Abstract: A method for calibrating electromagnetic radiation-based three-dimensional imaging includes: obtaining (501) a calibration imaging result at least partly on the basis of electromagnetic waves received from a calibration artifact, forming (502) calibration data on the basis of the calibration imaging result and a known thickness profile of the calibration artifact, and correcting (503), with the aid of the calibration data, an imaging result obtained at least partly on the basis of electromagnetic waves received from a sample to be imaged. The calibration artifact includes layers, for example Langmuir-Blodgett films, having pre-determined thicknesses and stacked on each other so as to achieve the pre-determined thickness profile of the calibration artifact. A three-dimensional imaging system configured to carry out the method.