High resolution protein localization using soft X-ray microscopy

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TitleHigh resolution protein localization using soft X-ray microscopy
Publication TypeJournal Article
Year of Publication2001
AuthorsMeyer-Ilse W, Hamamoto D, Nair A, Lelievre SA, Denbeaux G, Johnson L, Pearson AL, Yager D, Le Gros MA, Larabell CA
Journal TitleJournal of Microscopy-Oxford
Volume201
Pages395-403
Journal DateMar
ISBN Number0022-2720
Accession NumberISI:000167686600007
KeywordsCells, dynamics, embryos, green fluorescent protein, immunogold, matrix, organization, protein localization, rna splicing factor, soft X-ray microscopy, tomography, transcription, transmission
Abstract

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 mum thick and produce images approaching 30 nm resolution Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells, In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (lambda = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures, We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.

URL<Go to ISI>://000167686600007