<p>The materials and working method of a painting can reveal important information about our cultural history, as well as lend the conservator the necessary knowledge for treatment options. The removal of a cross-section sample reveals the three-dimensional (3d) structure of the painting and can be used to identify materials. However, cross-section samples are destructive and provide only local information. Nonlinear optical ultrafast pump-probe microscopy, originally developed for biomedical imaging, can provide high resolution 3d images with chemical contrast. In this dissertation, I adapt pump-probe microscopy to multiple materials and applications in cultural heritage research. Pump-probe dynamics were found to be sensitive to the ratio of the two chromophores present in the precious blue pigment lapis lazuli and its synthetic analogs, ultramarines blue and violet. Virtual pump-probe cross-sections were combined with nonlinear fluorescence contrast to study differences between the interactions of paper supports with inorganic crystalline pigments and organic dyes. Multiple early Italian paintings (The Crucifixion by Puccio Capanna, The Martyrdom of St. Alexander and The Body of Christ Supported by Angels attributed to Lorenzo Lotto) were imaged in-situ, in conjunction with traditional conservation science methods, as a part of a technical case study. Thus, pump-probe microscopy offers an important new tool for gaining fundamental insights into our cultural heritage.</p> / Dissertation