Wavefront-sensing-based autofocusing in microscopy.

Massive image acquisition is required along the optical axis in the classical image-analysis-based autofocus method, which significantly decreases autofocus efficiency. A wavefront-sensing-based autofocus technique is proposed to increase the speed of autofocusing and obtain high localization accuracy. Intensities at different planes along the optical axis can be computed numerically after extracting the wavefront at defocus position with the help of the transport-of-intensity equation method. According to the focus criterion, the focal plane can then be determined, and after sample shifting to this plane, the in-focus image can be recorded. The proposed approach allows for fast, precise focus detection with fewer image acquisitions compared to classical image-analysis-based autofocus techniques, and it can be applied in commercial microscopes only with an extra illumination filter.