Abberior Instruments
Julia Menzel1, Steffen Restel1, Florian Grimm2, Bastian Klußmann-Fricke1, Jan-Gero Schlötel1, Dennis Uhlenkamp1, Martin Meschkat1, Christian A. Wurm1,2
1Abberior Instruments, Germany. 2Abberior, Germany
Workshop Room 19
Super-resolution imaging techniques like STimulated Emission Depletion (STED) allow visualization of the small size of most subcellular organelles, structures, and dynamic interactions, which is essential for many research questions [1,2]. Technical developments like improved organic dyes or pulsed lasers were already able to reduce the required light dose on the sample. Nevertheless, most sample benefit from minimal laser light exposure while maintaining maximal resolution and signal intensity.
Breakthroughs in this respect are imaging techniques like Flexposure, also known as “Adaptive Illumination”. Flexposure is based on the principal of only illuminating places in the sample where light can make a positive contribution to the image [3]. Instead of illuminating every pixel with the STED laser, the illumination is adapted dynamically in a pixel-by-pixel fashion to the structure of the sample. Therefore, only positions emitting signal are illuminated with STED light, reducing the applied light dose significantly and improving imaging of sensitive samples (Fig 1A).
Beside the intensity of a fluorophore also the fluorescence lifetime can be measured to receive information about the fluorophore’s environment such as ion concentration or pH. In STED microscopy, fluorescence lifetime information is particularly beneficial, as it can be additionally harnessed to increase resolution - especially when imaging at reduced STED laser power to protect sensitive samples (Fig. 1B)
Beside imaging techniques, new developments in organic dyes provide further improvements for long term imaging in super-resolution. Exchangeable fluorescent probes bind to genetically encoded self-labelling protein tags with high affinity but can exchange regularly [4]. Therefore, the fluorophore is exchanged on a regular basis which can prolong imaging time (Fig. 1C).
In this workshop, participants will learn how sensitive samples benefit from optimized dye and imaging techniques. We will demonstrate on the abberior FACILITY LINE how STED imaging techniques like FLEXPOSURE and TIMEBOW are used for gentle imaging with reduced applied light dose to allow long-time imaging.
Figure 1A. Long-time imaging with FLEXPOSURE. Consecutive STED images of nuclear pore complexes imaged with and without Flexposure.
B. MATRIX STED and lifetime imaging. Differentiated Caco-2 cells were stained for Actin with Star Red (provided by D.Günzel, Charité Berlin, Germany).
C. Gentle STED multi-color LIVE Cell Imaging. Mammalian cells express a Halo-X fusion protein in the outer mitochondrial membrane visualized with Abberior LIVE 590 Halo-X (magenta) and actin stained with SiR actin (green).
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] Heine, J., Reuss, M., Harke B., Hell, S.W., 2017. Adaptive-illumination STED nanoscopy. PNAS 114 (37)
[4] Kompa, J., Bruins, J., Glogger M., Wilhelm, J., Frei, M.S., Tarnawski, M., D’Este, E., Heilemann, M., Johnsson, K., 2023. Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy. J. Am. Chem. Soc 145, 5