Corneal crosslinking (CXL) with UVA light is the primary treatment for keratoconus, a disease that affects cornea's stability, transparency and shape. UVA-CXL has limitations in penetration depth and unwanted irradiation on healthy tissue. As an alternative, a near-infrared femtosecond laser was used for targeted corneal crosslinking of fresh pigs’ corneas. Brillouin microscopy was implemented as a non-destructive method to determine the viscoelastic properties, by measuring the Brillouin shift. We compared the Brillouin shifts measured for UVA-CXL and fs-CXL treated corneas. Measurements were also performed on UVA-CXL pure bovine collagen I in order to correlate the changes observed in CXL cornea. An increase in Brillouin shift before and after crosslinking, for both UVA and femtosecond-CXL are measurable. We demonstrate the precision and efficacy of using femtosecond CXL in spatial targeted CXL at depth in corneal tissues.
Optical tweezers are powerful scientific tools capable of trapping and manipulating microscopic particles with a tightly focussed laser beam. They can be used to measure nm-sized movements as well as pN-forces in various applications . One of these applications is "dynamic force spectroscopy", where increasing forces are applied to e.g. receptor-ligand bonds to facilitate unbinding. Analysis of unbinding forces, or "rupture forces", can provide an overview of the thermodynamics and activation barriers during bond dissociation. Claudins are considered important component of tight junctions, which form a seal between epithelial cells to regulate paracellular transport. Clostridium perfringens enterotoxin (CPE) is a toxin that binds to certain claudins and disrupts the tight junctions. Understanding
of the specific interaction between CPE and receptor claudins could lead to several pharmaceutical applications. This work aims to measure the binding strength between c-CPE and claudins in living cells, using an optical tweezers system. The system was characterized and calibrated extensively, which is necessary for force measurements. The trap stiffness in both lateral directions was measured for 1μm silica particles in cell culture medium. Stiffness values up to kx ≈ 89.81 pN/μm and ky ≈ 129.28 pN/μm in cell culture medium were determined. To investigate the interactions between C-CPE and claudins, 1 μm silica particles were coated with C-CPE and trapped with the optical tweezers. MCF-7 cells, which are known to express receptor claudins were positioned towards the particle until contact, then retracted. The technique can detect unbinding events and measure corresponding rupture forces from 1 pN up to 56.7 pN in magnitude. Overall, this work shows that optical tweezers offer a versatile and precise non-contact method to probe the interaction of transmembrane proteins in living cells.
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