Estimation of the standard deviation in three-dimensional microscopy by spatial statistics

Usually, the calibration process for three-dimensional microscopy involves the use of a reference flat surface. The random fluctuations of the topographic image for this reference surface are used for determining the uncertainty of the microscope. When the sample material or the measuring conditions of the microscope are modified (such as the objective used in a confocal microscope, or the tip in an atomic force microscope), the measuring conditions vary and thus a new calibration is required. In this work, a technique based on spatial statistics methods (more specifically, the variogram function) is proposed to determine accurately the standard deviation for three-dimensional microscopy that does not require a reference flat surface and therefore eliminates the need for a previous calibration process of this parameter.