3D nanometrology of transparent objects by phase calibration of a basic bright-field microscope for multiple illumination apertures

Optical retrieval of the structure of transparent objects at the nanoscale requires adapted techniques capable of probing their interaction with light. Here, we considered a method based on calibration of the defocusing with partially coherent illumination and explored its phase retrieval capability over a wide range of illumination angles. We imaged: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis, (2) custom-made nano-steps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis, and (3) human cancer cells deposited on a glass substrate to assess the results of the calibration on complex transparent 3-dimensional samples. We first verified the model prediction in the spatial frequency domain and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to 0.5. Finally, we studied the dependence of the phase retrieval of a complex nanostructured object on the illumination aperture.