The current invention relates to the task of masking, i.e. determination of boundaries in an image of biological particles and/or elements or parts of biological particles in image cytometry. Methods for masking of a biological particle or element or region of a biological particle in a sample are provided which include staining a first element or region of the biological particle with a first fluorescent dye, staining a second element or region of the biological particle with a second fluorescent dye, recording an image of the fluorescent light signal emitted from the sample, and determining boundaries of the biological particle or element or region of the biological particle based on the fluorescent light signal in the image.