Method for collision-free positioning of a micromanipulation tool relative to a sample holder using a microscope, comprising the following steps: a) determining the focal position Z P of at least one point P on the surface of the sample holder by focusing a microscope lens with a first numerical aperture NA 1 on the at least one point; b) positioning the micromanipulation tool on the optical axis of the microscope; c) determining the focal position Z M of the micromanipulation tool by focusing the microscope lens or a second microscope lens with a second numerical aperture NA 2 that is smaller than the first numerical aperture NA 1 on the tip of the micromanipulation tool; d) calculating the lowering height Z A in consideration of a predetermined tolerance profile Δ Z by means of the following equation: Z A P = Z M − Z P − ΔZ with ΔZ = ΔZ M + ΔZ P wherein Δ Z M or Δ Z P is a predetermined tolerance, i.e. an inaccuracy assigned to an optical component or the entire optical system when determining the position of the manipulation tool or sample holder, wherein the tolerance Δ Z M of the manipulation tool and/or the tolerance Δ Z P of the sample holder is given by the depth of field d tief, i of the respective i-th lens used for determining the position thereof, according to the following equation: ΔZ M, P = d tief, i = λ ⋅ n NA i 2 + n M i ⋅ NA i ⋅ e i = 1,2,... wherein A is the wavelength of the observation light, n is the refractive index of the medium between the microscope lens and the sample holder, M i is the magnification of the i-th lens, and e is the resolution of a video camera used for the observation; e) positioning the micromanipulation tool at the point P by lowering it by the lowering height Z A. a) determining the focal position Z P of at least one point P on the surface of the sample holder by focusing a microscope lens with a first numerical aperture NA 1 on the at least one point; b) positioning the micromanipulation tool on the optical axis of the microscope; c) determining the focal position Z M of the micromanipulation tool by focusing the microscope lens or a second microscope lens with a second numerical aperture NA 2 that is smaller than the first numerical aperture NA 1 on the tip of the micromanipulation tool; d) calculating the lowering height Z A in consideration of a predetermined tolerance profile Δ Z by means of the following equation: Z A P = Z M − Z P − ΔZ with ΔZ = ΔZ M + ΔZ P wherein Δ Z M or Δ Z P is a predetermined tolerance, i.e. an inaccuracy assigned to an optical component or the entire optical system when determining the position of the manipulation tool or sample holder, wherein the tolerance Δ Z M of the manipulation tool and/or the tolerance Δ Z P of the sample holder is given by the depth of field d tief, i of the respective i-th lens used for determining the position thereof, according to the following equation: ΔZ M, P = d tief, i = λ ⋅ n NA i 2 + n M i ⋅ NA i ⋅ e i = 1,2,... wherein A is the wavelength of the observation light, n is the refractive index of the medium between the microscope lens and the sample holder, M i is the magnification of the i-th lens, and e is the resolution of a video camera used for the observation; wherein Δ Z M or Δ Z P is a predetermined tolerance, i.e. an inaccuracy assigned to an optical component or the entire optical system when determining the position of the manipulation tool or sample holder, wherein the tolerance Δ Z M of the manipulation tool and/or the tolerance Δ Z P of the sample holder is given by the depth of field d tief, i of the respective i-th lens used for determining the position thereof, according to the following equation: ΔZ M, P = d tief, i = λ ⋅ n NA i 2 + n M i ⋅ NA i ⋅ e i = 1,2,... wherein A is the wavelength of the observation light, n is the refractive index of the medium between the microscope lens and the sample holder, M i is the magnification of the i-th lens, and e is the resolution of a video camera used for the observation; e) positioning the micromanipulation tool at the point P by lowering it by the lowering height Z A.