Tomáš Lukeš successfully defended his Ph.D. thesis titled Super-resolution Microscopy Live Cell Imaging and Image Analysis at the Czech Technical University in Prague on April 18, 2017.
External fellowship advisor: Theo Lasser (École Polytechnique Fédérale de Lausanne, Switzerland)
Reviewers: Jörg Enderlein (Georg-August University Göttingen, Germany), Martin Hof (J. Heyrovský Institute of Physical Chemistry of the ASCR, v.v.i., Prague, Czech Republic), and Markus Sauer (Julius Maximilian University of Würzburg, Germany).
Abstract: Novel fundamental research results provided new techniques going beyond the diffraction limit. These recent advances known as super-resolution microscopy have been awarded by the Nobel Prize as they promise new discoveries in biology and live sciences. All these techniques rely on complex signal and image processing. The applicability in biology, and particularly for live cell imaging, remains challenging and needs further investigation. Focusing on image processing and analysis, the thesis is devoted to a significant enhancement of structured illumination microscopy (SIM) and super-resolution optical fluctuation imaging (SOFI) methods towards fast live cell and quantitative imaging. The thesis presents a novel image reconstruction method for both 2D and 3D SIM data, compatible with weak signals, and robust towards unwanted image artifacts. This image reconstruction is efficient under low light conditions, reduces phototoxicity and facilitates live cell observations. We demonstrate the performance of our new method by imaging long super-resolution video sequences of live U2-OS cells and improving cell particle tracking. We develop an adapted 3D deconvolution algorithm for SOFI, which suppresses noise and makes 3D SOFI live cell imaging feasible due to reduction of the number of required input images. We introduce a novel linearization procedure for SOFI maximizing the resolution gain and show that SOFI and PALM can both be applied on the same dataset revealing more insights about the sample. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of the sample through the estimation of molecular parameters. For quantifying the outcome of our super-resolution methods, the thesis presents a novel methodology for objective image quality assessment measuring spatial resolution and signal to noise ratio in real samples. We demonstrate our enhanced SOFI framework by high throughput 3D imaging of live HeLa cells acquiring the whole super-resolution 3D image in 0.95 s, by investigating focal adhesions in live MEF cells, by fast optical readout of fluorescently labelled DNA strands and by unraveling the nanoscale organization of CD4 proteins on a plasma membrane of T-cells. Within the thesis, unique open-source software packages SIM Toolbox and SOFI simulation tool were developed to facilitate implementation of super-resolution microscopy methods.
Deschout, H., Lukeš, T., Sharipov, A., Szlag, D., Feletti, L., Vandenberg, W., Dedecker, P., Hofkens, J., Leutenegger, M., Lasser, T., Radenovic, A. Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions (2016) Nature Communications, 7. https://doi.org/10.1038/ncomms13693
Girsault, A., Lukeš, T., Sharipov, A., Geissbuehler, S., Leutenegger, M., Vandenberg, W., Dedecker, P., Hofkens, J., Lasser, T. SOFI simulation tool: A software package for simulating and testing super-resolution optical fluctuation imaging (2016) PLoS ONE, 11 (9). https://doi.org/10.1371/journal.pone.0161602
Křížek, P., Lukeš, T., Ovesný, M., Fliegel, K., Hagen, G.M. SIMToolbox: A MATLAB toolbox for structured illumination fluorescence microscopy (2016) Bioinformatics, 32 (2), pp. 318-320. https://doi.org/10.1093/bioinformatics/btv576
Lukeš, T., Křížek, P., Švindrych, Z., Benda, J., Ovesný, M., Fliegel, K., Klíma, M., Hagen, G.M. Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation (2014) Optics Express, 22 (24), pp. 29805-29817. https://doi.org/10.1364/OE.22.029805
Kekrt, D., Lukeš, T., Klíma, M., Fliegel, K. 2D iterative MAP detection: Principles and applications in image restoration (2014) Radioengineering, 23 (2), pp. 618-631. www.radioeng.cz/fulltexts/2014/14_02_0618_0631.pdf
Lukeš, T., Kekrt, D., Fliegel, K., Klíma, M. Binarization of noisy microscopy images through signal reconstruction using iterative detection network (2014) 2014 IEEE International Conference on Image Processing, ICIP 2014, art. no. 7025802, pp. 3949-3952. https://doi.org/10.1109/ICIP.2014.7025802
Lukeš, T., Hagen, G.M., Krížek, P., Švindrych, Z., Fliegel, K., Klíma, M. Comparison of image reconstruction methods for structured illumination microscopy (2014) Proceedings of SPIE – The International Society for Optical Engineering, 9129. http://dx.doi.org/10.1117/12.2052621
Lukeš, T., Fliegel, K., Klíma, M. Objective image quality assessment of multiframe super-resolution methods (2013) Proceedings of 23rd International Conference, RADIOELEKTRONIKA 2013, art. no. 6530929, pp. 267-272. https://doi.org/10.1109/RadioElek.2013.6530929
Lukeš, T., Fliegel, K., Klíma, M. Performance evaluation of image quality metrics with respect to their use for super-resolution enhancement (2013) 2013 5th International Workshop on Quality of Multimedia Experience, QoMEX 2013 – Proceedings, art. no. 6603205, pp. 42-43. https://doi.org/10.1109/QoMEX.2013.6603205
Dean’s award: Tomáš Lukeš honored with dean’s award for a prestigious dissertation on June 14, 2017.