Identifying Optimum Limits of Concentration and Exposure Time of Trypan-Blue Dye to Stain Anterior Lens Capsule: Laser Spectroscopy Study
Narrative Responses:
Purpose
To elucidate the molecular changes in anterior lens capsule after staining with trypan blue dye. To observe the effect of different concentrations and different durations of exposure of dye on the capsule during phacoemulsification so that optimum limits of concentration and exposure time of the dye can be identified.
Methods
Raman laser spectrometry was utilized to get unique information about the molecular structure of anterior lens capsule. Unstained anterior lens capsules were collected after capsulorhexis from 20 patients (including five mature cataracts) for the experiments. Spectra for unstained and stained including mature cataract capsules were plotted. Capsules were exposed to time intervals of 10s, 15s, 30s, 60s for the concentrations of 0.012% and 0.06%. Spectra were obtained from capsules stained with different concentrations of dye (1 in 2, 3, 4, 5, 8 10, 50, 100 dilutions) at the end of 2 minutes. Computerised plotting of data was done.
Results
In unstained capsules, the secondary structure marker amide I band is seen at 1665-1675cm-1. Dye stained capsules showed broadening of amide I band and the appearance of higher wavenumber at 1690 cm-1 showing that the capsule proteins undergo uncoiling from tightly packed triple helix to randomly coiled state, thereby reducing its elasticity and increasing its stiffness. These structural changes were noted to start at a concentration of 0.006% (from 1 in 10 dilution and lesser dilutions) at the end of 2 minutes. The 1 in 5 dilution (0.012%) stained capsule showed these changes after 15 seconds of exposure time.
Conclusion
Trypan blue staining of anterior lens capsule makes it less elastic and more stiff. If the dye is used in a dilution of 1 in 5 or higher of the 0.06% solution (0.012%) and an exposure time of 10 seconds or less, no structural changes of the capsule were noted.