A modular, comprehensive microscopy platform for modern live cell imaging

Abstract

This thesis addresses the interdisciplinary application of spectromicroscopic techniques and the subsequent design of a comprehensive microscopic platform. In classical microscopy, the detected light intensity of a sample is the main source of information. Spectroscopy, i.e., the analysis of light-matter interactions, additionally analyses the physical properties of light and is thus an ideal complement to microscopy. It provides additional information that enables a much more comprehensive characterization of samples. Especially in the life sciences, the combination of imaging with spatially resolved spectroscopy (spectromicroscopy) is key to modern microscopy methods and serves as an important tool to gain ever deeper insights into the complexity of biological processes. Accordingly, the demands on a modern research microscope have steadily increased. By means of interdisciplinary research projects within the scope of this work, the successful application of several spectromicroscopy methods are demonstrated, including the detection of protein-protein interactions with FLIM-FRET (fluorescence lifetime imaging microscopy with Förster resonance energy transfer), contrast enhancement for autofluorescent samples with FIDSAM (fluorescence intensity decay shape analysis), the photophysical characterization of biological interaction markers with single-molecule spectroscopy, super-resolution imaging with single-molecule localization microscopy and single particle tracking of individual plasma membrane proteins in living cells. Using the knowledge gained from these projects, a microscope platform was designed and built to provide a unique combination of advanced light microscopy techniques for interdisciplinary research. This work offers the conceptual design as well as a detailed description of the microscope.