Sebastien Besner

Biomechanical characterization of keratoconus corneas ex vivo with Brillouin microscopy.

PURPOSE Loss of corneal strength is a central feature of keratoconus progression. However, it is currently difficult to measure corneal mechanical changes noninvasively. The objective of this study is to evaluate if Brillouin optical microscopy can differentiate the mechanical properties of keratoconic corneas versus healthy corneas ex vivo. METHODS We obtained eight tissue samples from healthy donor corneas used in Descemets stripping endothelial keratoplasty (DSEK) and 10 advanced keratoconic corneas from patients undergoing deep anterior lamellar keratoplasty (DALK). Within 2 hours after surgery, a confocal Brillouin microscope using a monochromatic laser at 532 nm was used to map the Brillouin frequency shifts of the corneas. RESULTS The mean Brillouin shift in the anterior 200 μm of the keratoconic corneas at the cone was measured to be 7.99 ± 0.10 GHz, significantly lower than 8.17 ± 0.06 GHz of the healthy corneas (P < 0.001). The Brillouin shift in the keratoconic corneas decreased with depth from the anterior toward posterior regions with a steeper slope than in the healthy corneas (P < 0.001). Within keratoconic corneas, the Brillouin shift in regions away from the apex of the cone was significantly higher than within the cone region (P < 0.001). CONCLUSIONS Brillouin measurements revealed notable differences between healthy and keratoconic corneas. Importantly, Brillouin imaging showed that the mechanical loss is primarily concentrated within the area of the keratoconic cone. Outside the cone, the Brillouin shift was comparable with that of healthy corneas. The results demonstrate the potential of Brillouin microscopy for diagnosis and treatment monitoring of keratoconus.

In vivo biomechanical mapping of normal and keratoconus corneas.

Corneal mechanical strength is critical to withstanding intraocular pressure and maintaining normal shape1,2. In keratoconus, the mechanical stability is compromised3, and this may lead to progressive morphological changes. Therefore, a noninvasive technique capable of accurately measuring the mechanical properties of the cornea may help understand the mechanism of keratoconus development and improve detection and intervention in keratoconus. We have previously developed Brillouin microscopy based on light scattering from inherent acoustic waves in tissues4 and shown that this technique can provide quantitative estimates of local longitudinal modulus5, which correlate to the Youngs/shear moduli of the cornea2,6. Using a clinically viable instrument, for the first time to our knowledge, here we mapped the elastic modulus of normal and keratoconus patients in vivo. We found distinctive biomechanical features that differentiate normal and keratoconic corneas and, therefore, have potential to serve as diagnostic metrics for keratoconus.

In Vivo Brillouin Analysis of the Aging Crystalline Lens

Purpose To analyze the age dependence of the longitudinal modulus of the crystalline lens in vivo using Brillouin scattering data in healthy subjects. Methods Brillouin scans were performed along the crystalline lens in 56 eyes from 30 healthy subjects aged from 19 to 63 years. Longitudinal elastic modulus was acquired along the sagittal axis of the lens with a transverse and axial resolution of 4 and 60 μm, respectively. The relative lens stiffness was computed, and correlations with age were analyzed. Results Brillouin axial profiles revealed nonuniform longitudinal modulus within the lens, increasing from a softer periphery toward a stiffer central plateau at all ages. The longitudinal modulus at the central plateau showed no age dependence in a range of 19 to 45 years and a slight decrease with age from 45 to 63 years. A significant intersubject variability was observed in an age-matched analysis. Importantly, the extent of the central stiff plateau region increased steadily over age from 19 to 63 years. The slope of change in Brillouin modulus in the peripheral regions were nearly age-invariant. Conclusions The adult human lens showed no measurable age-related increase in the peak longitudinal modulus. The expansion of the stiff central region of the lens is likely to be the major contributing factor to age-related lens stiffening. Brillouin microscopy may be useful in characterizing the crystalline lens for the optimization of surgical or pharmacological treatments aimed at restoring accommodative power.

Etalon filters for Brillouin microscopy of highly scattering tissues.

Brillouin imaging of turbid biological tissues requires an effective rejection of the background noise due to elastic scattering of probe laser light. We have developed a narrowband spectral notch filter based on a pair of a free-space Fabry-Perot etalon and a mirror. The etalon filter in a 4-pass configuration is able to suppress elastically-scattered laser light with a high extinction ratio of > 40 dB and transmit inelastically-scattered light in a frequency shift range of 2-14 GHz with only 2 dB insertion loss. We also describe a simple etalon that enables us to use semiconductor diode laser sources for Brillouin microscopy by removing spontaneous emission noise. Using a clinically-viable Brillouin microscope employing these filters, we demonstrate the first Brillouin confocal imaging of the sclera and conjunctiva of the porcine eye.

Shear Brillouin light scattering microscope.

Brillouin spectroscopy has been used to characterize shear acoustic phonons in materials. However, conventional instruments had slow acquisition times over 10 min per 1 mW of input optical power, and they required two objective lenses to form a 90° scattering geometry necessary for polarization coupling by shear phonons. Here, we demonstrate a confocal Brillouin microscope capable of detecting both shear and longitudinal phonons with improved speeds and with a single objective lens. Brillouin scattering spectra were measured from polycarbonate, fused quartz, and borosilicate in 1-10 s at an optical power level of 10 mW. The elastic constants, phonon mean free path and the ratio of the Pockels coefficients were determined at microscopic resolution.

Imaging of Keratoconic and normal human cornea with a Brillouin imaging system(Conference Presentation)

Keratoconus is a degenerative disorder of the eye characterized by human cornea thinning and morphological change to a more conical shape. Current diagnosis of this disease relies on topographic imaging of the cornea. Early and differential diagnosis is difficult. In keratoconus, mechanical properties are found to be compromised. A clinically available invasive technique capable of measuring the mechanical properties of the cornea is of significant importance for understanding the mechanism of keratoconus development and improve detection and intervention in keratoconus. The capability of Brillouin imaging to detect local longitudinal modulus in human cornea has been demonstrated previously. We report our non-contact, non-invasive, clinically viable Brillouin imaging system engineered to evaluate mechanical properties human cornea in vivo. The system takes advantage of a highly dispersive 2-stage virtually imaged phased array (VIPA) to detect weak Brillouin scattering signal from biological samples. With a 1.5-mW light beam from a 780-nm single-wavelength laser source, the system is able to detect Brillouin frequency shift of a single point in human cornea less than 0.3 second, at a 5μm/30μm lateral/axial resolution. Sensitivity of the system was quantified to be ~ 10 MHz. A-scans at different sample locations on a human cornea with a motorized human interface. We imaged both normal and keratoconic human corneas with this system. Whereas no significantly difference were observed outside keratocnic cones compared with normal cornea, a highly statistically significantly decrease was found in the cone regions.

A Fabry-Perot etalon-based notch filter for background cleaning in Brillouin microscopy(Conference Presentation)

In Brillouin scattering imaging, rejection of background noise due to elastic scattering and reflections from optical components is crucial. This is because Brillouin signal is weak, and the signal frequency shift compared with source laser line is very small. Therefore the line of interest is very easy to be contaminated. Whereas physical blocking of undesired component in a dispersed spectrum is used, to filter out background optically provides better stability. Conventional optical filter techniques, such as dielectric-stack filters, holographic volume filters, Lyot fitlers etc. normally have a stopband-width (bandstop filter) or edge-width (edge filter) ranging from a few nanometers to tens of nanometers despite high rejection rate. They cannot be implemented in Brillouin imaging due to the small wavelength shift (< 1 pm). We report a Fabry-Perot etalon-based notch filter for background cleaning in Brillouin imaging. The notch filer takes advantage of multiple reflections of the light beam with a Fabry-Perot etalon to achieve high rejection with narrow bandwidth. The theoretical rejection rate is multiple time of the rejection of one reflection of the etalon. We demonstrated a laser line suppression of > 40 dB while with < 40% power loss with experiments. Width of the stopband at -30 dB rejection level is ~ 1 GHz. This method is not wavelength specific. One etalon may be implanted to a wide spectrum of laser wavelengths. Furthermore, it does not require heating as gaseous notch filters. Our method can also be implemented to Raman scattering, fluorescent imaging and other imaging techniques in which line of interest is close to the laser source.