DOI: 10.1046/j.1365-2818.2001.00845.x. High resolution protein localization using soft X‐ray microscopy

High resolution protein localization using soft X‐ray microscopy

10.1046/j.1365-2818.2001.00845.x

Crossref journal-article

Wiley

Journal of Microscopy (311)

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.

Bibliography

MeyerâIlse, W., Hamamoto, D., Nair, A., LeliÃ¨vre, S. A., Denbeaux, G., Johnson, L., Pearson, A. L., Yager, D., Legros, M. A., & Larabell, C. A. (2001). High resolution protein localization using soft Xâray microscopy. Journal of Microscopy, 201(3), 395â403. Portico.

Authors 10

W. Meyer‐Ilse (first)
D. Hamamoto (additional)
A. Nair (additional)
S. A. Lelièvre (additional)
G. Denbeaux (additional)
L. Johnson (additional)
A. L. Pearson (additional)
D. Yager (additional)
M. A. Legros (additional)
C. A. Larabell (additional)

References 32 Referenced 147

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{'key': 'e_1_2_6_2_5_1', 'first-page': '95', 'article-title': 'Resolution determination in X‐ray microscopy: an analysis of the effects of partial coherence and illumination spectrum', 'volume': '8', 'author': 'Heck J.M.', 'year': '1998', 'journal-title': 'J. X-Ray Sci. Technol'} / J. X-Ray Sci. Technol / Resolution determination in X‐ray microscopy: an analysis of the effects of partial coherence and illumination spectrum by Heck J.M. (1998)
10.1006/adnd.1993.1013
10.1016/S0962-8924(98)01424-X
10.1017/S0033583500003139
Larabell C.A.(2000)Confocal microscopy of liveXenopusoocytes eggs and embryos.Methods in Molecular Biology Vol. 135;Developmental Biology Protocols Vol 1. (ed. by R. S. Tuan and C. W. Lo) pp. 175–182. Humana Press New Jersey. (10.1385/1-59259-685-1:175)
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10.1016/S0091-679X(08)61960-3
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Meyer‐Ilse W. Denbeaux G. Johnson L.E. Bates W. Lucero A.&Anderson E.H.(1999)X‐Ray Microscopy VI. XRM99 (ed. by W. Meyer‐Isle T. Warwick and D. Attwood) pp. 129–134. American Institute of Physics Melville New York.
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{'key': 'e_1_2_6_2_24_1', 'volume-title': 'Phase Contrast Studies of Hydrated Specimens with the X‐Ray Microscope at BESSY', 'author': 'Schmahl G.', 'year': '1994'} / Phase Contrast Studies of Hydrated Specimens with the X‐Ray Microscope at BESSY by Schmahl G. (1994)
Schmahl G. Rudolph D. Niemann B. Guttmann P. Thieme J.&Schneider G.(1996)X‐Ray microscopy.Naturwissenschaften 83 61–70. (10.1007/BF01141872)
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{'key': 'e_1_2_6_2_32_1', 'first-page': '245', 'article-title': 'X‐ray microscopic visualization of specific labeling of adhesive molecule CD36 and cytoadherence by Plasmodium falciparum infected erythrocytes', 'volume': '99', 'author': 'Yeung J.', 'year': '1998', 'journal-title': 'Res. Comms Mol. Pathol. Pharmacol'} / Res. Comms Mol. Pathol. Pharmacol / X‐ray microscopic visualization of specific labeling of adhesive molecule CD36 and cytoadherence by Plasmodium falciparum infected erythrocytes by Yeung J. (1998)

Dates

Type	When
Created	22 years, 5 months ago (March 12, 2003, 1:31 a.m.)
Deposited	1 year, 10 months ago (Oct. 17, 2023, 11:49 p.m.)
Indexed	4 hours, 15 minutes ago (Aug. 23, 2025, 9:45 p.m.)
Issued	24 years, 5 months ago (March 1, 2001)
Published	24 years, 5 months ago (March 1, 2001)
Published Online	23 years, 8 months ago (Dec. 21, 2001)
Published Print	24 years, 5 months ago (March 1, 2001)

Funders 0

None

BibTeX

@article{Meyer_Ilse_2001, title={High resolution protein localization using soft X‐ray microscopy}, volume={201}, ISSN={1365-2818}, url={http://dx.doi.org/10.1046/j.1365-2818.2001.00845.x}, DOI={10.1046/j.1365-2818.2001.00845.x}, number={3}, journal={Journal of Microscopy}, publisher={Wiley}, author={Meyer‐Ilse, W. and Hamamoto, D. and Nair, A. and Lelièvre, S. A. and Denbeaux, G. and Johnson, L. and Pearson, A. L. and Yager, D. and Legros, M. A. and Larabell, C. A.}, year={2001}, month=mar, pages={395–403} }

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