The Microscopy

Super-resolution microscopy

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)

 

Endesfelder U, Malkusch S, Flottmann B, Mondry J, Liguzinski P, Verveer PJ, Heilemann M (2011) Chemically induced photoswitching of fluorescent probes – a general concept for super-resolution imaging. Molecules, 16, 3106.

 

Heidbreder M, Endesfelder U, van de Linde S, Hennig S, Widera D, Kaltschmidt B, Kaltschmidt C, Heilemann M (2010) Subdiffraction Fluorescence Microscopy of Biomolecular Structure and Distributions with Quantum Dot Triexciton Imaging (QDTI). BBA Mol. Cell Research, 1803, 1224.

 

Heilemann M, van de Linde S, Schüttpelz M, Kasper R, Seefeldt B, Mukherjee A, Tinnefeld P, Sauer M (2008) Subdiffraction-Resolution Fluorescence Imaging with Conventional Fluorescent Probes. Angew. Chemie, 47, 6172.

 

 

 

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

 

Endesfelder U, Malkusch S, Fricke F, Heilemann M (2014) A simple method to estimate the average localization precision of a single‑molecule localization microscopy experiment. Histochem. Cell Biol. DOI 10.1007/s00418-014-1192-3.

 

Dietz MS, Fricke F, Krüger CL, Niemann HH, Heilemann M (2014) Receptor–Ligand Interactions: Binding Affinities Studied by Single-Molecule and Super-Resolution Microscopy on Intact Cells. ChemPhysChem 15, 671.

 

Flottmann B, Gunkel M, Lisauskas T, Heilemann M, Starkuviene V, Reymann J, Erfle H (2013) Correlative light microscopy for high-content screening. BioTechniques 55, 243.

 

Zessin PJM, Krüger CL, Malkusch S, Endesfelder U, Heilemann M (2013) A hydrophilic gel matrix for single-molecule super-resolution microscopy. Opt. Nanosc. 2, 4.

 

Malkusch S, Endesfelder U, Mondry J. Gelléri M, Verveer PJ, Heilemann M (2012) Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochem. Cell Biol. 137, 1.

 

 

 

The Biology

Membrane receptors

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

 

Fricke F, Malkusch S, Wangorsch G, Greiner JF, Kaltschmidt B, Kaltschmidt C, Widera D, Dandekar T, Heilemann M (2014) Quantitative single‑molecule localization microscopy combined with rule‑based modeling reveals ligand‑induced TNF‑R1 reorganization toward higher‑order oligomers. Histochem. Cell Biol. DOI 10.1007/s00418-014-1195-0.

 

Dietz MS, Fricke F, Krüger CL, Niemann HH, Heilemann M (2014) Receptor–Ligand Interactions: Binding Affinities Studied by Single-Molecule and Super-Resolution Microscopy on Intact Cells. ChemPhysChem 15, 671.

 

Dietz MS, Haße D, Ferraris DM, Göhler A, Niemann HH, Heilemann M (2013) Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells. BMC Biophysics 6, 6.

 

Heidbreder M, Zander C, Malkusch S, Widera D, Kaltschmidt B, Kaltschmidt C, Nair D, Choquet D, Sibarita JB, Heilemann M (2012) TNF-α influences the lateral dynamics of TNF receptor I in living cells. BBA Mol Cell Res. 1823, 1984.

 

 

 

Bacteria

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

 

Endesfelder U*, Finan K*, Holden SJ*, Cook PR, Kapanidis AN, Heilemann M (2013) Multiscale Spatial Organization of RNA Polymerase in Escherichia coli. Biophys. J. 105, 172.

 

 

 

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.

 

 

Collaborative projects

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)

 

Muranyi W, Malkusch S, Müller B, Heilemann M, Kräusslich HG (2013) Super-resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites dependent on the Envelope C-Terminal Tail. PLoS Pathogens 9, e1003198.

Malkusch S, Muranyi W, Müller B, Kräusslich HG, Heilemann M (2013) Single-molecule coordinate-based analysis of the morphology of HIV-1 assembly sites with near-molecular spatial resolution. Histochem. Cell Biol. 139, 173.

 

Lando D, Endesfelder U, Berger H, Dunne PD, McColl J, Klenerman D, Carr AM, Sauer M, Allshire R, Heilemann M, Laue ED (2012) Quantitative single molecule microscopy reveals that CENP-Acnp1 deposition occurs during G2 in fission yeast. Open Biology, 2, 120078.

 

 

 

The Chemistry

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.

 

Klehs K, Spahn C, Endesfelder U, Lee SF, Fürstenberg A, Heilemann M (2014) Increasing the Brightness of Cyanine Fluorophores for Single-Molecule and Superresolution Imaging. ChemPhysChem 15, 637.

 

 

 

Labeling

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)

 

Zessin PJM, Finan K, Heilemann M (2012) Super-resolution fluorescence imaging of chromosomal DNA. J. Struct. Biol. 177, 344.

Dutta S, Flottmann B, Heilemann M, Mokhir A (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Comm. 48, 9664.