Laser Spectroscopic Study on Effect of Time, Concentration, and Light Exposure of Anterior Lens Capsules Stained With Trypan-Blue Dye
Purpose
To examine the molecular changes in human anterior lens capsule on staining with trypan blue.
Methods
Thirty anterior lens capsules were obtained from 30 human eyes. Eight capsules were stained in-vivo. The rest were unstained, which were stained ex-vivo using different concentrations, different staining times and different light exposures. The samples were analyzed using an FT-Raman spectrometer. A diode-pumped Nd:YAG laser at 1064 nm with an output of 250 mW was used as the excitation source. 128 scans were collected for each spectrum. Spectra were obtained from a range of 400-4000 cm-1 with a resolution of 2 cm-1. Multiple peak fitting was done to determine position, intensity, width and the area under the peaks.
Results
The amide I band at around 1640 cm-1 and 1675 cm-1 corresponding to the regular and triple helix secondary structures were identified as markers for monitoring structure alterations in anterior lens capsules on trypan blue staining. On trypan blue staining at different concentrations (concentration of 0.06% and dilutions of 10, 50 and 100) the amide I band shifts to higher wavenumbers (1653 and 1703 cm-1) indicating commencement of disorders in structure. The structural disorder was also found to be dependent on the time of staining. Light exposure of more than 2 min causes substantial cross-linking in the tropocollagen fibers.
Conclusion
Trypan blue staining reduces the elasticity and increases the stiffness of anterior lens capsule. Concentration of dye, duration of staining and the time of light exposure were found to be crucial parameters in the structural alteration of the lens capsule.