Quantitative characterization of membrane-protein reversible association using FCS.

Functionally meaningful reversible protein-membrane interactions mediate many biological events. Fluorescence correlation spectroscopy (FCS) is increasingly used to quantitatively study the non-reversible binding of proteins to membranes using lipid vesicles in solution. However, the lack of a complete description of the phase and statistical equilibria in the case of reversible protein-membrane partitioning has hampered the application of FCS to quantify the partition coefficient (Kx). In this work, we further extend the theory that describes membrane-protein partitioning to account for spontaneous protein-membrane dissociation and reassociation to the same or a different lipid vesicle. We derive the probability distribution of proteins on lipid vesicles for reversible binding and demonstrate that FCS is a suitable technique for accurate Kx quantification of membrane-protein reversible association. We also establish the limits to Kx determination by FCS studying the Cramer-Rao bound on the variance of the retrieved parameters. We validate the mathematical formulation against reaction-diffusion simulations to study phase and statistical equilibria and compare the Kx obtained from a computational FCS titration experiment with the experimental ground truth. Finally, we demonstrate the application of our methodology studying the association of anti-HIV broadly neutralizing antibody (10E8-3R) to the membrane.

Poster Session: Experimental assessment of scleral anisotropy using multi-meridian air-coupled ultrasonic optical coherence elastography.

Scleral biomechanics plays a key role in the understanding of myopia progression. In this study, we characterized the elastic properties of sclera using an air-coupled ultrasonic (ACUS) optical coherence elastography (OCE) system. New Zealand rabbit eyes (n=7) were measured (<24hr postmortem) in four scleral locations: superior/inferior temporal (ST, IT), and superior/inferior nasal (SN, IN) maintaining an intraocular pressure of 15 mmHg. Elastic waves were induced in the sclera, and wave propagation velocity and shear modulus were measured along two directions: circumferential (superior-inferior) and meridional (nasal-temporal). Wave velocity in scleral tissue ranged from 6 to 24 m/s and shear modulus from 11 to 150 kPa. Velocity was significantly higher (p<.001) in the circumferential vs. meridional directions in the following locations: ST:15.83±2.85 vs 9.43±1.68 m/s, IT:15.00±3.98 vs 8.93±1.53 m/s; SN:16.79±4.30 vs 9.27±1.47 m/s; and IN:13.92±3.85 vs 8.57±1.46 m/s. The average shear modulus in the circumferential was also significantly higher (p<.001) than in the meridional direction for all locations: 65.37±6.04 vs 22.55±1.36 kPa. These results show that the rabbit sclera is mechanically anisotropic with higher rigidity in the circumferential direction compared to the meridional direction. ACUS-OCE is a promising non-invasive method to quantify the biomechanical changes in scleral tissue for future studies involving myopia treatments.

Poster Session: Estimation of scleral biomechanical properties from air-puff-coupled optical coherence tomography.

Progression of myopia is usually accompanied by axial overgrowth of the eyeball, which affects scleral biomechanics (BM). To study scleral biomechanics, we propose the use of air-puff deformation swept-source OCT imaging. Air-puff deformation imaging was performed at different sites of ex vivo porcine (n=5) and rabbit (n=3) eyes, (<24hr postmortem): Nasal/temporal equatorial and posterior sclera (NE, NP, TE, TP), superior (S) and inferior (I) sclera, and cornea (C). Intraocular pressure was kept at 15mmHg. Deformation data were used as input to inverse finite element model (FEM) algorithms to reconstruct BM properties. Experimental deformation amplitudes showed dependence on the animal model, with porcine scleras exhibiting greater inter-site variation (displacement of S, I was up to four times greater than that of N, T), while rabbit scleras exhibited at most 40% of displacement differences between all sites. Both models showed significant (p<.001) differences in the temporal deformation profile between sclera and (C), but similarities in all scleral locations, suggesting that the scleral temporal profile is independent of scleral thickness variations. The FEM estimated an elastic modulus of 1.84 ± 0.30 MPa (I) to 6.04 ± 2.11 MPa (TE) for the porcine sclera. The use of scleral air-puff imaging is promising for noninvasive investigation of structural changes in the sclera associated with myopia and for monitoring possible modulation of scleral stiffness with myopia treatment.

Effects of Different Scleral Photo-Crosslinking Modalities on Scleral Stiffness and Hydration.

Purpose: The purpose of this study was to evaluate the biomechanical and hydration differences in scleral tissue after two modalities of collagen cross-linking. Methods: Scleral tissue from 40 adult white rabbit eyes was crosslinked by application of 0.1% Rose Bengal solution followed by 80 J/cm2 green light irradiation (RGX) or by application of 0.1% riboflavin solution followed by 5.4 J/cm2 ultraviolet A irradiation (UVX). Posterior scleral strips were excised from treated and untreated sclera for tensile and hydration-tensile tests. For tensile tests, the strips were subjected to uniaxial extension after excision. For hydration-tensile tests, the strips were dehydrated, rehydrated, and then tested. Young's modulus at 8% strain and swelling rate were estimated. ANOVAs were used to test treated-induced differences in scleral biomechanical and hydration properties. Results: Photo-crosslinked sclera tissue was stiffer (Young's modulus at 8% strain: 10.7 ± 4.5 MPa, on average across treatments) than untreated scleral tissue (7.1 ± 4.0 MPa). Scleral stiffness increased 132% after RGX and 90% after UVX compared to untreated sclera. Scleral swelling rate was reduced by 11% after RGX and by 13% after UVX. The stiffness of the treated sclera was also associated with the tissue hydration level. The lower the swelling, the higher the Young's modulus of RGX (-3.8% swelling/MPa) and UVX (-3.5% swelling/MPa) treated sclera. Conclusions: Cross-linking with RGX and UVX impacted the stiffness and hydration of rabbit posterior sclera. The Rose Bengal with green light irradiation may be an alternative method to determine the efficacy and suitability of inducing scleral tissue stiffening in the treatment of myopia.

Estimation of scleral mechanical properties from air-puff optical coherence tomography.

We introduce a method to estimate the biomechanical properties of the porcine sclera in intact eye globes ex vivo, using optical coherence tomography that is coupled with an air-puff excitation source, and inverse optimization techniques based on finite element modeling. Air-puff induced tissue deformation was determined at seven different locations on the ocular globe, and the maximum apex deformation, the deformation velocity, and the arc-length during deformation were quantified. In the sclera, the experimental maximum deformation amplitude and the corresponding arc length were dependent on the location of air-puff excitation. The normalized temporal deformation profile of the sclera was distinct from that in the cornea, but similar in all tested scleral locations, suggesting that this profile is independent of variations in scleral thickness. Inverse optimization techniques showed that the estimated scleral elastic modulus ranged from 1.84 ± 0.30 MPa (equatorial inferior) to 6.04 ± 2.11 MPa (equatorial temporal). The use of scleral air-puff imaging holds promise for non-invasively investigating the structural changes in the sclera associated with myopia and glaucoma, and for monitoring potential modulation of scleral stiffness in disease or treatment.