We develop microscopic tools to visualize cellular structures well below the diffraction limit (~ 200 nm in the imaging plane).
- dSTORM imaging with photoswitchable synthetic dyes
- Concepts for quantitative super-resolution imaging
- New concepts for fluorescence fluctuation in SOFI imaging
- Quantum dot triexciton imaging (QDTI)
Development of application methods
- Binding constants of ligand-receptor-interactions on single cells determined by single-molecule imaging
- Coordinate-based colocalization analysis for single-molecule localization microscopy (SMLM)
- Cluster analysis for SMLM
- Drift and chromatic correction in 3D SMLM
- Localization precision of SMLM by nearest neighbor analysis (NeNA)
- Correlated electron microscopy and super-resolution microscopy
- Correlated high-throughput and high-resolution microscopy
The activation of cellular signaling events occurs at the cell membrane. This involves ligand-receptor interactions as well as oligomerization of receptor proteins. We are studying the association and activation of various membrane receptors using single-molecule microscopy, single-molecule spectroscopy and super-resolution imaging. Specifically, we are interested in the receptors MET, TNF-R1 and TLR4. Furthermore, we develop specific single-molecule tools to address these questions.
- Dimerization of MET upon InlB binding studied with single-molecule photobleaching
- Binding constants of ligand-receptor-interactions on single cells
- Organization of TNF-R1 studied at the single-cell level
- Single-molecule tracking of TNF-R1 in live cells
Because of their small size, the structural organization of bacterial cells has been so far inaccessible to light microscopy methods. We apply super-resolution and single-molecule methods to study cellular processes in bacteria. We are specifically interested in the organization of the bacterial chromosome, as well as in bacterial transcription.
- Nanoscale organization of RNA polymerase in E. coli
- Super-resolution imaging of the E. coli chromatin
Spahn C, Endesfelder U, Heilemann M (2014) Super-resolution imaging of Escherichia coli nucleoids reveals highly structured and asymmetric segregation during fast growth. J. Struct. Biol. DOI 10.1016/j.jsb.2014.01.007
Structural organization of DNA and RNA in eukaryotes
- Super-resolution imaging of chromosomal DNA
|Zessin PJM, Finan K, Heilemann M (2012) Super-resolution fluorescence imaging of chromosomal DNA. J. Struct. Biol. 177, 344.|
We are engaged in a number of collaborations with other research groups, and work on more biological topics.
- Structural organization of HIV proteins at the nanoscale (in cooperation with HG Kräusslich, University of Heidelberg)
- Quantification of kinetochore proteins in S. pombe (in collaboration with ED Laue, Cambridge)
Photochemistry of organic dyes
- Increasing the brightness of synthetic fluorophores in heavy water
- Photoswitching of synthetic dyes in phalloidin-dye-conjugates (in collaboration with T Kuner, Heidelberg)
Nanguneri S, Flottmann B, Herrmannsdörfer F, Kuner T, Heilemann, M. (2014). Single-molecule super-resolution imaging by tryptophan-quenching-induced photoswitching of phalloidin-fluorophore-conjugates. Microsc. Res. Techn. DOI: 10.1002/jemt.22349.
Single-molecule localization microscopy specifically demands for high labeling densities and the incorporation of suitable photoswitchable fluorophores. We develop and establish new labeling schemes, e.g. based on click chemistry or novel protein tags such as the TMP tag.
- Visualization of eukaryotic and bacterial DNA and RNA with click chemistry
- Unnatural amino acids and click chemistry labeling in E. coli
- Fluorogenic nucleic acid probes (in collaboration with A. Mokhir, University of Erlangen)