Method for localizing a region of interest in a sample and micromachining the sample using a charged particle beam

The invention relates to a method and apparatus for localization of a region of interest with a fluorescent entity inside a sample and for micromachining said sample in. an integral fluorescence microscope/charged particle 5 beam apparatus. The optics of the fluorescence microscope for imaging the sample onto a detector comprises an astigmatic optical component. The method comprises the steps of: - determine a position of a focal plane of the 10 fluorescence microscope with respect to a reference plane in said integral apparatus; - obtaining an image of the fluorescent entity in the sample using the fluorescence microscope; - determine a position. of the fluorescent entity 15 with respect to a focal plane of the fluorescence microscope, by evaluation of a degree of astigmatism and/or an ellipticity of a fluorescence intensity profile of the image of the fluorescent entity; and - micromachining' the sample around. the determined 20 position using a charged particle beam.