Duration 5min, Q&A 3min
Laser Spectroscopy Study of the Molecular Structure of Anterior Lens Capsule and the Structural Changes on Staining with Trypan Blue Dye
To identify whether trypan blue dye staining of the anterior lens capsule brings about changes in its molecular structure and its implications in elastic properties during cataract surgery.
Human anterior lens capsules (obtained during phacoemulsification) were collected. Age and cataract-type matched 10 samples each of trypan blue stained & unstained capsules along with anterior capsules of 10 trypan blue stained mature cataracts were transported to the lab in balanced salt solution & analysed by Raman Laser Spectroscopy within 6-8 hrs.
Anterior lens capsules were studied by laser Raman spectrometer equipped with a CCD detector and laser of wavelength 532 nm. A 50x objective (NA = 0.45) was used to focus the laser beam on both peripheral and core regions of the sample. Spectral acquisition window was set from 500 to 3500 cm-1. Raman band assignments were performed on the obtained spectra (with a resolution of 1 cm-1 achieved with 1800 grooves/mm holographic grating) by using spectrum from commercially available pure type IV collagen as reference. For dye incorporated lens capsule samples FT-Raman setup was used with a laser line of 1064 nm to collect Raman spectra.
Results and Conclusion
Results: Raman spectra of unstained capsule showed secondary structure marker amide I band at around 1670 cm-1 corresponding to the collagen IV component. The disulphide bridges of the 7S domains showed characteristic peaks from 500-550 cm-1 and C-S stretching modes appear at around 720 cm-1. Stained capsule showed broadening of amide I band and appearance of higher wavenumber components at around 1690 cm-1. The C-S stretching mode shifts to lower wavenumber 714 cm-1. In fibrotic capsules of mature cataracts, appearance of lower wavenumber components in the amide I band at around 1658 cm-1 indicated the presence of the collagen I fibers
Conclusion: Trypan blue staining weakens hydrogen bonding between the fibrils, changes the polar environment around carbonyl group of the peptide bonds and changes the organization of collagen IV, resulting in altered physical characteristics of the capsule like decrease in elasticity and mechanical strength which is more so in mature cataracts. So in phacoemulsification of mature cataracts , while performing capsulorhexis and during nucleus splitting, extra caution is required to avoid inadvertent tears in the capsule margin if the capsule is stained with trypan blue.
[ Keyword ]
Anterior Lens Capsule / Trypan Blue Dye / Molecular structure / Mechanical Characteristics / Raman Laser Spectroscopy
[ Conflict of Interest ]