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. 2015 Nov 5;10(11):e0142309. doi: 10.1371/journal.pone.0142309

Fig 3. Illustration of Underestimation Measurement Bias by the Automated Image Analysis Protocol.

Fig 3

A) IVCCM image obtained using the Rostock corneal module of the Heidelberg Tomograph III using the 300 μm field of view lens B) Image from panel A traced for parameter quantification using CCMetrics, the software used in the manual protocol of IVCCM image analysis. Red lines indicate fibres, blue lines indicate branches and green dots indicate branching points. C) Image from Panel A post-analysis by ACCMetrics, the software used in automated IVCCM image analysis, showing which nerves were captured by the automated quantification process. The arrows were included to indicate nerves that were not quantified by ACCMetrics despite being traced for quantification using the manual protocol. This inability of ACCMetrics to detect fainter nerve fibres is representative of the entire dataset and explains the measurement (underestimation) bias inherent in using automated IVCCM image analysis protocols.