Steffen Restel¹, Julia Menzel¹, Florian Grimm², Bastian Klußmann-Fricke¹, Jan-Gero Schlötel¹, Dennis Uhlenkamp¹, Martin Meschkat¹, Christian A. Wurm^1,2

¹Abberior Instruments, Germany. ²Abberior, Germany

Workshop Room 19

In microscopy, the quality of images is heavily influenced by the optical properties of the sample. Aberrations, caused by inconsistencies within the sample or inadequate immersion, can significantly impact the ability to focus and obtain high-quality images. This issue is particularly important for super-resolution microscopy techniques such as STimulated Emission Depletion (STED) microscopy. Multi-color STED microscopy is widely used to resolve nano-scale organizations in fixed and live cells [1,2] as well as in tissue [3]. In STED microscopy, precise and accurate focusing of both the excitation and depletion lasers within the sample is crucial. This can be achieved through adaptive optics, which is a powerful tool for aberration correction.

The abberior RAYSHAPE employs adaptive optics by using a deformable mirror to dynamically redirect aberrated light, thereby restoring the ability to focus [4]. This enables the acquisition of high-quality images of challenging and complex biological samples, including organoids, whole mount preparations of organs, plant tissue, tissue sections, and samples from expansion microscopy (ExM). Without RAYSHAPE, the excitation laser power would need to be increased with deeper focus levels to compensate for the loss of signal caused by aberrations. In contrast, RAYSHAPE allows for the preservation of both resolution and brightness deep within thick samples, while enabling imaging at low light levels with improved confocal, 2D and 3D STED resolution. This correction of aberrations with RAYSHAPE is dynamic across a wide range of z-levels and sets it apart from traditional mechanical aberration corrections, providing clear advantages in image quality and general imaging flexibility.

In this workshop, participants will learn the advantage of dynamic aberration correction provided in the abberior FACILITY LINE. We will demonstrate how complex samples benefit from RAYSHAPE to ensure high quality super-resolution STED imaging throughout the entire depth of the sample.

Figure 1: Confocal imaging with RAYSHAPE correction . xz section of a stage 17 Drosophila embryo stained for chitin (abberior LIVE 610, green) and DNA (abberior LIVE 550, cyan).

References:
[1] Hell, S.W., Wichmann, J., 1994. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780
[2] Saal, S.J., Hell, S.W., Jakobs, S., 2017. Fluorescence nanoscopy in cell biology. Nat Rev. 18
[3] Urban, N.T., Willig K.I., Hell, S.W., Nägerl, V., 2011. STED Nanoscopy of Actin Dynamics in Synapses Deep Inside Living Brain Slices. Biophys J. 7, 101(5)
[4] Gould, TJ., Burke, D., Bewersdorf, J., and Booth M.J., 2012. Adaptive optics enables 3D STED microscopy in aberrating specimens. Optics Express 20, 19

abberior STED workshop: Dynamic aberration correction in STED microscopy