3D Transformation Matrix Calculation and Pixel Intensity Normalization for the Dual Focus Tracking System

We provide details of the dual-focal 3D tracking and pixel intensity calibration (DuFT) system used to record gene expression in targeted areas on the skin of freely moving mice. The accuracy of the 3D position calculation was determined by placing a scintillator on the calibration disk at various known locations within the recording cage. The height of the disk was varied from the bottom (Z = 0 cm) to the top (Z = 30 cm). The distance from the central axis range from near the center (R = 1 cm) to near to edge (R = 5 cm). The mean deviation between the known and calculated position was .31 ± 0.16 mm. The maximum deviation was less than .86 mm. The results indicate that the location of a scintillator within the recording cage imaged with two cameras can be calculated with submillimeter accuracy. We hope that our methods can be applied to improve automatic (even real-time) tracking of various animals in vivo.