Wavefront curvature sensing and control in microscopy

The purposes are multiples: creation of a wavefront curvature sensor, use of the curvature sensing to control a multi-actuator lens developed at CNR-IFN of Padua (18 piezoelectric actuators) and use of the multi-actuator lens to generate an astigmatic wavefront in order to obtain a 3D image by a single 2D image of the sample in examination. The wavefront curvature sensor has demonstrated a good linearity at different amplitudes of several aberrations. Successively, we have proved that the multi-actuator lens can be controlled with curvature sensing. In this sense, with out of focus images it is possible to directly set the voltage to the actuators’ lens in order to obtain a flat wavefront. Finally, we have demonstrated that the multi-actuator lens can be used in a fluorescence microscope to generate an astigmatic wavefront in order to stretch fluorophores in the x or y direction. We have taken several images at different depths, we have calculated the x and y dimensions of them and fitted with the gaussian waist. After these operations, we have recorded an image of a sample with astigmatic wavefront and use the dimensions of the fluorophores to infer the z-coordinate of the molecule. Then we have compared the z-coordinate calculated in this way with the real z-coordinate, calculated by using a z-scan piezoelectric actuator. The residuals between these two methods were less than 60 nanometres. This preliminar analysis can be used in a future development, especially in single molecule microscopy.