Correlating Soft X-Ray Cryo-Tomography With Super-Resolution Cryo-Microscopy

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During my Fellowship, I will work on correlative cryogenic super-resolution microscopy in the research group spearheading the development of correlative soft x-ray cryo-tomography (SXT) and fluorescence cryo-microscopy. During the past decade, the Larabell group (UCSF) has developed the world's first soft x-ray cryo-microscope for cell biology together with the first fluorescence cryo-microscope (cryo-FM) for correlative studies. The correlation of data - measured from the same cell using these two imaging modalities - generates a holistic view of the cell, with molecules and cellular processes being visualized directly within their near-native cryo-preserved ultrastructural context. In SXT, cryogenic intact cells of up to 15 micrometers in thickness are illuminated with 'water window' X-rays (2.3-4.4 nm), which are absorbed 10 times more strongly by carbon-containing biomolecules than by water. This leads to quantitative high-contrast images of the cellular ultrastructure without contrast-enhancing staining. The existing cryo-FM is diffraction-limited, and, therefore, can only localize processes at a spatial resolution significantly lower than that achievable with SXT. To improve this localization accuracy, a super-resolution version of the cryo-FM has to be developed. Since, unlike other sub-diffraction techniques, structured illumination microscopy (SIM) does not require fluorophores with specific photoswitchable properties, it represents an attractive super-resolution strategy for cryogenic conditions. Linear SIM can achieve a twofold lateral resolution improvement compared to conventional light microscopy. My role in the group will be to work on the implementation of a structured illumination cryo-microscope, and correlate its data with SXT images using self-written image processing algorithms and standard tomogram reconstruction software. After first proof-of-principle experiments, I will apply the novel correlative super-resolution imaging technique in a collaboration to cultivate a research program aimed at understanding the role played by nuclear organization and chromatin compartmentalization in gene regulation of mouse olfactory receptors. The combination of SXT and cryo-SIM will represent a ground-breaking advance in cell imaging, and will allow unprecedented interpretation and analyses of molecular organization and ultrastructural architecture of cryo-preserved intact cells at sub-diffraction accuracy. In short, my fellowship work in the Larabell group will give me technical skills and scientific insights at the highest level, and of great potential value to the fields of cell biology and microscopy. The technologies I will be developing are also applicable in electron microscopy, an area of scientific strength that would be advanced significantly by the availability of a cryo-SIM for correlative imaging studies.