AN IMAGING SYSTEM WITH MULTI-MODALITY APPLICATIONS (SIM/STED/STORM) APTITUDES FOR SUPER-RESOLUTION IN SPACE AND TIME

The life science & engineering (LS&E) microscopy center applies tools from various fields ranging from optical engineering through signal processing to molecular biology. The center operates strategically is in the interface of engineering and life science applications, and widespread use in the increasingly technology for the field of life science. A major effort of the center revolves around super-resolution microscopy and related applications. Super resolution microscopy revolutionized the approach to study molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. When developing new methods for super resolutions microscopy, it is imperative to compare to the existing state-of-the-art, which often, in our case, means existing super-resolution methods such as STORM, STED or SIM.

Our main imaging challenge is to construct a multimodality SR microscopy system that can interchange between the different methods of SR on the same sample. The center attempts to design a multiuser microscopy system that can achieve multi-modality and multi-resolution imaging both in space and in time. The multimodal SR system is based on Zeiss Elyra7 microscope that supplies- both Structured Illumination Microscopy (SIM²), a method that can supply ‘real-time’ imaging for dynamic samples, and single molecule localization modalities PALM\STORM, that are inherently slow, but can supply a ‘ground truth’ measurement with high precision of super resolution. In addition, the system includes the Abberior STEDYCON system that can be used for STED imaging, or for laser manipulation and/or activation such as FRAP experiments combined with the SIM modality right after for fast live super resolution imaging.

Our results demonstrate that our tailor made multi-modality microscope can be used for laser manipulation applications combined with spatial and temporal super resolution imaging. Cells treated with a short labeled peptide were imaged using SIM² and STED setups. The SIM² and STED images were co-registered in nanometers scale by using the IMARIS software. Then, the STED setup was applied to achieve bleaching of a defined region of interest. Immediately after, SIM was used again to observe live cell dynamics and follow intensity changes at speed of 40 frames per second. The SIM and STED imaging were performed without needing to move the sample between microscopes. Our combined super-resolution microscopes allowed to exploit the advantage of the gentle and fast SIM technique for live cell imaging with the ability of a laser scanning microscope, provided by STED, for laser manipulation applications.