Quantitative Einzelmolekül-Mikroskopie der Aggregation von ErbB Rezeptoren
Zusammenfassung der Projektergebnisse
The aim of the project was to investigate how the stoichiometry of aggregates of cell surface receptors influence the degree of malignancy in different kinds of cancers. Emphasis was put here on the ErbB receptor family and its known affiliates. Different levels of ErbB receptors have particularly been found in several types of breast cancer with varying degree of malignancy. The working hypothesis of the project thus was that not only over-expression of said receptors determines the severity of the disease but that interactions between the same and other receptor families have an even more significant impact on the severity. To this end, recently developed super-resolution microscopy techniques have been used, paired with powerful image analysis and intelligent data post-processing. Here, focus was on the related super-resolution approaches STORM and PALM. Consequently, the project consisted of an experimental and a computational component. The first part addressed all requirements related to cell biological experiments. This included basic wet lab techniques, several biochemical investigation and manipulation approaches, optical microscopy setups, and imaging procedures for super-resolution microscopy. The second part was dedicated to basic image analysis algorithms, advanced post-processing techniques to determine quantitative results, mathematical modeling and simulation of the dynamics of cell surface receptors and their surroundings, and efficient data storage and management solutions. Main results of the project are: i) The establishment of super-resolution imaging of cell surface receptors using various techniques. This includes traditional STORM and PALM experiments as well as novel imaging techniques based on DNA origami. ii) The development of basic and advanced image analysis tools for processing single-molecule imaging data sets and performing intelligent postprocessing. The latter solutions focused on extracting the number of individual receptor molecules from super-resolution imaging data. Although this approach was promising when applied to in silico data sets, it basically did not master data sets from actual single-molecule experiments. The developed algorithm nevertheless provides a significant ansatz to quantitatively analyze current and future single-molecule experiments.
Projektbezogene Publikationen (Auswahl)
- Computational approaches to count single molecules using super-resolution microscopy; Gordon Research Conference on Single Molecule Approaches to Biology, 2012, Mt. Snow, West Dover
U Schmidt, JL Werbin, PK Sorger, and G Danuser
- Single molecule counting using STORM/PALM; 56 Biophysical Society Meeting 2012, San Diego
U Schmidt, JL Werbin, PK Sorger, and G Danuser
