Mapping Diffusion Properties in Living Cells

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

FPGA implementation of a 32x32 autocorrelator array for analysis of fast image series

With the evolving technology in CMOS integration, new classes of 2D-imaging detectors have recently become available. In particular, single photon avalanche diode (SPAD) arrays allow detection of single photons at high acquisition rates (\geq 100 kfps), which is about two orders of magnitude higher than with currently available cameras. Here we demonstrate the use of a SPAD array for imaging fluorescence correlation spectroscopy (imFCS), a tool to create 2D maps of the dynamics of fluorescent molecules inside living cells. Time-dependent fluorescence fluctuations, due to fluorophores entering and leaving the observed pixels, are evaluated by means of autocorrelation analysis. The multi-{\tau} correlation algorithm is an appropriate choice, as it does not rely on the full data set to be held in memory. Thus, this algorithm can be efficiently implemented in custom logic. We describe a new implementation for massively parallel multi-{\tau} correlation hardware. Our current implementation can calculate 1024 correlation functions at a resolution of 10{\mu}s in real-time and therefore correlate real-time image streams from high speed single photon cameras with thousands of pixels.Comment: 10 pages, 7 figure

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements