Soft x-ray tomography (SXT) is ideally suited to imaging sub-cellular architecture and organization, particularly in eukaryotic cells. SXT is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50 nm, or better. In soft x-ray tomography (SXT) cells are imaged using photons from a region of the spectrum known as the ‘water window’. This results in quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. Cells are therefore visualized very close to their native, fully functional state. The utility of SXT has recently been enhanced by the development of high numerical aperture cryogenic light microscopy for correlated imaging. Taking this multi-modal approach now allows labeled molecules to be localized in the context of a high-resolution 3-dimensional tomographic reconstruction of the cell.
We used soft x-ray tomography (SXT) – a high-resolution, quantitative imaging technique – to measure cell size and organelle volumes in yeasts. Cell size is a key factor in initiating cell division in yeasts, whereas the number and volume of the organelles has a profound impact on the function and viability of a cell. Consequently, determining these cell parameters is fundamentally important in understanding yeast biology. SXT is well suited to this type of analysis. Specimens are imaged in a near-native state, and relatively large numbers of cells can be readily analyzed. In this study, we characterized haploid and diploid strains of Saccharomyces cerevisiae at each of the key stages in the cell cycle, and determined if there were relationships between cellular and organelle volumes. We then compared these results with SXT data obtained from Schizosaccharomyces pombe, the three main phenotypes displayed by the opportunistic yeast pathogen Candida albicans, and from a coff1-22 mutant strain of Saccharomyces cerevisiae. This comparison revealed that volumetric ratios were invariant irrespective of yeast strain, ploidy or morphology, leading to the conclusion these volumetric ratios are common in all yeasts.
The opportunistic pathogen Candida albicans can undergo phenotypic switching between a benign, unicellular phenotype and an invasive, multicellular form that causes candidiasis. Increasingly, strains of Candida are becoming resistant to antifungal drugs, making the treatment of candidiasis difficult, especially in immunocompromised or critically ill patients. Consequently, there is a pressing need to develop new drugs that circumvent fungal drugresistance mechanisms. In this work we used soft X-ray tomography to image the subcellular changes that occur as a consequence of both phenotypic switching and of treating C. albicans with antifungal peptoids, a class of candidate therapeutics unaffected by drug resistance mechanisms. Peptoid treatment suppressed formation of the pathogenic hyphal phenotype and resulted in striking changes in cell and organelle morphology, most dramatically in the nucleus and nucleolus, and in the number, size, and location of lipidic bodies. In particular, peptoid treatment was seen to cause the inclusion of lipidic bodies into the nucleus.
Summary We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of ‘super-resolution’ techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.
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