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Roll-to-roll (R2R) flexible electronics manufacturing technologies can enable continuous production of flexible electronic devices with a potential significant cost reduction. One of the critical applications of R2R flexible electronic technologies is the manufacturing of flexible display backplanes. In the manufacturing process, multi-layer device registration and overlay alignment is critical. Typically used flexible polymer substrates, though, compared with conventional glass substrates, have relatively large dimensional instability. From the authors' previous work, baseline data using a R2R photolithography system has been reported for fabrication, registration and overlay of micron-sized patterns on both unsupported discrete plastic substrates and carried by a web. Micron-sized features with 1micron overlay accuracy have been achieved on unsupported discrete 5 mil (125 micron) thick substrates of Dupont Melinex® ST507 polyethylene terephthalate (PET) coated with photoresist , . In this paper, a vector model based on experimental results is designed to investigate and map the substrate deformation and overlay alignment in a R2R photolithography process. The vector model quantifies the significance of distortion offsets caused by elastic deformation in the overlay process on R2R based substrates. Furthermore, the relationship between the placement of the alignment fiducials and the local overlay offsets is also initially investigated. Finally, the overlay alignment processes and registration capability are applied to dimensionally stable flexible glass substrates.