Using super resolution imaging to investigate the photodynamic inactivation of pathogenic microorganisms

• Applicant: Dr. Anita Gollmer
• Subject Area: Dermatology ; Medical Physics, Biomedical Technology ; Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2013 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 235013909

The increasing spread of multiresistant microorganisms has become a major problem not only in medicine but also food industry. Besides the development of novel antibiotics, other methods for controlling pathogenic microorganisms have been extensively studied. One of these methods is the photodynamic inactivation of microorganisms (PDI). The microorganisms are incubated with dyes (photosensitizers, PS). After a short time of incubation, PS are excited by light. This leads to the production of highly reactive singlet oxygen (1O2) directly at the microorganism, which oxidatively damages cellular structures and consequently the microorganisms are killed.At present it is unclear which chemical PS structures can cause effective killing in certain microorganisms. In order to use PDI effectively for many microorganisms (bacteria, fungi, spores), the knowledge about the subcellular localization of chemically different PS and their effects is of great importance (structure-action mechanism). This is commonly accomplished by means of fluorescence microscopy and by spectroscopic and biochemical detection methods. Compared to eukaryotic cells, bacteria and spores are considerably smaller (~ 1 - 5 microns), which prevents the use of conventional light microscopy. Therefore, super resolution microscopy techniques (PALM / STORM) are used that allow imaging beyond the light-microscopic resolution limit.As a first step, photo-switchable dye conjugates are developed for the PALM / STORM application. The PS and their conjugates will be studied spectroscopically in cell suspensions (1O2 luminescence, PS phosphorescence, quantum yields). Since the photon emission curve critically depends on the respective adjacency of 1O2, the kinetics of the detected signals (luminescence, phosphorescence) provide initial information about the localization of the PS and the photo-switchable dye conjugates in the microorganisms.Second, the subcellular localization of the PS and their dye conjugates will be visualized using the PALM / STORM technique. Morphological changes of various cell organelles, which are stained with photo-switchable dyes shall be monitored and analyzed in real-time in microorganisms incubated with PS. In addition, the PDI induced morphological changes will be examined with a transmission electron microscope (TEM).By correlating the results from spectroscopic studies, TEM and PALM/ STORM, it should be possible to fathom the significance of the physicochemical properties of different PS molecular structures for the effectiveness of the killing of microorganisms. The results of the project will provide the needed scientific basis for further development and optimization of photodynamic inactivation of microorganisms.

• DFG Programme: Research Grants