Korrelative Kryo-Röntgentomographie und höchstauflösende Kryo-Fluoreszenzmikroskopie
Biophysik
Zusammenfassung der Projektergebnisse
An important goal in structural biology is to image specific molecules and complexes directly within their native cellular context. This requires preparation and microscopy techniques that allow both the visualization of the sub-cellular architecture of a cell and – at the same time – the unambiguous localization of molecular complexes within this cell. For best functional and structural characterization of macromolecules, the ideal imaging setup provides protein-specific information without interfering with the native structure of the cell and covers the entire resolution range from micrometer down to nanometer with isotropic resolution. An appropriate tool to approach this goal is correlative cryo-microscopy. Correlative microscopy images a specimen with two complementary modalities that – combined – create a highly informative, holistic view of the cell. While fluorescence microscopy (FM) provides high molecular specificity visualizing the spatial distribution of fluorescently labeled molecule, it lacks the information about surrounding structures. This ultrastructural context can be provided by soft x-ray tomography (SXT) which allows to visualize the subcellular details of intact frozen-hydrated cells of up to 15 μm in thickness at a resolution of about 50 nm depending on the zone plate used as objective lens. Despite of the increasing number of correlative studies involving electron and light microscopy, the full potential of correlative imaging has not yet been exploited. Exploiting the full potential includes (i) establishing and optimizing alternative cryo-correlation techniques, such as correlative SXT, that are based on a different contrast mechanism than charged particle microscopy and can visualize different sub-cellular structures in intact cells that are cryo-immobilized in a near-native state, (ii) improving FM for correlative imaging by working at cryo-conditions and optimizing the optical setup thereof, and (iii) combining correlative imaging with super-resolution fluorescence techniques to identify macromolecular complexes with sub-diffraction accuracy. The ultimate goal of correlative imaging is to meet all three needs and combine them as correlative superresolution cryo-microscopy. I planned to pursue this goal by correlating SXT with superresolution cryo-fluorescence microscopy (cryo structured Illumination microscopy, cryo-SIM) with a high numerical aperture (NA) cryo-lens. SIM was chosen to achieve super-resolution since it does not require particular fluorophore properties and is expected to work normally under cryo-conditions. Due to delays in the completion of the cryo-SIM setup and especially due to the shortening of my fellowship because of a new job, commissioning of the cryo-SIM setup had to be postponed and I focused instead on establishing a quantifiable biological test system (chromochloris zofingiensis algae) for the cryo-correlation of several imaging modalities. Lipid bodies in chromochloris zofingiensis algae were visualized, quantified and correlated using SXT, cryo-FM and room-temperature SIM. With chromochloris zofingiensis as a biological test system, I established multimodal (cryo-)microscopy, sample preparation, automation and optimization, analysis, quantification and manual correlation of multimodal datasets. For the correlation, algal lipids were stained with BODIPY which turned out to be a functional stain for both cryo-FM and room-temperature SIM. Lipids strongly absorb soft X-rays and are unambiguously delineated in soft x-ray tomograms of frozen-hydrated cells due to their distinct LAC values. After sequential imaging by both cryo-FM and SXT with cells in a near-native frozen-hydrated state, lipid bodies of the same cell were correlated using fluorescent polystyrene microspheres as joint fiducial markers that were visible in both imaging modalities. Multimodal imaging of lipid bodies in chromochloris zofingiensis did not only contribute to method development for correlative microscopy, but also gave unprecedented insights into the lipid distribution and densities in chromochloris zofingiensis algae. This is of great interest for biofuel production since microalgae have potential to help meet demands for energy and food without exacerbating environmental problems. We could show that the addition of an organic carbon source to their growth medium led to a significant increase in lipid and starch content. This switch was reversible within 48 hours; the removal of organic carbon quickly restored original cell organelle volumes, providing evidence for metabolic changes. Thus, this study also provided insights into manipulating algal cells to enhance production of high-value carotenoids and biofuel feedstocks.
