3D BIPLANE MICROSCOPY

The invention provides a microscopy system configured for tracking microscopic particles in 3D, the system comprising a sample comprising luminescence particles; a sample stage for mounting the sample; at least one light source configured to illuminate an area of the sample, the at least one light source causing luminescence light from primarily one tracked particle of the luminescence particles; a beam-steering device configured to selectively move a light beam to illuminate different areas of the sample such that the luminescence light is detected; a beam-splitting device located in a detection light path that splits the luminescence light into at least two paths, the beam-splitting device creating at least two detection planes that correspond to different object planes in the sample at least one camera positioned to detect simultaneously the at least two detection planes, the number of object planes being represented in the camera by the same number of recorded regions of interest; and a controller programmable to combine a signal from the recorded regions of interest, determine a 3D trajectory of the particle at each time point of a recorded data sequence, move the beam-steering device to illuminate the different areas of the sample in accordance with corresponding positions of the one tracked particle.