Comparative study of three-dimensional localization accuracy in conventional, confocal laser scanning and axial tomographic fluorescence light microscopy

Abstract

For many biological applications, precise and accurate 3D object localizations and 3D-distance measurements are necessary. Point spread functions of artificial objects of subwavelength dimensions have been measured in order to characterize the image forming properties as well as to localize extended objects in both conventional and confocal florescence light microscopy with and without the axial tomographic technique. With the axial tomographic technique it is possible to tilt the object in such a way, that substructures are located in the same focal plane. The distance of two points measured under this optimal perspective fits best to the real 3D-distance. In this case, optical sectioning is unnecessary, if only distance measurements have to be performed.