Measuring the area of tear film break-up by image analysis software

Tear film breakup time (BUT) test only examines the first break in the tear film, but subsequent tear film events are not monitored. We present a method of measuring the area of breakup after the appearance of the first breakup by using open source software. Furthermore, the speed of the rupture was determined. 84 subjects participated in the study. 2 μl volume of 2% sodium fluorescein was instilled using a micropipette. The subject was seated behind a slit-lamp using a cobalt blue filter together with a Wratten 12 yellow filter. Then, the tear film was recorded by a camera attached to the slit lamp. 4 frames of each video was extracted, the first rupture (BUT_0), breakup after 1 second (BUT_1), rupture after 2 seconds (BUT_2) and breakup before the last blink (BUT_F). Open source software of measurement based on Java (NIH ImageJ) was used to measure the number of pixels in areas of breakup. These areas were divided by the area of exposed cornea to obtain the percentage of ruptures. Instantaneous breakup speed was calculated for second 1 as the difference between BUT_1 - BUT_0, whereas instant speed for second 2 was BUT_2 - BUT_1. Mean area of breakup obtained was: BUT_0 = 0.26%, BUT_1 = 0.48%, BUT_2 = 0.79% and BUT_F = 1.61%. Break speed was 0.22 area/sec for second 1 and 0.31 area/sec for second 2, showing a statistical difference between them (p = 0.007). Post BUT analysis may be easily monitoring with the aid of this software.