Giuliano Scarcelli

Brillouin Optical Microscopy for Corneal Biomechanics

PURPOSE The mechanical properties of corneal tissue are linked to prevalent ocular diseases and therapeutic procedures. Brillouin microscopy is a novel optical technology that enables three-dimensional mechanical imaging. In this study, the feasibility of this noncontact technique was tested for in situ quantitative assessment of the biomechanical properties of the cornea. METHODS Brillouin light-scattering involves a spectral shift proportional to the longitudinal modulus of elasticity of the tissue. A 532-nm single-frequency laser and a custom-developed ultrahigh-resolution spectrometer were used to measure the Brillouin frequency. Confocal scanning was used to perform Brillouin elasticity imaging of the corneas of whole bovine eyes. The longitudinal modulus of the bovine corneas was compared before and after riboflavin corneal collagen photo-cross-linking. The Brillouin measurements were then compared with conventional stress-strain mechanical test results. RESULTS High-resolution Brillouin images of the cornea were obtained, revealing a striking depth-dependent variation of the elastic modulus across the cornea. Along the central axis, the Brillouin frequency shift varied gradually from 8.2 GHz in the epithelium to 7.5 GHz near the endothelium. The coefficients of the down slope were measured to be approximately 1.09, 0.32, and 2.94 GHz/mm in the anterior, posterior, and innermost stroma, respectively. On riboflavin collagen cross-linking, marked changes in the axial Brillouin profiles (P < 0.001) were noted before and after cross-linking. CONCLUSIONS Brillouin imaging can assess the biomechanical properties of cornea in situ with high spatial resolution. This novel technique has the potential for use in clinical diagnostics and treatment monitoring.

Brillouin Microscopy of Collagen Crosslinking: Noncontact Depth-Dependent Analysis of Corneal Elastic Modulus

PURPOSE Corneal collagen crosslinking (CXL) is designed to halt the progression of keratoconus and corneal ectasia by inducing corneal stiffening. However, it currently is difficult to monitor and evaluate CXL outcome objectively due to the lack of suitable methods to characterize corneal mechanical properties. We validated noncontact Brillouin microscopy to quantify corneal mechanical properties before and after CXL. METHODS CXL was performed on fresh porcine eyes using various presoaking times and light doses, with or without epithelial debridement. From Brillouin maps of corneal elastic modulus, stiffness and average modulus of anterior, middle, and posterior stroma were analyzed. Corneal stiffening index (CSI) was introduced as a metric to compare the mechanical efficacy of a given CXL protocol with respect to the standard protocol (30-minute riboflavin presoak, 3 mW/cm² ultraviolet illumination for 30 minutes). RESULTS Brillouin corneal stiffness increased significantly (P < 0.001) by epi-off and epi-on CXL. The increase of Brillouin modulus was depth-dependent, indicating that anterior stromal stiffening contributes the most to mechanical outcome. The increase of anterior Brillouin modulus was linearly proportional to the light dose (R² > 0.98). Compared to the standard epi-off procedure, a typical epi-on procedure resulted in a third of stiffness increase in porcine corneas (CSI = 33). CONCLUSIONS Brillouin microscopy allowed imaging and quantifying CXL-induced mechanical changes without contact in a depth-dependent manner at high spatial resolution. This technique may be useful to evaluate the mechanical outcomes of CXL procedures, to compare different crosslinking agents, and for real-time monitoring of CXL in clinical and experimental settings.

Bioimaging of hyaluronic acid derivatives using nanosized carbon dots.

Fluorescent nanosized carbon dots (Cdots) are an emerging bioimaging agent with excellent chemical inertness and marginal cytotoxicity in comparison to widely used semiconductor quantum dots. In this work, we report the application of Cdots for real time bioimaging of target specific delivery of hyaluronic acid (HA) derivatives. Polyethylene glycol (PEG) diamine-capped Cdots were synthesized by the pyrolysis of citric acid in a hot solvent. The synthesized Cdots showed strong fluorescence under UV excitation with emission properties dependending on the excitation wavelength. HA-Cdot conjugates were synthesized by amide bond formation between amine groups of Cdot and carboxylic groups of HA. After confirmation of the negligible cytotoxicity of Cdots and HA-Cdot conjugates, in vitro bioimaging was carried out for target specific intracellular delivery of the HA-Cdot conjugates by HA receptor-mediated endocytosis. Furthermore, in vivo real-time bioimaging of Cdots and HA-Cdot conjugates exhibited the target specific delivery of HA-Cdot conjugates to the liver with abundant HA receptors. Taken together, we could confirm the feasibility of HA derivatives as a target-specific drug delivery carrier for the treatment of liver diseases and Cdots as a promising bioimaging agent.

In vivo Brillouin optical microscopy of the human eye

We report the first Brillouin measurement of the human eye in vivo. We constructed a Brillouin optical scanner safe for human use by employing continuous-wave laser light at 780 nm at a low power of 0.7 mW. With a single scan along the optic axis of the eye, the axial profile of Brillouin frequency shift was obtained with a pixel acquisition time of 0.4 s and axial resolution of about 60 μm, showing the depth-dependent biomechanical properties in the cornea and lens.

Noncontact three-dimensional mapping of intracellular hydromechanical properties by Brillouin microscopy

Current measurements of the biomechanical properties of cells require physical contact with cells or lack subcellular resolution. Here we developed a label-free microscopy technique based on Brillouin light scattering that is capable of measuring an intracellular longitudinal modulus with optical resolution. The 3D Brillouin maps we obtained of cells in 2D and 3D microenvironments revealed mechanical changes due to cytoskeletal modulation and cell-volume regulation.

Collagen Cross-Linking Using Rose Bengal and Green Light to Increase Corneal Stiffness

PURPOSE Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application. METHODS Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment. RESULTS RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability. CONCLUSIONS RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.

Multistage VIPA etalons for high-extinction parallel Brillouin spectroscopy.

We demonstrate a high-resolution high-extinction parallel spectrometer for Brillouin spectroscopy of turbid samples. Cascading multiple VIPA etalons in the cross-axis configuration allowed us to achieve a high extinction ratio of up to 80 dB with sub-GHz resolution. Using a three-stage VIPA, we obtained the Brillouin spectra from Intralipid solutions at concentrations up to 10%.

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.

Two-photon interference with thermal light

We report an experiment that extends the concept of two-photon probability amplitude to the general case of independent photons. The experiment also shows interesting practical applications regarding high resolution interference patterns with thermal sources.

Distant clock synchronization using entangled photon pairs

We report a proof-of-principle experiment on distant clock synchronization. Besides the achievement of picosecond resolution at 3km distance, this experiment demonstrated a concept for high-accuracy nonlocal timing and positioning based on the quantum feature of entangled states.