Quantitative Assessment of Hyaline Cartilage Elasticity during Optical Clearing using Optical Coherence Elastography Chih-Hao Liu 1, Manmohan Singh 1, Jiasong Li 1, Zhaolong Han 1, Chen Wu 1, Shang Wang 2, Rita Idugboe 1, Raksha Raghunathan 1, Valery P. Zakharov 3, Emil N. Sobol 4, Valery V. Tuchin 3,5,6, Michael Twa 7, and Kirill V. Larin 1,3,5,6+ 1 Department of Biomedical Engineering, University of Houston, 3605 Cullen Boulevard, Houston, Texas USA 2 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA 3 Department of Electrical Engineering, Samara State Aerospace University, Samara, Russia 4 Department of Physics, Moscow State University, Moscow, Russia 5 Department of Optics and Biophotonics, Saratov State University, Saratov, Russia 6 Interdisciplinary Laboratory of Biophotonics, Tomsk State University, Tomsk Russia 7 College of Optometry, University of Houston, 505 J.Davis Armistead Bldg., Texas USA + Corresponding author:

Motivation [1] M. G. Stewart, T. L. Smith, E. M. Weaver et al., “Outcomes after nasal septoplasty: results from the Nasal Obstruction Septoplasty Effectiveness (NOSE) study,” Otolaryngology--Head and Neck Surgery, 130(3), (2004). [2] E. Sobol, A. Sviridov, V. Svistushkin et al., "Feedback controlled laser system for safe and efficient reshaping of nasal cartilage." 7548, 75482H-75482H-5. [3] E. Sobol, A. Sviridov, A. Omel’chenko et al., “Laser reshaping of cartilage,” Biotechnology and Genetic Engineering Reviews, 17(1), (2000). [4] D. E. Protsenko, A. Zemek, and B. J. F. Wong, “Temperature dependent change in equilibrium elastic modulus after thermally induced stress relaxation in porcine septal cartilage,” Lasers in Surgery and Medicine, 40(3), (2008). Laser septochondrcorrection (LSC) (non-destructive surgery) advantage Safe(bloodless, painless) non-invasive Less complication compared with traditional septoplasty surgery [1,2] Stress relaxation process Permanent deformation Change from Bound water to free water state Biomechanical property changes [3] Fig: Scheme of Laser septochondrcorrection procedure Fig: Optimal condition for laser reshaping window [3] Fig: Stress relaxation mechanism [4]

Motivation Optical clearing technique An approach to monitor the change of tissue optical properties (structural information) OCT signal slope [1] However…The elasticity changes of biological tissues during clearing process haven’t been studied yet Optical coherence elastography (OCE) Biomechanical property measurement Cornea[2], soft-tissue tumor[3], cardiac muscle[4] In this work we report the first use of OCE to monitor the elasticity changes during optical clearing process. Speckle variance analysis OCE detection Uniaxial mechanical testing Fig. Visualization of the elastic wave propagation in ex vivo rabbit cornea [1] K. V. Larin, M. G. Ghosn, A. N. Bashkatov et al., “Optical clearing for OCT image enhancement and in-depth monitoring of molecular diffusion,” IEEE Journal of Selected Topics in Quantum Electronics, 18(3), (2012). [2] S. Wang, and K. V. Larin, “Shear wave imaging optical coherence tomography (SWI-OCT) for ocular tissue biomechanics,” Optics letters, 39(1), (2014). [3] S. Wang, J. Li, R. K. Manapuram et al., “Noncontact measurement of elasticity for the detection of soft-tissue tumors using phase-sensitive optical coherence tomography combined with a focused air-puff system,” Optics letters, 37(24), (2012). [4] S. Wang, A. L. Lopez, Y. Morikawa et al., “Noncontact quantitative biomechanical characterization of cardiac muscle using shear wave imaging optical coherence tomography,” Biomedical Optics Express, 5(7), (2014).

Material and method Sample preparation Two samples were width-wise extracted from the same nasal septum cartilage OCE measurement Uniaxial mechanical testing Optical clearing agent 1X PBS 20% glucose Clearing period 0-20 min: 1X PBS min: 20% glucose 1.3cm 1cm Fig: The used cartilages during the optical clear experiment

Phase-stabilized swept source OCT (PhS-SSOCT) Broad band swept laser:1310nm Scan range: 150nm Scan rate: 30k Hz The axial resolution: ~11 µm Phase stability: 16 µm Scan distance: 6.25mm (n=251) OCT signal Phase: Elastic wave velocity Intensity: Speckle variance Uniaxial mechanical compression testing Fig: diagram of mechanical compression testing Fig: Schematic diagram of PhS-SSOCT

Quantification of elasticity from OCE Displacement profile Where λ 0 was the central wavelength of the laser source, and was the phase of OCT signal, and n was the refractive index. Elasticity quantification: Time delay t Cross-correlation analysis Elastic group Velocity can be expressed as: Young’s modulus [1]: where ρ=1100 kg/m 3 was the density of the tissue, ν=0.5 was the Poisson ratio [1] Shang Wang, J. Li, S. Vantipalli et al., “A focused air-pulse system for optical-coherence-tomography-based measurements of tissue elasticity,” Opt. Lett., 10(7), (2013). Fig: (left) OCE setup with OCE measurement positions. (left) typical displacement profile Corresponding to the red point in (left)

Speckle variance computation Speckle variance [1] Study the fluid kinetics during the clearing process Procedure Perform a linear fit on the OCT A- line signal The linear fit was then subtracted from the OCT signal The speckle variance was determined by a standard deviation of the slope removed OCT signal [1]C.-H. Liu, J. Qi, J. Lu et al., “Improvement of tissue analysis and classification using optical coherence tomography combined with Raman spectroscopy,” Journal of Innovative Optical Health Sciences, 8(2), (2014). Fig: (left) A typical OCT A-line intensity profile with a linear fit (right) Slope-removed OCT A-line intensity profile with standard deviation bounds.

Result Speckle variance Kinetic glucose diffusion min OCE elasticity 0-20 min (water absorbance) 20-30min (Bound to free water state) min (water diffused back) Fig: (upper) Speckle variance, as quantified by the standard deviation of the slope-removed A-line intensity profile. (lower) Young’s modulus as estimated by equation (2) utilizing the elastic wave group velocity as measured by PhS-SSOCE. The cartilage sample was immersed in 1X PBS for 20min, then in 20% glucose for 120min. Fig: Stress relaxation mechanism [1] [1] E. Sobol, A. Sviridov, A. Omel’chenko et al., “Laser reshaping of cartilage,” Biotechnology and Genetic Engineering Reviews, 17(1), (2000).

Result Quantitative value difference Anisotropy of the biomedical properties [1] Fig. (upper) Elasticity as measured by PhS-SSOCE and uniaxial mechanical testing. (lower) Uniaxial mechanical compression testing. The cartilage sample was immersed in 1X PBS for 20 minutes, then in 20% glucose for 120 minutes. [1] B. J. F. Wong, K. K. H. Chao, H. K. Kim et al., “The Porcine and Lagomorph Septal Cartilages: Models for Tissue Engineering and Morphologic Cartilage Research,” American Journal of Rhinology, 15(2), (2001).

Conclusion The elasticity of the cartilage Decrease Sample dehydration caused by glucose solution. Increase Sample hydration by the water diffused back to the cartilage during mechanical compression test The elasticity trend obtained by PhS-SSOCE uniaxial compression test The results demonstrate the feasibility of utilizing OCE to detect and monitor the biomechanical properties during optical clearing. In Future, Viscosity change characterize the water content of the cartilage. are in agreement

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