Optical aberrations limit the practical working distance and resolution in optical microscopes. They originate mainly from refractive index variations within the sample object and their effect typically increases as a function of imaging depth. Their effect is most evident in super-resolution systems, due to the high numerical aperture objectives they require. Optical aberrations can be corrected with adaptive optical elements -- and to an extent, with post-processing methods, such as image deconvolution.The current state-of-the-art adaptive optics systems can be divided into two categories: (1.) direct wavefront measurement based and (2.) sensorless. In (1.) typically a Shack-Hartmann (SH) wavefront sensor is used to directly quantify the phase variations caused by the optical aberrations that can then be corrected with a deformable mirror (DM) or a spatial light modulator (SLM). In (2.) no wavefront sensor is used, but instead one iteratively applies different aberrations on the DM/SLM and tries to find settings that maximise the image quality, based on some metric, such as image brightness. The problem with (1.) is that a separate wavefront sensor is required, and the SH sensor typically requires multi-photon excitation, as otherwise out-of-focus background noise will preclude the wavefront measurement. The problem with (2.) is that iterative optimization takes a lot of time, and it is also detrimental to the sample due to the large number of images required.
The aim of the AdaptiveSTED project was to develop a third (3.) kind of an aberration correction scheme for laser-scanning optical microscopy (LSM) that would not require a separate wavefront sensor, nor the iterative calibration. The idea was born out of the realization that when one replaces the typical single-element detector in an LSM with a detector array, it is possible to collect information of the microscope’s effective point-spread-function at every single sampling position -- directly from the recorded fluorescence signal. A second important benefit of using an array detector, it is also possible to improve the microscope’s optical resolution and signal-to-noise ratio (SNR) by image scanning microscopy (ISM) pixel reassignment. A second important aim of the AdaptiveSTED project was to develop an adaptive image reconstruction method for ISM, to ensure optimal image quality even in challenging imaging conditions.
