Total internal reflection fluorescence (TIRF) has come of age, but a reliable and easy-to-use tool for calibrating evanescent-wave penetration depths is missing. We provide a test-sample for TIRF and other axial super-resolution microscopies for emitter axial calibration. Our originality is that nanometer(nm) distances along the microscope’s optical axis are color-encoded in the form of a multi-layered multi-colored transparent sandwich. Emitter layers are excited by the same laser but they emit in different colors. Layers are deposited in a controlled manner onto a glass substrate and protected with a non-fluorescent polymer. Decoding the penetration depth of the exciting evanescent field, by spectrally unmixing of multi-colored samples is presented as well. Our slide can serve as a test sample for quantifying TIRF, but also as an axial ruler for nm-axial distance measurements in single-molecule localization microscopies, supercritical-angle fluorescence, and related super-resolution.