High resolution protein localization using soft X‐ray microscopy
- 1 March 2001
- journal article
- research article
- Published by Wiley in Journal of Microscopy
- Vol. 201 (3), 395-403
- https://doi.org/10.1046/j.1365-2818.2001.00845.x
Abstract
Soft X‐ray microscopes can be used to examine whole, hydrated cells up to 10 µm 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 (λ = 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.Keywords
This publication has 28 references indexed in Scilit:
- Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp✪The Journal of cell biology, 2000
- Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: II. TomographyJournal of Microscopy, 2000
- Maskless, parallel patterning with zone-plate array lithographyJournal of Vacuum Science & Technology B: Microelectronics and Nanometer Structures, 1999
- Soft x-ray microscopyTrends in Cell Biology, 1999
- THE GREEN FLUORESCENT PROTEINAnnual Review of Biochemistry, 1998
- RöntgenmikroskopieThe Science of Nature, 1996
- Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains.The Journal of cell biology, 1995
- New high‐resolution zone‐plate microscope at beamline 6.1 of the ALSSynchrotron Radiation News, 1995
- X-Ray Interactions: Photoabsorption, Scattering, Transmission, and Reflection at E = 50-30,000 eV, Z = 1-92Atomic Data and Nuclear Data Tables, 1993
- Fine structural organization of the interphase nucleus in some mammalian cellsJournal of Ultrastructure Research, 1969