High-Resolution 4Pi Fluorescence Microscopy for Studies of Sub-Cellular Dynamics in Live Cells - HighLight 2004
Zusammenfassung der Projektergebnisse
Optical microscopy is currently the only experimental method that allows researchers to monitor bio-molecular processes in living cells in all three spatial dimensions as a function of time. Therefore, in our original grant application in 2004, we had proposed to study functional dynamics within live cells using an ultra-high resolution 4Pi microscope, which promised to offer a 5- to 7-fold enhanced resolution in the axial direction due to an interferometric arrangement of two opposing objective lenses. Specifically, we had proposed (1) studies of the formation and plasticity of synapses of neuronal cells, (2) visualization of bacterial cell differentiation and interaction with host cells, (3) receptor triggering by ligands and elucidation of signal transduction pathways, (4) vesicle shedding at the trans-Golgi work, and (5) detailed mechanistic investigations of virus-host interaction pathways. We had planned to address these topics in collaboration with a number of groups from Biology and Medicine at Ulm University, so that our group from the Department of Biophysics, Ulm, would provide the technical expertise in high resolution microscopy as well as fluorescent marker development and application. The 4Pi microscope, which was set up in our laboratory in June 2005, was a poorly conceived setup, and our laboratory went through a painful debugging procedure during the following 2 – 3 years, which kept key personnel busy. The sequence of failures and subsequent down times often lasting for several weeks made it impossible to stick to a reasonable schedule for the various collaborations. After several years, the instrument finally reached a certain level of reliability. However, a high level of expertise and long-time experience is needed to run the system and even then, it is quite an effort to produce good 4Pi images with an acceptable amplitude of side lobes. Consequently, the 4Pi microscope was eventually withdrawn from the market. As yet, we have published altogether four papers in which the 4Pi microscope played a key role. Two of them addressed specific 4Pi microscopy analyses on fixed cells. Other work in our laboratory also benefited in part from the availability of the system. However, the 4Pi system could never be applied to the very topic of the project originally planned, i.e., live-cell imaging, because system performance was not sufficient for 4Pi imaging using (comparatively low numerical aperture) water immersion objectives which are required for live-cell imaging.
Projektbezogene Publikationen (Auswahl)
- Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation. J. Biol. Phys. 33 (2007) 433-443
Glaschick, S., Röcker, C., Deuschle, K., Wiedenmann, J., Oswald, F., Mailänder, V., & Nienhaus, G. U.
- Two-photon Excitation and Photoconversion of EosFP in Dual-color 4Pi Confocal Microscopy. Biophys. J. 92 (2007) 4451-4457
Ivanchenko, S., Glaschick, S., Röcker, C., Oswald, F., Wiedenmann, J., & Nienhaus, G. U.
- mRuby, a Bright Monomeric Red Fluorescent Protein for Labeling of Subcellular Structures. PLoS ONE 4 (2009) e4391
Kredel, S., Oswald, F., Nienhaus, K., Deuschle, K., Röcker, C., Wolff, M., Heilker, R., Nienhaus, G. U. , & Wiedenmann, J.
- The Inability to Disrupt the Immunological Synapse distinguishes HIV-1 from most other Primate Lentiviruses. J. Clin. Invest. 119 (2009) 2965-2975
Arhel, N., Clauss, K., Nienhaus, G. U., & Kirchhoff, F.
