Leonardo had a very significant effect on the relationship between science and art, and starting from what was still a fifteenth-century concept, he made his art the mirror of nature which must be thoroughly studied and researched before it can be properly portrayed. This was the reason for his dedicated study of botany, anatomy and, in particular, optics, in which the eye was considered the link between the image and the soul. According to him, "Painting is the composition of light and shade mixed together with the different qualities of all its colours, simple and composite" (Treatise on Painting). His paintings can be analysed from this point of view: with the fumato technique, space acquires a depth of perspective, which he himself called "airy", because it is influenced by the filtering of air; light and shadow therefore blend to produce a harmony of form and a perfection that characterises the whole of his artistic output.
Leonardo's painting is brought to life by his preoccupation with chiaroscuro rather than with colour. His treatment of colour, however, is skilled and here too, he anticipated the laws which science and art would only codify and apply in the centuries to come. With frescoes, he preferred tempera and strove to solidify the paint with new glutens, as in the famous Last Supper in the Monastery of Santa Maria delle Grazie in Milan. Taking the moments after Christ said, "One of you will betray me", Leonardo expresses all the amazement at this statement in the faces of the disciples, except for Judas, who sits with his elbow on the table, staring at Christ. One of the most important of masterpieces in the whole of painting is undoubtedly "the lady on the balcony" which Leonardo painted in Rome on the commission of Giuliano de Medici, and for which there are no surviving preparatory studies.
Celebrated by Vasari as a portrait of Monna Lisa Gherardini, wife of Francesco del Giocondo of Florence, the Mona Lisa is at a level higher than the background of a wide empty landscape with which she is in perfect harmony. The fact that the subject and the landscape are in total unity suggests that man forms part of nature without conflict or opposition. The drawing in the Biblioteca Reale in Turin universally known as a self-portrait of Leonardo has, more than anything else, influenced the popular idea of him as the aged genius with a thick white beard. In fact, critics disagree on the date of the drawing and also on its subject. An English critic, Robert Payne, has suggested that it may be a portrait of Leonardo's father, while a German critic has cast doubts on its authenticity and attributed it to a nineteenth-century artist, Giuseppe Bossi. The majority of critics, however, tend to regard it as a self-portrait of Leonardo, perhaps slightly idealised in relation to the reality, while continuing to have doubts as to the dating.