A calibration apparatus for a tip-enhanced Raman microscope includes a substrate; a two-dimensional Raman scatterer that is mounted on an upper surface of the substrate; and a well-defined topographic structure that is formed at the upper surface of the substrate. The topographic structure may include convex geometric shapes such as triangles and squares arranged in one or more periodic lattices. Calibration is via adjusting a focal length of a laser beam until a signal from a spectrometer repeatedly exhibits a stepped response when a focal point of the laser beam traverses an edge of a two-dimensional Raman scatterer, then adjusting the relative lateral positions of a scanning probe microscope probe tip and the focal point until the signal from the spectrometer and a signal from the scanning probe microscope repeatedly change within an acceptable time delay while the focal point and the probe tip traverse edges of the topographic structure.