Multipoint focus-detecting method for measurement of biological nonsmooth surface topography

To measure the 3D characteristic of biological nonsmooth surface, a multipoint focus-detecting method based on the light splitting effect of crossed grating was developed. The surface profile is obtained by analyzing the variation in spot size on it rather than in light intensity used in conventional confocal method. Thus the influence of surface reflectivity variation could be reduced. The system is simple and the lateral resolution could be adjusted by changing the moving step of the stage on which the sample lies. Currently the axial uncertainty is about 20 μm. The profile of fish scales was measured and the uncertainties were analyzed.