TY  - GEN
A1  - Deschamps, Joran
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/34241/
N2  - Advances in light microscopy have allowed circumventing the diffraction barrier, once thought to be the ultimate resolution limit in optical microscopy, and given rise to various superresolution microscopy techniques. Among them, localization microscopy exploits the blinking of fluorescent molecules to precisely pinpoint the positions of many emitters individually, and subsequently reconstruct a superresolved image from these positions. While localization microscopy enables the study of cellular structures and protein complexes with unprecedented details, severe technical bottlenecks still reduce the scope of possible applications. In my PhD work, I developed several technical improvements at the level of the microscope to overcome limitations related to the  photophysical behaviour of fluorescent molecules, slow acquisition rates and three-dimensional imaging.
I built an illumination system that achieves uniform intensity across the field-of view using a multi-mode fiber and a commercial speckle-reducer. I showed that it provides uniform photophysics within the illuminated area and is far superior to the common illumination system. It is easy to build and to add to any microscope, and thus greatly facilitates quantitative approaches in localization microscopy.
Furthermore, I developed a fully automated superresolution microscope using an open-source software framework. I developed advanced electronics and user friendly software solutions to enable the design and unsupervised acquisition of complex experimental series. Optimized for long-term stability, the automated microscope is able to image hundreds to thousands of regions over the course of days to weeks. First applied in a system-wide study of clathrin-mediated endocytosis in yeast, the automated microscope allowed the collection of a data set of a size and scope unprecedented in localization microscopy.
Finally, I established a fundamentally new approach to obtain three-dimensional superresolution images. Supercritical angle localization microscopy (SALM) exploits the phenomenon of surface-generated fluorescence arising from fluorophores close to the coverslip. SALM has the theoretical prospect of an isotropic spatial resolution with simple instrumentation. Following a first proof-of-concept implementation, I re-engineered the microscope to include adaptive optics in order to reach the full potential of the method.
Taken together, I established simple, yet powerful, solutions for three fundamental technical limitations in localization microscopy regarding illumination, throughput and resolution. All of them can be combined within the same instrument, and can dramatically improve every cutting-edge microscope. This will help to push the limit of the most challenging applications of localization microscopy, including system-wide imaging experiments and structural studies.
CY  - Heidelberg
AV  - public
Y1  - 2024///
TI  - Towards quantitative high-throughput 3D localization microscopy
KW  - Open-source
ID  - heidok34241
ER  -