eXtreme Resolution
Microscopy (XRM)

Research

1. Instrumentation: Microscope development

The development of novel nanoscopy methods for the analysis of biological macromolecular complexes ("biomolecular machines") in their natural environment is a major question of present biological and biophysical research. Hence, novel light-optical methods are required, allowing to study such biological structures within the living cells, even when their dimension is far below the conventional resolution limit of some hundreds of nanometers.
In our research division, two new and complementary superresolution microscopy methods have been developed:

a) Spectral Precision Distance Microscopy (SPDM)

SPDM is a variant of Single Molecule Localization Microscopy (SMLM). In combination with multispectral labeling techniques, this proceedure allows to analyze the topology of biomolecular machines down to a few nanometers.

b) Structured Illumination Microscopy (SIM).

Presently, two approaches for implementing structured illumination have been realized: Axially Structured Illumination (ASIM) and Laterally Structured Illumination (LSIM). In our labs, both these implementations have been realized. While in LSIM, a truely enhanced optical resolution is achieved, the setup of an ASIM microscope has considerably less stringent requirements, and offers its own advantages. We have implemented ASIM in the form of Spatially Modulated Illumination Microscopy. With this microscope, sizes of fluorescent objects as low as a few tens of nanometers can be determined with high precision.

These microscopes are used for the biophysical analysis of important structural parameters of functional revelance in a domain so far inaccessable in living cells. The structural data obtained by these light optical methods are interpreted in the framework of theoretical modelling of the cell structure. For this, it is necessary to quantify these models and to derive by means of scientific computing from these models consequences which are subject to experimental verification. Here, numerical simulations are used. As an example, a dynamic model of the genomic structure of the human nucleus has been developed, which provides numerous predictions with respect to the 4D geometry, the thermal induced mobility, and the impact of radiation. Ways to achieve these aims is by a combination of the methods of biophotonics, biophysics, biocomputing and information processing.

2. Photophysics of Fluorescence Labelling

In fluorescence microscopy, different transitions of the dye molecules give rise to a multitude of contrasting mechanisms. In linear excitation regime (conventional microscopy), the generated (detected) fluorescence intensity is proportional to the illumination intensity. This is no longer the case for advanced, superresolution microscopy techniques. We are investigating the transition behaviour of fluorophores in order to optimize the resulting micrsocope image in terms of resolution and contrast.

3. Biological and Biomedical Applications

Applications range from Chromatin folding and accessibility to transcription factors, to epigenetic regulation, to radiation damage, to ageing, to susceptibility to environmental factors and pathogens.

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Publications

Please find a list of publications on our webpage at the Kirchhoff Institute for Physics