Microscopy method for determining the position of an emitter object in a three dimensional space which comprises: a) illuminating an emitter object so as to cause an emission of scattered light or fluorescent light from the emitter object; b) focusing the emitted light in a primary focused optical beam through an objective lens; c) splitting the primary optical beam in a first secondary beam and in a second secondary beam; d) directing the first and the second secondary beam through a varifocal lens, having an optical axis, along respective optical paths such that the first secondary beam impinges on the varifocal lens along a direction corresponding to said optical axis and the second secondary beam impinges decentralized on the varifocal lens at an offset distance Δd from the optical axis and along a direction parallel to the same, in which the varifocal lens has an electronically controllable focal length, e) electronically controlling the focal length of the varifocal lens changing the focal length through a range of focal length values so as to move the respective focal positions of the first and second beam along said optical axis through said range of focal length values in a predetermined travel time, f) simultaneously acquiring a first image and a second image of the emitter object in an integration time greater than or equal to the travel time of the focal positions simultaneously detecting the first secondary beam in-axis and the decentralized second secondary beam in respective first detection area and second detection area arranged on an image plane (x, y); g) analyzing the first and the second image for determining a first object position on the first image and a second object position on the second image and determining a relative displacement Δr in the image plane (x, y) of the position of the object in the two images, and h) determining an axial position z p of the emitter object along an axis z perpendicular to the image plane on the basis of the relative displacement Δr. a) illuminating an emitter object so as to cause an emission of scattered light or fluorescent light from the emitter object; b) focusing the emitted light in a primary focused optical beam through an objective lens; c) splitting the primary optical beam in a first secondary beam and in a second secondary beam; d) directing the first and the second secondary beam through a varifocal lens, having an optical axis, along respective optical paths such that the first secondary beam impinges on the varifocal lens along a direction corresponding to said optical axis and the second secondary beam impinges decentralized on the varifocal lens at an offset distance Δd from the optical axis and along a direction parallel to the same, in which the varifocal lens has an electronically controllable focal length, e) electronically controlling the focal length of the varifocal lens changing the focal length through a range of focal length values so as to move the respective focal positions of the first and second beam along said optical axis through said range of focal length values in a predetermined travel time, f) simultaneously acquiring a first image and a second image of the emitter object in an integration time greater than or equal to the travel time of the focal positions simultaneously detecting the first secondary beam in-axis and the decentralized second secondary beam in respective first detection area and second detection area arranged on an image plane (x, y); g) analyzing the first and the second image for determining a first object position on the first image and a second object position on the second image and determining a relative displacement Δr in the image plane (x, y) of the position of the object in the two images, and h) determining an axial position z p of the emitter object along an axis z perpendicular to the image plane on the basis of the relative displacement Δr.