively lost if t h e analysis is limited t o t h e a t t a c h e d cells only."12
F u r t h e r r e s e a r c h h a s to b e d o n e t o elucidate t h e origin o f light s c a t t e r i n g by cells, in o r d e r t o a d d r e s s t h e q u e s t i o n o f t h e reason o f light s c a t t e r i n g c h a n g e s in d y i n g cells. C o n v e r s e l y , O C T m e a s u r e m e n t s as p r e s e n t e d in t h i s study could be helpful in identifying t h e s c a t t e r e r s in cells.
C O N C L U S I O N
T h e r e s u l t s o n c u l t u r e d cells a p r e s e n t e d in this s t u d y i n d i c a t e t h e ability o f O C T t o d e t e c t a n d differentiate b e t w e e n viable, a p o p t o t i c a n d n e c r o t i c cells, b a s e d o n their optical p r o p e r t i e s . T h i s f u n c t i o n a l s u p p l e m e n t to h i g h - r e s o l u t i o n O C T i m a g i n g c a n b e o f g r e a t clinical b e n e f i t , e n a b l i n g o n line m o n i t o r i n g o f t i s s u e s , feedback in c a n c e r t r e a t m e n t . F u r t h e r m o r e , O C T i m a g i n g o f a p o p t o s i s in t h e vascular wall m i g h t e n h a n c e the ability o f O C T t o d e t e c t t h e v u l n e r a b l e p l a q u e .
ACKNOWLEDGEMENTS
T h i s w o r k w a s p a r t o f 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 der M a t e r i e ( 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 ) ' , and 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 ( g r a n t 9 9 . 1 9 9 ) .
O C T IMAGING OF APOPTOSIS AND NECR< )SIS
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