In 1520 Florence, Italy, a young woman named Isabella met a painter named Piero who asked to use her as inspiration for his masterpiece. Falling in love with Piero, Isabella agreed to allow him to paint her, even letting Piero use some of her blood and a ground up part of her finger in the painting's pigment. As their work continued, Isabella's love for and obsession with Piero grew and when she finally confessed her feelings to him, he rejected her, claiming to only see her as his muse. When Isabella later returned, she nearly destroyed the painting before finding Piero having sex with another woman. Instead of destroying the painting, Isabella brutally murdered Piero. After Isabella was burned at the stake for her crimes, her ghost became tied to the painting due to her blood and piece of her finger being used to create it. The painting passed into the possession of Isabella's family and eventually to the Catholic Church.
In 2015, the painting was sent to St. Philomena's Church in Worcester, Massachusetts, bringing along the vengeful spirit of Isabella who started murdering unfaithful men. Isabella related her history and the history of the painting to Sister Mathias who eventually realized Isabella was the killer. While Dean believed Isabella was likely tied to her journal, Sam believed there was more to it than that. Sam read from Isabella's journal and learned of her blood and finger being used in the painting's pigment and realized Isabella was actually tied to the painting. Sam searched through the still-unopened crates from the monastery in Tivoli which had sent Isabella's family's things to the church and eventually located the painting. Before Isabella could kill Dean, Sam salted and burned the painting, destroying Isabella.