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Visualizing specific nuclear proteins in eukaryotic cells using soft X-ray microscopy
Abstract
Specific nuclear proteins in immunogold labeled Drosophila melanogaster cells were visualized by applying soft X-ray microscopy. In addition, first experiments were performed to localize two different labeled nuclear proteins in the same X-ray micrograph by using immunofluorescence microscopy for distinguishing the proteins.
In this chapter, we outline some of the properties of X-ray microscope systems in operation today, and highlight some of their
present applications. We will not discuss the history of X-ray microscopes prior to about 1975 but instead refer the reader
to a series of conference proceedings known as “X-ray Optics and X-ray Microanalysis,” which began in 1956. Originally these
had valuable material on X-ray microscopy but this diminished after about 1970. The first five were at Cambridge (1956) (Cosslett
et al., 1957), Stockholm (1959) (Engström et al., 1960), Stanford (1962) (Pattee et al., 1963), Orsay (1965) (Castaing et
al., 1966) and Tubingen (1968) (Molenstedt et al., 1969). We also recommend the historical perspectives by A. Baez (Baez,
1989, 1997) and the book by Cosslet and Nixon (1960). There is a recognisable thread of continuity between today’s status
of the field and efforts that began slowly around 1975 (Niemann et al., 1976; Parsons, 1978; Kirz and Sayre, 1980c; Parsons,
1980) and blossomed with the availability of synchrotron light sources and nanofabrication technologies; this thread can be
traced in part via the proceedings of another conference series that began in 1984 (Schmahl and Rudolph, 1984a) and has continued
until today (Sayre et al., 1988; Michette et al., 1992; Aristov and Erko, 1994; Thieme et al., 1998b; Meyer-Ilse et al., 2000b;
Susini et al., 2003). Zone-plate X-ray microscopes now exist at roughly two dozen international synchrotron radiation research
centers (see Table 13–3), and commercial lab-based instruments are also available. Three types are in especially widespread
use. Transmission X-ray microscopes (TXMs) specialize in the rapid acquisition of 2D images using high flux sources, and in
the collection of tilt sequences of projection images for 3D imaging by tomography. Scanning transmission X-ray microscopes
(STXMs) specialize in the acquisition of reduced dose images and point spectra with high energy resolution for elemental and
chemical state mapping, and require high source brightness. Scanning fluorescence X-ray microprobes (SFXMs) are similar to
STXMs except that fluorescence X-rays are collected by energy-resolving detectors for trace element mapping. All three approaches
are now working below 100 nm resolution, to the point of reaching 15 nm resolution in some demonstrations (Chao et al., 2005).
While many of the new technical developments continue to be pursued by specialists in X-ray optics and microscopy, much of
presentday activity comes from scientists in other fields of research who are using X-ray microscopes to address their particular
questions. This chapter is mainly aimed at scientists from the latter group as well as those from the other communities represented
in the content of this series of books.
Soft x-ray microscopy is now routinely capable of imaging biological specimens with resolutions that are five times better than the best visible light microscopes (⩽50 nm). However, for biological labeling the only options developed for x-ray microscopy have been silver enhanced gold probes that can be used with both scanning and wide field CCD microscopes, such as XM-1 at the Advanced Light Source (ALS), and luminescent lanthanide probes that necessitate a scanning microscope (SXM). To add to the arsenal of useful x-ray biological probes, we have begun the development of labels that rely on the L-edge absorption lines of vanadium. Vanadium is especially attractive as a biological contrast reagent because it has two strong absorption lines at energies that range from ∼512 to 525 eV just below the oxygen K-edge, which makes it an ideal material for imaging in the water window. In this report, we present our initial findings on the application of vanadium for biological labeling. Fixed NIH 3T3 cells grown on silicon nitride windows were incubated with vanadyl sulfate and in some cases basified with triethylamine. After vanadium treatment of the cells, they were thoroughly rinsed and then imaged using XM-1 above and below the vanadium 516 eV resonance. Vanadium staining was clearly visible around and in the cells. These findings suggest that bioconjugated vanadium clusters could provide sufficient x-ray contrast to be used as biological probes. © 2000 American Institute of Physics.
The resolution of transmission X-ray microscopes (TXMs) using zone plate optics is presently about 30 nm. Theory and experiments presented here show that this resolution can be obtained in radiation sensitive hydrated biological material by using shock frozen samples. For this purpose the interaction of X-rays with matter and the image formation with zone plates is described. For the first time the influence of the limited apertures of the condenser and the zone plate objective are in included in calculations of the image contrast, the photon density and radiation dose required for the object illumination. Model considerations show that lowest radiation dose and high image contrast are obtained in optimized phase contrast which exploits absorption as well as phase shift. The damaging effect of the absorbed X-rays is quantitatively evaluated by radiation-induced kinetics showing that cryogenic samples are structurally stable. To verify these theoretical models the TXM was modified to allow imaging of frozen-hydrated samples at atmospheric pressure. Details inside cells and algae as small as 35 nm are visible at 2.4 nm wavelength in amplitude contrast mode. At this resolution the cryogenic samples show no structural changes. As predicted, optimized phase contrast shows structures inside the frozen-hydrated objects with high contrast. Stereo-pair images of algae reveal the 3D organization of the organelles. Element analysis and micro-tomography of whole cryogenic cells are possible.
Transmission soft X-ray microscopy is now capable of achieving resolutions that are typically 5 times better than the best-visible light microscopes. With expected improvements in zone plate optics, an additional factor of two may be realized within the next few years. Despite the high resolution now available with X-ray microscopes and the high X-ray contrast provided by biological molecules in the soft X-ray region (lambda = 2-5 nm), molecular probes for localizing specific biological targets have been lacking. To circumvent this problem, X-ray excitable molecular probes are needed that can target unique biological features. In this paper we report our initial results on the development of lanthanide-based fluorescent probes for biological labeling. Using scanning luminescence X-ray microscopy (SLXM, Jacobsen et al., J. Microscopy 172 (1993) 121-129), we show that lanthanide organo-polychelate complexes are sufficiently bright and radiation resistant to be the basis of a new class of X-ray excitable molecular probes capable of providing at least a fivefold improvement in resolution over visible light microscopy. Lanthanide probes, able to bind 80-100 metal ions per molecule, were found to give strong luminescent signals with X-ray doses exceeding 10(8) Gy, and were used to label actin stress fibers and in vitro preparations of polymerized tubulin.
X-raymicroscopy is applied to detect specific proteins in whole cell nuclei of Drosophila melanogaster using immunogold labeling and silver enhancement. As a model for a small subnuclear structure the Drosophila dosage compensation protein MSL-1 was chosen. It associates with a number of other proteins to form a hetero-multiprotein complex, which elevates the transcriptional activity of the single X chromosome in males. This phenomenon is known as dosage compensation and is essential for the survival of male flies. The distribution of the Drosophila dosage compensation complex was studied by X-ray microscopy, because though the complex is expected to function by remodeling the structure of chromatin, its exact mode of action is not yet known. Many similar protein complexes are associated with different aspects of chromatin-mediated gene regulation in all eucaryotic organisms and can also be studied with the approach presented in this work. The distribution of MSL-1 protein in the nuclei of fixed D. melanogaster culture cells is visualized using the Göttingen X-ray microscope at the electron storage ring BESSY I. In addition to conventional and confocal laserscan fluorescence microscopy, X-ray microscopic investigations were performed at room as well as at cryogenic temperatures. The label can clearly be identified in the X-ray micrographs and shows detailed structure in the cell nuclei. Currently, X-ray micrographs show details in the cell nuclei about five times smaller than those in visible light micrographs.
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G. Denbeaux, W. Meyer-Ilse, X-ray Microscopy, AIP
Conference Proceedings, Vol. 507, 2000, p. 184.
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