AdaptiveSTED · Real-time automatic aberration correction for easy high-resolution imaging in complex specimens, by STED and other point-scanning microscopy techniques

Real-time automatic aberration correction for easy high-resolution imaging in complex specimens, by STED and other point-scanning microscopy techniques

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.

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