The absence of any metal elements and pigment particles in the blue paint indicated the use of an organic, vegetable-derived dye. Research in pigment history literature found that the probable blue colouring agent was the medieval dye Turnsole (folium) (or a similar lichen-based indicator dye). It is stated to have the property of changing colour with acid or alkali, red in acid and blue in alkali (similar to litmus). To verify this, in view of the recorded colour change a chemical test was performed on samples of the blue paint. The result was that "a slow colour change occurred in dilute acetic acid solution, the blue colour being lost to be replaced by an orange colour, while some finite red particles not previously visible leached out from the destroyed medium". As Turnsole has never before been recorded in a painting - only in medieval manuscript illumination - it was considered that further analysis was appropriate. Consequently, analysis of fragments of the blue paint by High Performance Liquid Chromatography was undertaken at the Laboratory for Materials and Techniques, the Royal Institute for Cultural Heritage, Brussels by a scientist, expert in the identification of early dyes and pigments. This test supported the likelihood of theTurnsole finding, so that all technical evidence acquired to date points to the use of the Turnsole dye.

Paint samples were also subjected to laser Raman spectroscopic analysis. This analysis found clear evidence of the use of the yellow pigment massicot in the painting. This lead-derived pigment was employed by artists from antiquity but it was not used later than the seventeenth century, when it was superseded by Naples Yellow in the artist's palette. The analysis also showed evidence of Prussian Blue, considered to be the subject of a later restoration.

The Medium: Laser Raman spectroscopic analysis was not able to specifically identify the medium, but the Raman spectra were able to exclude the mediums of oil, resin, gum, glair and animal glue, a possibility therefore being a glue of vegetable origin.

Read the Tondo Paint Materials Report from the UK Forensic Science Service.