Abstract
When imaging through tissue, the optical inhomogeneities of the sample generate aberrations that can prevent effective Stimulated Emission Depletion (STED) imaging. This is particularly problematic for 3D-enhanced STED. We present here an adaptive optics implementation that incorporates two adaptive optic elements to enable correction in all beam paths, allowing performance improvement in thick tissue samples. We use this to demonstrate 3D STED imaging of complex structures in Drosophila melanogaster brains.
Highlights
Far-field super-resolution fluorescence microscopy is transforming biological science by permitting the observation of structures and processes that were inaccessible using microscopes operating at conventional resolutions [1, 2]
In order to perform 3D stimulated emission depletion (STED) imaging, we started with the AO correction, derived during confocal imaging, on the deformable mirror (DM) and performed an additional round of correction on the spatial light modulators (SLMs) using the Fourier Ring Correlation (FRC) metric with a threshold of 0.6 and the correction parameters described for system alignment
We have explicitly demonstrated this enhanced sensitivity to aberrations in the case of 3D STED - in aberration conditions that still allowed an acceptable image to be generated by the confocal imaging mode, we saw very poor quality imaging from the STED mode
Summary
Far-field super-resolution fluorescence microscopy is transforming biological science by permitting the observation of structures and processes that were inaccessible using microscopes operating at conventional resolutions [1, 2]. Three-dimensional (3D) resolution enhancement can be implemented using a different phase plate that creates a so-called “bottle beam” in which a zero intensity point is surrounded in all directions by higher intensity [5] This latter configuration offers better 3D imaging ability but is more difficult to implement in practice. It is clear that full aberration correction in the STED microscope requires compensation of specimen-induced aberrations in all three of these paths and, due to the polarization dependence of the SLM it was not possible to correct all three beam paths with SLM only techniques We demonstrate such an AO that permits effective 3D STED imaging of biological structures in tissue specimens. This development represents an essential step in enabling super-resolution imaging at greater depth
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