Modal sensorless adaptive optics relies on the use of an image quality metric to estimate the amplitude of
aberrations, and of a well-suited set of aberration modes to describe the aberration. This set is chosen so that
aberration of one mode does not influence correction in another mode. In this paper, we show how these modes
can be derived experimentally, and investigate the influence of imperfect crosstalk removal on the accuracy of
correction. We show that the resulting error can be mitigated using appropriate algorithms that can incorporate
knowledge of the influence of the modes on the metric and, if available, partial knowledge of the aberrations.
Finally, we derive from these results the minimum time required for correction in various situations.
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.
We investigate the parameters governing the accuracy of correction in modal sensorless adaptive optics for
microscopy. In this paper we focus on the case of two-photon excited fluorescence. Using analytical, numerical
and experimental results, we show that using a suitable number of measurements, accurate correction can
be achieved for up to 2 rad rms initial aberrations even without optimisation of the correction modes. We
demonstrate that this correction can be achieved using low light levels to minimise photobleaching and toxicity,
and we provide examples of such optimised correction.
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