The initial steps that Johannes Vermeer took to create Girl with a Pearl Earring (c. 1665) are, partially or completely, hidden beneath the painted surface. Vermeer's painting technique involved applying underlayers that vary in colour and thickness, on top of which he applied the upper paint layer(s). To investigate the painting's genesis, the Girl was examined using non-invasive scientific imaging as part of the project The Girl in the Spotlight (2018). This was complemented by the re-examination of existing paint cross-sections using microscopic analytical techniques, including focused ion beam-scanning transmission electron microscopy (FIB-STEM). The weave of the canvas, visible in the X-radiograph, was examined using a new computer-assisted thread-level canvas analysis algorithm to measure the thread spacing and thread angles. The canvas was primed with a warm light grey ground, although probably not by Vermeer himself. The tinted ground provided neutral base tone upon which he began to lay in areas of light and shade in his composition, and some outlines around the figure. Multispectral infrared reflectography( MS-IRR) images in four spectral bands spanning the spectral range from 900 to 2500 nm were collected of the painting at high spatial resolution (50 µm/pixel). Visible 3D digital microscopy visualised the underlayers where Vermeer left them visible around the perimeter of the figure as a contour, and where the upper paint layers are thin. These infrared reflectograms and visible micrographs identified three distinct preparatory phases of Girl with a Pearl Earring: the ground, fine black outlines, and underlayers. Some pentimenti were also detected, including a change in the position of her ear. The combination of examination methods showed that the materials beneath the surface played an important role in establishing the three-dimensional space, fall of light, and the eventual colour in different areas of the painting.