Single Molecule Detection and Tracking in the Near-Infrared:Solution to the Background Problem in Ultrasensitive Biological Microscopy

Major aim of this project was to extend the technique of single molecule detection towards biological applications. One essential prerequisite for studies of cellular processes on a single molecule level is to solve the problem of cellular autofluorescence, a property of cells to emit fluorescence when illuminated by lasers. By utilizing laser sources in the near infrared in combination with suitable fluorophores, our group was able to overcome this hurdle and succeed in the investigation of cellular processes on a single molecule level. Thus, our novel technique not only allows detection of single proteins and lipids on cells but also opens the possibility to study their dynamics. Our first investigations aimed on the mobility of single fluorescent-labeled lipids in the plasma membrane of human muscle cells. In contrast to the homogenous lipid-matrix model of the plasma membrane, our results revealed well-defined lipid domains in the membrane. Although this domain formation was already postulated in scientific literature, direct visualization of lipid-domains failed until our study. Furthermore, our approach utilizing single molecule microscopy opens the possibility to characterize lipid-mobility both within and out of these domains. This kind of studies concerning the function of cellular components will represent the end of a process that has just started with sequencing the human genome. While understanding the detailed function of the whole proteome of a cell can only be a long-term goal, the next step in proteomics is the investigation of protein expression levels in different cell lines. Especially the variance of protein expression due to diseases has to be addressed. Since the setup developed by our group within this project combines ultra-sensitivity with the capability of quantification, it represents a perfect tool for protein expression studies. In addition, the ultra-sensitivity of our setup will be generally applicable in diagnostics of minimal residual diseases, characterized by only trace amounts of pathogens or cancer cells in the blood. The existing setup is shared with the Austrian bio-community within the framework of the `Schindler Memorial Center for Single Molecule Microscopy` and is already well accepted and utilized by groups throughout the world.