Faculty: Gerhard Schütz
High resolution optical microscopy techniques have revolutionized the life sciences in the last years. Fluorescently labelled proteins can be imaged with a lateral resolution of ~20 nm and a z-resolution of ~40 nm (1). Up to now, however, application of these techniques was limited to cell culture systems, which were easy to handle (Figure). In this project we would like to take the challenge and open up also histological sections to such new investigations.
There are several reasons to select serial ultrathin sections of mouse brain as the sample of choice:
i) neurons show a complex structural organization on length scales of a few nanometers up to centimeters, so that they are an interesting sample per se for the new microscopy techniques;
ii) ultrathin section have a thickness of ~1µm, so that background fluorescence will be low enough to enable single molecule imaging;
iii) with Hans Ulrich Dodt, an internationally renowned neurologist is faculty member of this consortium;
iv) our group has long-lasting collaboration with the group of Harald Sitte (Medical University Of Vienna) within an FWF-funded SFB on neurotransmitter transporters, which would be a good complementation of this project.
We propose to use the method termed dSTORM (direct stochastic optical reconstruction microscopy), which was introduced in 2008 by Markus Sauer (2). In this method, the sample is labelled with organic dyes that show pronounced blinking under specific redox conditions. In this way, only a small subfraction of all fluorophores is active and visible at any instant of time; the majority remains in a dark state. On a single frame, the molecules are thus imaged as individual well-separated spots. The trick is to determine the position of these spots with a mathematical algorithm, which allows for localization precision of a few tens of nanometers. By recording thousands of images, all fluorophores will be once captured in their active states, yielding the positions of all tagged molecules in the sample.
Measurement conditions, sample preparation and fluorescence dyes will be optimized for dSTORM analysis with respect to highest resolution. Target proteins will be membrane transporters, and G-protein coupled receptors at the post-synaptic membrane. As standards for orientation we will stain membrane proteins in a second colour by immune-histochemical labelling. Artificially introduced astigmatism will be employed to calculate the 3-dimensional position of single molecule emitters to an accuracy of ~40 nm (3). We will use this approach in order to resolve high resolution 3D images of the thin sections. Spectral information is intended to be aligned with unbiased molecular information by subsequent analysis of the tissue section by MALDI MSI.
1. Sengupta, P., S. Van Engelenburg, and J. Lippincott-Schwartz. 2012. Visualizing cell structure and function with point-localization superresolution imaging. Dev Cell 23:1092-1102.
2. Heilemann, M., S. van de Linde, M. Schuttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer. 2008. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chem Int Ed Engl 47:6172-6176.3. Huang, B., W. Wang, M. Bates, and X. Zhuang. 2008. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319:810-813.
4. Dodt, H. U., U. Leischner, A. Schierloh, N. Jahrling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgansberger, and K. Becker. 2007. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods 4:331-336
5. Ertürk, A., K. Becker, N. Jahrling, C. P. Mauch, C. D. Hojer, J. G. Egen, F. Hellal, F. Bradke, M. Sheng, and H. U. Dodt. 2012. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc 7:1983-1995.
6. Chung, K., J. Wallace, S. Y. Kim, S. Kalyanasundaram, A. S. Andalman, T. J. Davidson, J. J. Mirzabekov, K. A. Zalocusky, J. Mattis, A. K. Denisin, S. Pak, H. Bernstein, C. Ramakrishnan, L. Grosenick, V. Gradinaru, and K. Deisseroth. 2013. Structural and molecular interrogation of intact biological systems. Nature 497:332-337.
- Close collaborations with the Medical University of Austria, a through a Special Research Program granted by the FWF (SFB35) and the National Institute of Drug Abuse (Baltimore) are documented.
- Protocols for fluorescence labelling of thin sections will be established and new fixation protocols for transforming intact tissue into an optically transparent and macromolecule-permeable construct, which preserves the native molecular information shall be implemented in collaboration with H. U. Dodt (TU Vienna).
- Multimodal Imaging will be approached by analyzing the identical samples after the optical investigation also via mass spectrometry to gather unbiased molecular information (collaboration with M. Marchetti-Deschmann (TU Vienna).
- Access to superresolution STED-microscopy as a complementary imaging technique will be available in collaboration with C. Eggeling (Oxford University).
- In order to obtain a global perspective of the obtained single slice data, images recorded from subsequent sections have to be aligned. To this end, algorithms will be developed in collaboration with R. Sablatnig (TU Vienna).