Combining force and fluorescence microscopy for the manipulation and detection of single cells, viruses, and proteins

Abstract

Total Internal Reflection Fluorescence Microscopy (TIRFM) and Atomic Force Microscopy (AFM) are both successfully applied in biophysics and biology. AFM provides the possibility to image and to mechanically manipulate the samples on a nanometer scale. TIRFM allows the observation of single fluorescent molecules close to the surface at a high signal-to-noise ratio and, consequently, improves the temporal and spatial localization of events of interest. The ability to use both microscopy techniques simultaneously allows to study specific biochemical changes (by using fluorescence markers) that are induced by mechanical stimulation (AFM) and gives access to new methodical approaches.<br /> This thesis describes the construction of a combined TIRFM-AFM instrument, its possibilities, and limitations, e.g. time correlated mechanical manipulation and fluorescence detection, effects of the tip inside the excitation and emission volume, as well as noise interactions of both systems. To investigate the tip effects, the fluorescence of single dye molecules linked to ribosomes was characterized. The background light from the AFM cantilevers during single molecule detection was determined as well as the quenching potential of the tip.<br /> The TIRFM-AFM setup was applied to investigate biological samples from the micrometer length scale of living cells down to the nanometer length scale of viruses and actin protein filaments.<br /> 1) On the cell scale the TIRFM capabilities were used to identify three sub-types of a Parkinson-like cell line in a mixed population which could not be separated by cell-biological procedures. After identification, the AFM was applied to probe the mechanical properties of the individual sub-types. The results indicate a sub-type specific interaction between the fluorescently labeled Leucine-rich repeat kinase2 (LRRK2) and parts of the cytoskeleton which lead to a different mechanical response for each of the sub-types.<br /> 2) On the virus scale AFM-induced mechanical fatigue experiments were performed on the Adenovirus. The goal was to disassemble the virus capsid and to visualize the release of the genome in real time by specific fluorescence labeling of Deoxyribonucleic acid (DNA). We compared the genome release of the wild type mature adenovirus with that of a non-infectious immature-like mutant. The measurements show that the immature genome remains compact after the shell is opened up by AFM. This suggests that the maturation step is essential for rendering the virus to be infective.<br /> 3) On the protein level we studied a newly discovered protein that is involved in the organization of the actin-myosin fibers in muscle cells of C. elegans. In vitro, the protein called DreBriN-like family homolog 1 (DBN-1) induces bundling of actin filaments. These bundles tend to form loops. TIRFM allowed to target these bundles and loops by fluorescence labeling of actin. Subsequent high resolution AFM imaging revealed a decoration of the actin filaments with the 'cross-linking' DBN-1 proteins.<br /> As an additional project an 'optical AFM' was developed. This instrument is based on optical tweezers and specifically designed to overcome the noise limitations of AFM at low forces. The trapped bead is moved in the direction perpendicular to the microscope cover slide so that cells can be indented in a similar fashion as with AFM. It was constructed to perform force spectroscopy experiments on cells at very low forces.