Dynamic laser speckles as applied to study of the laser-mediated structural changes of the collagenous biotissues Liana V. Kuznetsovaa, Dmitry A. Zimnyakova, Alexander P. Sviridovb, Stepan A. Baranovb, Natali Y. Ignatievab a Optics Department, Saratov State University, Moscowskaya st., 155, Saratov, 410012, Russia b Institute of Laser and Information Technologies of Russian Academy of Sciences Troitsk, Moscow region, 142190, Russia

ABSTRACT The potential for monitoring of the laser-mediated thermal modification of tissue by means dynamic laser speckles methods is examined. The specific case of delivering of speckle-modulated light backscattered by cartilage with the use of a fiber-optic bundle probe is considered. The proposed technique is experimentally compared with the conventional laser speckle contrast analysis. Both techniques similarly display the basic features of laser-mediated alterations in tissue but the bundle-based technique provides the monitoring of tissue modification with more details and more pronounced correlation between the speckle contrast and the tissue temperature. The possibility of evaluation of the fundamental physical-chemical parameters of tissue modification (in particular, the activation energy) from the temperaturedependent dynamics of the speckle contrast is demonstrated. Keywords: laser speckles, collagenous tissue, structural changes, speckle contrast, activation energy.

1. INTRODUCTION Recent advances in optical biomedical technologies are particularly related to the development and clinical application of the effective speckle methods of tissue diagnostics. These methods are successfully used for characterization of the dynamic and structure properties of the non-stationary scattering media of biological nature. Among other speckle techniques the full-field method pioneered by Fercher and Briers1-3 and known as Laser Speckle Contrast Analysis (LASCA) is widely used now for monitoring of blood perfusion in living tissues4-7 because of the relative simplicity of instrumentation and data processing algorithms. Also, the LASCA technique provides the statistical robustness of the estimates of diagnostical parameters due to the simultaneous processing of a great number of statistically independent time series of speckle intensity fluctuations in the various detection points. In turn, because of the relatively short time intervals necessary for the accumulation of the statistically reliable initial data, this method can be applied for the analysis of relatively high-speed processes in living tissues, which are induced by changes in the tissue physiological state or by various external factors. In particular, the high potential of the full-field speckle technique was demonstrated in the case of monitoring of laser-mediated thermal modification of collagenous tissue such as cartilage8 . The contrast of speckle-modulated images of the treated zone of tissue, which are partially blurred due to the exposure time comparable with the speckle decorrelation time, appears the adequately sensitive indicator of characteristic changes in the cartilage structure at various stages of modification. Application of fiber-optic units (in particular, a fiber-optic bundle) in the optical system of a full-field speckle instrument can be considered as the further step from the studies towards the clinical applications of the speckle monitoring of the complex time-varying dynamics of living tissues. In particular, such bundle-based speckle monitor due to mechanical flexibility of the light-delivering channel can be combined with the fiber-optics IR laser system for thermal treatment of living tissues and used as the feedback control system of treatment process. Besides, the incorporation of a multiple-

Complex Dynamics and Fluctuations in Biomedical Photonics III, edited by Valery V. Tuchin Proc. of SPIE Vol. 6085, 608509, (2006) · 1605-7422/06/$15 · doi: 10.1117/12.659326

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channel fiber-optic unit in the speckle-based instrument can provide the additional potential for the processing and analysis of dynamic speckle patterns due to specific filtering, sampling, and phase-modulating properties of such unit. Note that the important problem arising in this case is the noise-like disturbance of spatial-temporal speckle dynamics, which can appear due to unexpected movements of the bundle. This problem can be particularly avoided by providing mechanical stability of the speckle instrument during the time required to capture a set of speckle-modulated images. The goal of this work is the study of peculiarities of the speckle contrast analysis with the use of a bundle-based full-field instrument applied for monitoring of the thermal modification of collagenous tissue (cartilage).

2. EXPERIMENTAL TEXHNIQUE AND RESULTS The full-field speckle analyzer with the bundle-based light delivery unit (Fig. 1) was used for monitoring of lasermediated modification of cartilage. Laser beam from a single-mode He-Ne laser (1) (632.8 nm, linear polarization, 5 mW output power) was expanded by 50× telescopic system (2) and fell on the surface of a sample (3) of ex vivo bovine nasal septum cartilage. The samples under study were taken from freshly slaughtered animals and prepared as square pieces with 25×25 mm2 lateral dimensions and 1.5 mm thickness. The diffuse reflected light was collected by the imaging lens 4 (the focal length of 16 mm and diameter of 6 mm) and speckle-modulated image of the area of interest on the sample surface was formed on the input tip of a fiber-optic bundle (5). The used bundle with the regular hexagonal packing of single fibers (the fiber diameter is 25 µm and the separation between the fibers in a row is ~3 µm) was 1.2 m in length. Light from the output tip was detected by a CCD sensor (6) (SONY ICX415 chip-based unit, 25 fps frame rate, 10-bit analog-to-digit conversion) co-axially placed at the distance of 300 mm from the output tip. Thermal treatment of the samples was carried out by radiation of an Erbium fiber-optic laser (7) ( λ = 1.56 µm, adjustable output power up to 5 W) arranged with multimode quartz fiber (8) (0.6 mm in diameter) as a radiationdelivering unit. The diameter of the treatment zone on the sample surface was approximately equal to 15 mm. The wavelength of 1.56 µm provides the bulk absorption of laser radiation by tissue8.

1

PC

pm Fig. 1. Scheme of experimental setup for monitoring of laser-mediated modification of cartilage: 1 – He-Ne laser; 2 – telescopic system – beam expander; 3 – sample under study; 4 – image-transferring element: 5 – fiber-optic bundle; 6 – CCD camera; 7 – Erbium laser; 8 – light-delivering fiber; 9 – thermograph.

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PC

1PC Fig. 2. Scheme of the experimental setup with the LASCA modality; 1 – He-Ne laser, 2 – telescopic system, 3 – object, 4 – CCD camera, 5 – Erbium laser, 6 – light-delivering fiber, 7 – thermograph.

For comparison, the similar experiments were carried out with the conventional LASCA modality (Fig. 2) arranged with the CCD sensor of the same type and high-quality imaging lens (LMZ13A5M). The magnification of the tissue surface image at the CCD sensor was ~ 0.4×. Simultaneously with the capture of the sequences of dynamic speckle patterns, the spatial-temporal distributions of the tissue surface temperature were monitored with the use of a thermograph (IRTIS-200 type, Russia). Typical profiles of the instantaneous temperature on the sample surface, which corresponds to different stages of cartilage modification, are illustrated by Fig. 3.

I

70

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60 50 40 30 15

-10

-5

0

5

10

15

x mm Fig. 3. The temperature distributions across the surface of treatment zone at various stages of cartilage modification; 1 – initial phase of modification (tissue heating), 2 – quasistationary phase of modification.

Due to the backscattering geometry of detection and contributions of multiply scattered diffuse components, the detected light is characterized by the significantly more broadband intensity fluctuations in comparison with the case of transillumination monitoring of thermally treated ex vivo cartilage8 . The remarkable blur of speckle patterns occurs at

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the frame rate of 25 fps. Consequently the value of VT~ was estimated for each frame in the recorded sequence without any additional time averaging with a moving window. Fig. 4 displays the dependencies of the temperature in the central region of the treatment zone (a) and the normalized speckle contrast VT~ (t ) VT~ (0) (b) on the time lapse t in the course of the treatment procedure provided with the different values of the output power of IR radiation. Similarly to previously reported results on the transillumination speckle monitoring of cartilage thermal modification8 , the loop-like behavior of VT~ (t ) VT~ (0) occurs with the changes in tissue temperature. Fig. 4, c shows the similar dependence obtained with the LASCA modality.

3. DISCUSSION OF RESULTS The comparison of VT~ (t ) dependencies obtained with the LASCA modality and the bundle-based analyzer allows us to emphasize the expressed non-monotonic behavior of the exposure-dependent speckle contrast in the first case. The “stationary” phase of thermal modification (which corresponds to plateau-like region of T (t ) curve) and the phase of thermal relaxation (after switching off the Erbium laser) are featured by the remarkable oscillations in VT~ (t ) (Fig. 4, c). Such non-monotonic behavior is related to macroscopically heterogeneous spatial distributions of modulating speckles in the image plane, which are associated with inhomogeneities of the cartilage structure. Similar statistical inhomogeneities of image-modulating speckles varying in the course of tissue modification were observed in the case of transillumination monitoring8. On the contrary, dynamic speckle patterns obtained with the use of the bundle-based modality exhibit significant suppression of the contrast oscillations due to the specific conditions of speckle formation (such as random phase modulation of partial optical signals sampled by the bundle and their mixing in the diffraction zone). This gives the opportunity for more detailed analysis of peculiarities in behavior of the exposure-dependent speckle contrast at the various stages of thermal treatment. In particular, the existence of inverse peaks on VT~ (t ) dependencies (marked by black arrows in Fig. 4, b) and dramatic changes in the contrast behavior at the “quasistationary” phase of modification (the trend to increase with the increasing tissue temperature) can be emphasized. The latter peculiarity is obviously manifested for data obtained with the higher values of treatment power (curve 3 in Fig. 4, b) and is correlated with previously reported loop-like behavior of VT~ (T ) in the transillumination mode8. The presumable interpretation of such behavior is related to the change in a predominating mechanism of dynamic light scattering in the thermally treated tissue at the temperatures above 70° C (from generation of new scattering centers due to partial denaturation of the basic components of cartilage structure towards migration of accumulated denaturation products in the treatment zone). Note that in the case of the too high value of treatment power (curve 3 in Fig. 4, a) the heat consumption in the treated tissue volume occurs insufficient and overheating of the treated tissue takes place (see marked region on curve 3 in Fig. 4, a). This situation is characterized by the remarkably less values of VT~ at the stage of thermal modification itself (the time interval from about 20 s to about 40 s, Fig. 4, b) in comparison with more “smooth” regimes of the tissue treatment (curves 1 and 2).

~

Considering the decrement of the exposure-dependent speckle contrast ∆VT~ (t ) = VT~ (t ) − VT~ (t + T ) , we can assume the

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contrast decrement with the increasing tissue temperature allow us to consider the decay in

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at the first stage of tissue

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modification as a thermally activated process with a certain value of the activation energy.

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Fig. 4. a – the dependencies of the tissue temperature in the central region of treatment zone on the time lapse; b – the dependencies of V ~ on the time lapse obtained with the bundle-based full-field speckle analyzer; c – the dependence of V ~ on the time lapse T

T

obtained with the LASCA modality.

Fig. 5 displays the values of ln(∆VT~ ) estimated for the pairs of sequential frames and plotted against the instantaneous inverse absolute temperature in the central part of treatment zone. This plot corresponds to two sets of experimental data obtained with the conventional LASCA modality and the bundle-based speckle analyzer. For the relatively narrow interval of T the plotted data can be approximated with the reasonable accuracy by the linear dependencies of −1 ln(∆VT~ ) on T , which are also shown in Fig. 5. This allows us to assume the Boltzmann-like dependence of the inverse speckle decorrelation time on the absolute temperature of modified tissue: τ c−1 ≈ K1 exp(− Ea RT ) , where the normalization parameter K1 depends on various factors such as the frequency factor of the basic thermally activated process with the activation energy of E a and the transfer properties of used speckle-imaging system. Note that both data sets are characterized by the close values of activation energy (61 ± 5) kJ/mol for data obtained with the LASCA

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modality and (64 ± 6) kJ/mol in the case of application of the bundle-based speckle analyzer, respectively) but significantly differ in the values of the normalization parameter ( K1, LASCA K1,bundle ≈ 2.9 ). This difference is obviously caused by difference in the transfer properties of the conventional LASCA system and the bundle-based speckle analyzer. In the latter case the influence of the transfer properties of light-delivering channel is more expressed because of the cascade transformation of speckle patterns and less quality of the image-transferring component in comparison with the examined LASCA modality.

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The inverse absolute temperature,(°K)' Fig. 5. The values of ln( ∆VT~ ) obtained with the bundle-based speckle analyzer (1-3) and the LASCA modality (4) and plotted against the values of the inverse absolute temperature of the tissue in the central region of treatment zone. The output power of Erbium laser is: 4.5 W (1), 4.2 W (2), 4.8 W (3), 4.5 W (4).

Let us consider some probable mechanisms of the laser-induced cartilage modification, which cause dynamic light scattering in the treated tissue. One mechanism can be related to the temperature-dependent changes in the rate of scatter diffusion in the treated volume. For many condensed media, the dependence of the diffusion coefficient on the temperature is described as follows: D = D0 exp( − E 0 RT ) , where E 0 is the activation energy of transition of scattering particle from one stable position to another one. Among other factors, the contrast decrement is determined by

~

~

the average displacement of scatters L over the observation time T (i.e., ∆VT~ ∝ L ∝ DT ∝ exp(− E0 2 RT ) ). Thus, this consideration leads to the Boltzmann dependence of the contrast decrement on the characteristic temperature of tissue. Another probable mechanism is related to the thermally induced alterations in tissue structure, which are accompanied by the generation of new effective scattering sites in the treated volume. Assuming the mono-molecular process of thermally induced transformation of one of the basic components of cartilage and the insignificant changes in its ~ concentration during the observation time T , we can obtain that the contrast decrement is proportional to the

~

~&

temperature-dependent transformation rate ∆VT~ ∝ N& . With the used assumptions, the value of N will be controlled by

Boltzmann factor exp(− E a 2 RT ) . Thus, both considered mechanisms lead to the Boltzmann-like dependence of

∆VT~ on T .

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The obtained effective value of activation energy, which controls the temperature-dependent speckle dynamics at the initial stage of thermal treatment, can correspond to the thermally induced transfer of tissue liquid, which causes the changes in local configurations of scattering sites, or to the low-energy modification of collagen matrix. Note that the thermal denaturation of collagen itself can be rather excluded from the considered mechanisms, which cause the pronounced structural changes at the initial phase of modification. Typically, the expressed thermal denaturation of collagen in cartilage tissue occurs at the temperatures above ~70 °C9 . However, the thermally induced dynamics of collagen matrix in tissue can be related to the partial denaturation of proteoglycan aggregates (PGA)10 or to the conformational transitions in glycosaminoglycans as one of the basic PGA components. It is worth to note that each disaccharide unit in the glycosaminoglycan macromolecule is stabilized by three hydrogen bonds11 with the total energy of ~ 60-75 kJ/mol12 , that is close to the found value of the effective activation energy.

4. CONCLUSIONS We have examined the potential of speckle contrast monitoring of laser-mediated cartilage modification by means of the developed full-field speckle analyzer, which is featured by application of the fiber-optic bundle as the light-transferring unit and by analysis of timeintegrated dynamic speckles in the diffraction zone. These features provide the suppression of low-frequency noise-like fluctuations of the exposure-dependent speckle contrast, which are typical in the case of application of the conventional LASCA technique. Therefore, the more detailed picture of the thermally induced alterations in cartilage structure can be obtained with the use of the developed speckle analyzer. It was found that the changes in the speckle contrast decrement due to increase in the tissue temperature up to Η 65°C can be approximated by the Boltzmann law with the effective activation energy 61 – 64 kJ/mol, which is close to the energy of conformational transitions in glycosaminoglycan macromolecules. Above 65°C, the treatment of the tissue is accompanied by the dramatic alterations in thermally induced speckle dynamics due to the change of dynamic scattering mechanism.

ACKNOWLEDGMENT This work was supported by grants from the Russian Foundation for Basic Research ## 04-02-16533, 04-02-97203 and 04-02-16743.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

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10. A.M. Jamieson, J. Blackwell, H. Reihanian, H. Ohno, R. Gupta, D.A. Carrino, A.I. Caplan, L.H. Tang, and L.C. Rosenberg, “Thermal and solvent stability of proteoglycan aggregates by quasielastic laser light-scattering”, Carbohydrate Research, 160, p. 329-341, 1987. 11. J.E. Scott, “Secondary and tertiary structures of hyaluronan in aqueous solution. Some biological consequences,” In: Science of Hyaluronan Today. V.C. Hascall and M. Yanagishita, eds., 1998. http://www.glycoforum.gr.jp/science/hyaluronan/hyaluronanE.html 12. G.C. Pimentel and A.L. McClennan, The Hydrogen Bond, W.H. Freeman, San Francisco, 1960.

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