Abstract: Microscopy method and apparatus for determining the positions of emitter objects in a three-dimensional space that comprises focusing scattered light or fluorescent light emitted by an emitter object, separating the focused beam in a first and a second optical beam, directing the first and the second optical beam through a varifocal lens having an optical axis such that the first optical beam impinges on the lens along the optical axis and the second beam impinges decentralized with respect to the optical axis of the varifocal lens, simultaneously capturing a first image created by the first optical beam and a second image created by the second optical beam, and determining the relative displacement of the position of the object in the first and in the second image, wherein the relative displacement contains the information of the axial position of the object along a perpendicular direction to the image plane.

Abstract: Microscopy method and apparatus for determining the positions of emitter objects in a three-dimensional space that comprises focusing scattered light or fluorescent light emitted by an emitter object, separating the focused beam in a first and a second optical beam, directing the first and the second optical beam through a varifocal lens having an optical axis such that the first optical beam impinges on the lens along the optical axis and the second beam impinges decentralized with respect to the optical axis of the varifocal lens, simultaneously capturing a first image created by the first optical beam and a second image created by the second optical beam, and determining the relative displacement of the position of the object in the first and in the second image, wherein the relative displacement contains the information of the axial position of the object along a perpendicular direction to the image plane.