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Identifying optimum limits of concentration and exposure time of trypan blue dye to stain anterior lens capsule: laser spectroscopy study

Poster Details

First Author: M.Mathen INDIA

Co Author(s):    S. Siddantha   C. Narayana           

Abstract Details


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.


Narayana nethralaya Hospital, Bangalore, India


Phacoemulsification was performed in a tertiary institute. Anterior lens capsules were collected and stored in balanced salt solution. They were transported within 6 to 8 hours to the laboratory equipped with Raman Laser spectroscope to perform the experiments required to persue the purpose of the study. Unstained anterior lens capsules were collected after capsulorhexis from 20 patients (including five mature cataracts) for the experiments. Anterior lens capsules were studied by laser Raman spectrometer equipped with a CCD detector and laser of wavelength 532 and 1064 nano meters. An objective was used to focus the laser beam on both peripheral and core regions of the sample. Spectral acquisition window was set and Raman band assignments were performed on the obtained spectra by using spectrum from commercially available pure type four collagen as reference. Raman laser spectrometry was utilized to get unique information about the molecular structure of anterior lens capsule. 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.


In unstained capsules, the secondary structure marker amide I band is seen at 1665-1675 cm-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.


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. So during surgery if surgeon prefers to retain the same elasticity of the capsule like in the unstained form, one could use the 0.012% dilution of the dye with an exposure time of less than 10 seconds. In the contrary, if the surgeon prefers to have a less elastic capsule to perform the capsulorhexis, one could use the undiluted (0.06%) form of the dye for the same exposure time. The less elastic and stiffer nature of the capsule has to be kept in mind to avoid inadvertent tears of the capsule while preforming capsulorhexis and while splitting the nucleus especially in mature and harder cataracts where trypan blue dye is almost always used. FINANCIAL INTEREST: NONE

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