OST determination from close-up images (post-processing)

FLIR Tools (FLIR Systems, Inc.) and BFIC (Batch Thermal Images Converter, Dave Spilka) software were used for extraction of OST data from close-up images and changes of colour scheme post acquisition (Fig. 3). A thermal image was loaded and the ocular area, which normally is the warmest area in the image, was marked with a rectangular shape using the corresponding marking tool. The hottest spot (maximum temperature) in the rectangle, which is automatically indicated, must be located within the ocular surface (area between the two eyelids) and was defined as ocular surface temperature (OST). If the hottest spot was not located within the ocular surface, the marking rectangle was altered until this criterion was fulfilled. The value of the hottest spot was transferred to MS Excel. BFIC has a batch procedure for this purpose. OST was determined from 3 to 5 focused images individually for each eye. The OST of a mouse was the mean of all determined OSTs of both eyes, if not stated otherwise.

Post-processing for OST determination from focused close-up images. Only focused images are included to analysis. The ocular area is marked with a rectangle. Hottest temperature in this area is indicated with a red triangle. This red triangle must be within the ocular area since this indicates the OST for the area. Otherwise the shape of the rectangle has to be changed until this criterion is fulfilled.