Calibration of an adaptive microscope using phase diversity

Accurate control over the phase and amplitude modulation in an adaptive microscope is essential to the quality of aberration correction that can be achieved. In this paper we present a robust and compact method for characterising such amplitude and phase modulation in the pupil plane of the focussing objective. This method, based on phase diversity, permits calibrating the microscope as a whole and thus avoids errors in the alignment of the wavefront shaping device after calibration and the resulting imprecision in the induced modulation: by acquiring three 2D images of the point spread function at different distances from the focal plane, we show that the electric field distribution at the pupil plane can be retrieved using an iterative algorithm. We have applied this technique to the characterisation of the phase modulation induced by a deformable mirror when conjugated with the entrance pupil of different objectives, which permits accurate evaluation of the performance of the mirror for subsequent aberration correction.