Three-dimensionally localizing light emitting molecules of unknown orientation and unknown Z-position

摘要

To the end of three-dimensionally localizing light emitting marker entities of unknown orientation and unknown position in a sample, the light emitted by each single marker entity is imaged in at least two different ways onto at least one detection plane which corresponds to a focal plane (13) in the sample resulting in at least two images of the marker entity. Virtual x- and y-positions of the marker entity in parallel to the focal plane (13) are separately determined from the emitted light intensity distribution over each image of the marker entity. Further, the z-position of the marker entity normal to the focal plane is determined from the emitted light intensity distributions over the images of the marker entity. The real x- and y-positions of the marker entity in parallel to the focal plane (13) are determined based on its virtual x- and y-positions and on its z-position.