Mapping Diffusion Properties in Living Cells

Abstract

The function of living cells is based on chemical reactions. It has been shown that the velocity of these reactions is limited by the molecular transport in the cell. Therefore also the spatial organization of a cell plays a major role. In order to investigate such transport processes, fluorescence correlation spectroscopy (FCS) is often used in combination with fluorescently labeled proteins. In FCS a small subvolume of the cell (~1µm³) is observed with a laser-based microscope. The fluctuations of the fluorescence, emitted from this subvolume, are acquired. An autocorrelation analysis of these fluctuations reveals the concentrations and diffusion coefficients of the labeled particles. Usually, FCS is implemented using a confocal microscope, which can observe only a single spot at any time. For this thesis, FCS was extended to an imaging method, by combining it with light sheet fluorescence microscopy (SPIM). This relatively new widefield microscopy technique allows to observe an arbitrarily positionable, thin plane (diameter: 1-3µm) in the cell. By using a fast electron-multiplying charge-coupled device camera, the combination of SPIM and FCS allowed to map the motion also of relatively small autofluorescent proteins in living cells. At first, the setup of a light sheet microscope is described. This microscope was designed and optimized for SPIM-FCS measurements in living cells. Several test measurements show the applicability of SPIM-FCS to in vitro samples and to all larger compartments of a living cell (nucleus, cytoplasm, cellular membrane). Afterwards, the usability of several commercially available cameras as image sensor for SPIM-FCS measurements is assessed. At the time of writing, EM-CCD cameras offer the best trade-off between photosensitivity and achievable temporal resolution (~ 500µs). In addition to these linear cameras, also the use of single-photon avalanche diode (SPAD) arrays is investigated. These offer a significantly better temporal resolution (1-10µs) than current EM-CCD cameras, which would render them the ideal image sensor for SPIM-FCS. However, they do not yet reach the photo-sensitivity of EM-CCDs. Two different SPAD arrays were characterized in detail and first successful SPIM-FCS measurements of solute fluorescent molecules could be demonstrated. In a second step, SPIM-FCS was extended by a cross-correlation analysis (SPIM-FCCS), which allowed for the first time to map the interactions of differently labeled cytosolic molecules in living cells. For this purpose, the cross-correlation function between the fluorescence fluctuations from two different color channels is analyzed. A non-zero amplitude of this cross-correlation function is found only, if the differently labeled molecules interact and move together. Finally, the methods developed during this project were applied to different cellular systems. The mapping of the mobility of inert tracer molecules of different sizes allowed to measure the viscosity of the cytoplasm in different cells. A position-dependence of this mobility could only be found in the nucleoli. In addition, an important step in the remodelling cycle of the keratin intermediate filament system was investigated. As a third application, SPIM-F(C)CS measurements of different chromatin-associated proteins demonstrated the dynamics in the cellular nucleus. Mobility maps of labeled histone proteins revealed the organization of chromatin in interphase nuclei. In addition, the activity of the nuclear receptor RXR and a transcription factor were mapped.