Entering the Nano-Cosmos of the Cell by Means of Spatial Position Determination Microscopy (SPDM): Implications for Medical Diagnostics and Radiation Research

During the last 20 years fluorescence light microscopy has made an enormous progress towards fluorescence nanoscopy in order to elucidate the nanostructural organization of cellular machineries beyond classical limits of resolution in light microscopy. One of these novel techniques is Spatial Position Determination Microscopy (SPDM), an approach of molecular localization microscopy based on the application of specific fluorescence labelling of cellular structures by means of dyes that undergo reversible photobleaching resulting in blinking effects during image acquisition. This blinking allows spectral separation of individual molecules and thus precise localization and distances measurements far below the diffraction limited resolution of an objective lense. Here, the principles of this technique are presented and the imaging power is demonstrated by several examples of nano-analyses in various cell systems. It is shown how receptor clusters of Her2/neu change their morphology in different tumour cell types depending on the degree of overexpression. From the distribution of the m164 protein, nano-structural information of the endoplasmatic reticulum and the nuclear membrane can be obtained after murine cytomegalovirus infection. SPDM analysis of the distribution of nucleosomes in cell nuclei in comparison to computer models can give insights into the nano-architecture of cell nuclei and its conformation changes after radiation exposure and during DNA repair processes. Finally, first approaches of SPDM and Combinatorial Oligonucleotide Fluorescence In Situ Hybridization (COMBOFISH) indicate that nano-analyses of chromatin conformation of gene target sites will become feasible.