Imaging Living Cells with an X-ray Laser

Sammanfattning: Imaging living cells at a resolution higher than the resolution of optical microscopy is a significant challenge. Fluorescence microscopy can achieve a degree of super-resolution via labeling cellular components with a fluorescent dye. Reaching nanometer or sub-nanometer resolution requires high-energy radiation with significantly shorter wavelength than that of optical light. X-rays and electrons have the requisite wavelengths and could be suitable for such studies; however, these probes also cause significant radiation damage. A dose in excess of 100,000,000 Gray (Gy, J/kg) would be required to reach nanometer resolution on a cell, and no cell can survive this amount of radiation. As a consequence, much of what we know about cells at high resolution today comes from dead material.Theory predicts that an ultra-short and extremely bright coherent X-ray pulse from an X-ray free-electron laser can outrun key damage processes to deliver a molecular-level snapshot of a cell that is alive at the time of image formation. The principle of ‘diffraction before destruction’ exploits the difference between the speed of light (the X-ray pulse) and the much slower speed of damage formation. The femtosecond pulse ‘freezes’ motion in the cell at physiological temperatures on the time scale of atomic vibrations, offering unprecedented time resolution and a plethora of new experimental possibilities.This thesis describes the first test experiments on imaging living cells with an X-ray laser. I present results in three essential areas of live cell imaging. (i) We have used an aerosol injector to introduce live cyanobacteria into the X-ray focus, and recorded diffraction patterns with extremely low background at very high hit rates. (ii) We demonstrated scattered signal beyond 4 nm resolution in some of these experiments. (iii) The thesis also describes image reconstruction, using a new fully automated pipeline that I developed during my studies. The reconstruction of diffraction patterns was successful for all patterns that did not have saturated pixels. The new software suite, called RedFlamingo, selects exposures with desired properties, can sort them according to sample size, shape, orientation, exposure, the number and type of objects in the beam during the exposure, their distance from each other, and so forth. The software includes validation tools to assess the quality of the reconstructions.