**3. Results**

#### *3.1. Vis-NIR Multispectral Reflectography*

Enhanced instrumental resolution was ensured by raster acquisition performed with oversampling (4x) in both x and y scanning directions, while proper image deconvolution was applied to the datacube. False-color imaging, which was performed on the high-resolution images following the processing described above, revealed traces of the preliminary drawing that outlines the steep cliff on the left of the waterfall and the fortress on the promontory. Details of the rocky wall can be observed in Figure 2, which displays the FC images obtained by combining the NIR -at 1292 nm, the red, and the green images (Figure 2b,d), and PC2, PC3, and PC4 images (Figure 2c,e). According to XRF analysis of the same areas [27], the sketch was drawn with a lead stylus, which is consistent with findings pertaining to other drawings by the same author. The use of a lead stylus is particularly evident in the reported NRG-FC images, where a dark line is visible beneath the red of the main drawing. The same lines appear bright orange in the PC-FC images. Leonardo used to draw with different metal points: lead, which is soft and easy to erase, was used to sketch out drawings that were then refined with a pen; silver and metal alloys were preferred for figure sketches and studies, often completed with lead white highlights [30].

**Figure 2.** Traditional false-color and trichromatic RGB PC processing reveals the presence of the underdrawing in lead stylus on the recto. (**a**) White dashed rectangles on the RGB image indicate the regions where the preliminary sketch was observed. NRG-FC at 1292 nm (**b**,**d**) and PC-FC (**c**,**e**) images offer a clearer outline of the sketches made with the metal point.

The preliminary sketch in metal point was completed with another tool, possibly a dip pen, in two successive steps [31]. Multispectral analyses showed the presence of two distinct drawings, one outlining the main composition with pale brown lines and the other defining more intense-colored details. The different spectral behavior of the two materials is especially evident in the NIR image at 1600 nm (Figure 3a), where all the main elements of the landscape become transparent except for the outline of the rocks, the fine strokes defining the vegetation in the foreground, and the maelstrom at the bottom of the waterfall. XRF identified common chemical elements in the two types of ink distribution, namely iron, copper, and sulphur. This is consistent with iron-gall ink, and, more specifically, with hydrated green and blue vitriol (FeSO4·nH2O and CuSO4·nH2O, respectively), which were typically added to tannic acid solutions to obtain ink [27,32]. The different absorption properties of the two drawings suggest that Leonardo may have used a more diluted, and therefore IR transparent, ink for the main composition, and a more concentrated one for defining specific details [27]. Reflectance spectra (Figure 3d) acquired in the three points shown in Figure 3c confirm the similarities between the two ink compositions. The marked rising in reflectance beyond the red region (inflection point occurring at ca. 730 nm), which accounts for the brownish tone perceived in the visible, is compatible with metal-gall inks [33]. In the analyzed drawing, the overall degree of reflectance appears lower as the ink's hue becomes darker, with a rise occurring in the NIR. Conversely, the lower hiding power of the more diluted iron-gall ink mixture inevitably determines a higher contribution of the paper support, which, in its turn, affects the position of the inflection point in the resulting reflectance spectrum [34]. While there are studies relating the ink's hue to its metal content [35], such a hypothesis requires further verification, especially when considering that artists would often add organic carbon to the mixture to enhance its dark tone. Moreover, recipes for iron-gall inks are numerous and varied, and may include very diverse components and impurities, each undergoing various degradation processes resulting in color changes. In our case, browning and darkening may have resulted from

oxidation of ink components into quinonoid structures, as well as degradation products of the Arabic gum in the ink and cellulose in the paper support [32].

**Figure 3.** (**a**) NIR image at 1600 nm showing the absorbing properties of the sketch drawn with one of the two inks; magnified detail of the fortress in the NIR (**b**) and RGB (**c**) images; (**d**) reflectance spectra of the ink in three different points indicated by the red, blue, and black circles in (**c**).

The use of a similar drawing technique was identified on the verso of the sheet (Figure 4). The main sketch, namely the stream with a bridge and a barely traced outline of hills in the distance, is executed with a dry-point technique [31]. The FC image at 950 nm (Figure 4a) allows for a distinction between this drawing (greenish in appearance) and a few darker details redefined with ink. The latter becomes increasingly transparent in the NIR spectral range and disappears completely at 1600 nm (Figure 4b), consistent with what is observed on the recto, whereas the rocks and the flowing stream in the foreground remain clearly visible. The material used for the main composition, appearing grey in the visible, was identified as lampblack, a dark material typically obtained from the combustion of oils and candles [31]. The soft and grainy appearance of the drawing lines suggests the use of the pastel technique, which originated in France and is believed to have been introduced in Italy by Leonardo himself [36]. The two studies of figures in the upper part of the sheet, both disappearing at 1600 nm, are pen-drawn with brown ink, with indistinct traces of an under-drawing, possibly made with hematite-based red chalk [27], a technique traditionally known as sanguine. These sketches, as well as the bust of a draped figure drawn with the same material, are particularly evident in the detail of the FC-PC image in Figure 4c (light blue traits and yellow traits, respectively).

**Figure 4.** Multispectral analysis of the verso: (**a**) NRG-FC image at 950 nm evidencing the lampblack pastel drawing (greenish to the eye) and the dark details redefined with iron-gall ink (white dashed rectangles indicating the regions magnified in (**c**,**d**); (**b**) NIR image at 1600 nm; (**c**,**d**) Details of the FC-PC image (PC1-3) showing the sketches of male figures in brown ink appearing light blue, and the bust of a female figure in hematite appearing yellow (**c**); geometric studies in lead point, appearing bluish (**d**).

Fine traces of geometric studies (Figure 4d), executed with a very fine lead point [31], can be seen in the details of the FC-PC image. The superimposed sequence of drawing lines made visible by multispectral imaging suggests that the geometric patterns were the first to be drawn and were then covered by the black pastel landscape and the other ink elements at a later point in time.

#### *3.2. Laser Scanning Microprofilometry*

Morphological analysis of the paper surface allowed for the identification of the typical wavy texture impressed in the sheet during the papermaking process. Papermaking between the 15th and the 16th centuries in Europe involved the use of stamping mills consisting of rows of wooden pestles or mallets, which were caused to rise and fall by means of a series of cams to reduce the linen or, more rarely, cotton fabrics to pulp [37,38]. The pulp was suspended in a vat of water. A papermaking mold, i.e., a wooden framework on which a screen made of wires was either placed or strung, was dipped into the vat and scooped up the pulp, thereby trapping the latter within the fine porous screen of the mold. The wires acted as a sieve, filtering out the pulp as the water drained through [39], causing the typical impressions of the wires running sideways ("laid lines") and from top to bottom ("chain lines") on the final sheet. Then, the sodden sheet was transferred onto a wool felt. Alternating wool felts and freshly formed sheets are built up to form a "post", which was eventually transferred to a screw press to remove the excess water, thus impressing the wavy surface texture of the wool felt into the paper. The paper sheets so obtained were finally separated and taken to a loft to dry. The topographic map of the recto (Figure 5a,c), acquired with a sampling step of 50 μm, highlights the presence of

seven chain lines with an interval of 3.5–3.7 cm among each, as well as a dense sequence of fine laid lines running parallel to the longer side of the sheet. Interestingly, the NIR reflectance images revealed the presence of seven lines drawn in the same position of the chain lines, which were interpreted as a preliminary grid (by way of example, see NIR image at 1292 nm in Figure 5b,d) [30]. The material used to trace the grid absorbs the Vis-NIR radiation in a manner similar to the above-mentioned preparatory drawing, corroborating the lead point hypothesis [18]. Another series of spaced lines etched parallel to the long side of the sheet appears to have been imprinted freehand by Leonardo with what seems to have been a blind stylus, considering both the width and irregularity of the strokes, and the absence of drawing traces.

**Figure 5.** Visualization of the preparatory grid (yellow lines) in the micrometric topographic map (19 × 28 cm) rendered as an image (**a**) and in the NIR image at 1292 nm (**b**). The magnification of the region (19 × 9 cm) highlighted by the white dashed rectangle allows for a clearer identification of the drawn grid lines (pointed out by the yellow arrows in (**c**,**d**)), as well as the fine horizontal laid lines (red arrows in (**c**)) impressed in the paper by the felt.

The 3D model also highlighted the presence of deep incisions at the bottom of the sheet near the profile of the raised terrain in the foreground (Figure 6). These traces may be a by-product of the transferring method used, namely the interposition of transparent paper to transfer the preparatory drawing or assemble sketches to compose a whole scene [40]. In his *Trattato della Pittura*, Leonardo often reports using transparent paper, or even flat glass, not only to copy the preparatory drawing but also to verify the correspondence between the final work and the model previously copied by direct observation [41]. This particular method is still under-researched, due to both the almost total loss of the original materials and the analytic difficulty of detecting its traces. In fact, scholars have often interpreted drawings transferred by means of transparent paper as indirect incisions resulting from cartoon transposition, or even carbon-copying [30].

**Figure 6.** Topographic map of the drawing superimposed on the recto RGB image acquired with the multispectral scanner. The area highlighted by the dashed rectangle (7 × 11 cm) (**a**) is magnified in (**b**); the white arrows indicate the incisions profiling the main elements in the foreground, attributed to the method of transferring the sketch with the use of transparent paper.

## *3.3. Spectral-Domain Optical Coherence Tomography (Sd-OCT)*

OCT tomocubes (5 × <sup>5</sup> × 0.45 mm3, voxel size 3.5 <sup>μ</sup>m3) allowed for the areal and crosssectional visualization of micrometric features related to the artistic technique possibly used by Leonardo. Of particular interest are the traits engraved in the white background with no trace of colored material and no relation to the drawn landscape (Figure 7a,b). Some of these lines are interpreted as superficial scratches ascribed to mechanical damage [31], while others, likely drawn with a blind stylus, seem to outline graphic forms, such as a triangle and an indefinite sketch. The latter's OCT tomocube (Figure 7b) shows a circular concavity, 1.5 mm in diameter and 40–45 μm-deep, which may indicate the use of a pointed tool in this area. Another possible explanation is that they were impressed in the paper while they were being drawn on another superimposed sheet, which would account for their hardly understandable location in the sky above the landscape. Further investigation and comparison with other drawings may clarify this point.

**Figure 7.** OCT analysis of the blind traits in the background. (**a**) RGB image detail showing the region of interest (ROI, black rectangle); (**b**) topographic map superimposed on the RGB image showing the magnified ROI (the white square showing the area measured with OCT); (**c**) tomocube of the circular impression—light blue rectangle highlighting the position of the z-y section reported below.
