Cryo X-ray microscopy with high spatial resolution in amplitude and phase contrast
Introduction
High-resolution X-ray microscopes use zone plates (ZPs) – circular transmission gratings with radially increasing line density – as X–ray optics 1, 2, 3. The numerical aperture NA=mλ/(2 drN) of a ZP is given by the maximum diffraction angle, which in turn is given by the outermost zone width drN, the wavelength λ and the diffraction order m. For incoherent object illumination, the obtainable resolution δ in the first diffraction order is according to the Rayleigh criterion approximately given by the outermost zone width drN and is independent of the wavelength λ:High-resolution ZPs are developed by many groups 4, 5, 6. At 2.4 nm wavelength X-ray objectives with drN=19 nm have already resolved features of 25 nm size 7, 8. This resolution is approximately 10–fold better than that obtained with conventional visible light microscopy.
The kind of object structures which become visible in a microscopical image depend on the interaction process of the radiation with the sample: for visible and UV light this process is dominated by the interaction with the valence electrons of the atoms. Their electronic states are strongly influenced by chemical bonds in molecules, whereas X-rays mainly interact with inner-shell electrons of atoms. Therefore, information about the energy levels in atoms and molecules can be obtained, if the transitions of inner-shell electrons into unoccupied electronic states of molecules are studied with wavelengths near X-ray absorption edges with high spectral resolution. This can be done with X-ray microscopy; it is highly sensitive to elements and bonds between atoms, e.g. σ- and π-bonds.
By comparison, in the transmission electron microscope (TEM) elastic and inelastic scattering of electrons is used for the image formation. If the inelastically scattered electrons are detected by electron energy loss spectroscopy (EELS), the elemental distribution can be obtained because the energy loss is determined by the atomic species. If only the elastically scattered electrons are detected, i.e. inelastically scattered electrons are filtered, phase contrast images can be obtained by defocusing and making use of the spherical aberrations of electron optical lenses [9]. In practice, such phase contrast images of unstained biological material in vitreous ice show lower contrast than images of dried samples. In addition, the contrast transfer function depends critically on the object's thickness [10]. Therefore, this method is mainly used for imaging thin cryo sections of about 50–100 nm thickness or thin macromolecules and viruses embedded in vitreous ice. In these cases the obtainable resolution in the TEM is about 1 nm and mainly limited by radiation damage 11, 12. For specimens up to 1 μm thickness, e.g. whole bacteria, the interpretation of phase contrast TEM images becomes much more difficult and the resolution obtained in the images is comparable to the resolution actually achieved using X-ray microscopes [13]. Furthermore, it is very difficult to acquire three-dimensional (3D) information in the TEM from objects thicker than 1 μm, e.g. whole cells, because then different sections of an object have to be prepared and imaged separately.
However, X-ray microscopy allows to image much thicker hydrated specimens. In the water window wavelength range between the inner-shell absorption edges of oxygen and carbon (2.34–4.37 nm wavelength) samples of about 10 μm thickness can be studied [14]. Even thicker samples of about 100 μm thickness can be investigated at wavelengths around 0.3 nm [15]. The small numerical aperture of X-ray optics leads to longer depth of focus compared to high-resolution visible light objectives. For these reasons X-ray micro-tomography is possible, which can reveal the 3D structure of whole, unsectioned cells in their natural hydrated state. Tomography with scanning X-ray microscopes was first demonstrated using a microfabricated gold pattern [16]. Recently, tomographic reconstructions of mineralized bacterial sheaths were obtained from TXM images recorded at 2.4 nm wavelength [17]. X-ray micro-tomography of hydrated cells will give new possibilities to study biological structures. For this purpose the best imaging conditions and the most suited preparation techniques have to be evaluated in order to avoid significant damage of the cells caused by X-rays. This report focuses on the new technique of X-ray imaging of frozen hydrated biological samples. The different contrast modes are discussed and the required radiation dose and relevant radiation damage processes are determined. In the experimental part a new cryo stage is described and X-ray images are presented, which verify the theory and demonstrate the capabilities of cryo X-ray microscopy in biological and medical research.
Section snippets
Theory
Imaging by X-rays can be divided into two main steps: (1) X-rays interact with the matter of the object and (2) an image is generated by a ZP used in one selected focusing order of diffraction.
Image contrast
The image contrast of model protein structures embedded in ice was calculated for amplitude and phase contrast. The results are shown in Fig. 3 for infinite ZP aperture. High amplitude contrast is obtained in the water window, which is due to the strong X-ray absorption of cellular structures containing carbon and nitrogen compared to the lower absorption of water. To visualize fine structures in hydrated cells with higher contrast, the different phase shift of X-rays in cellular structures and
Radiation damage of frozen-hydrated objects
The diffraction of X-rays in the object depends on the elemental distribution which should remain constant during the exposure time so as to avoid structural changes visible in the image. In general a radiolysis model describing the effect of absorbed X-rays on the molecule distribution of a sample has to include both the X-ray induced kinetics and the transport of matter. The concentration distributions [Mi](r, τ) of species Mi in space r and time τ can be described by a set of partial
Cryo transmission X-ray microscope
The X-ray optical arrangement and the cryogenic object stage adapted to the TXM are shown in Fig. 10. In conjunction with a pinhole a condenser ZP acts as a dispersive and focusing element, which monochromatizes the incoming polychromatic radiation from an electron storage ring (e.g. BESSY) and focuses it directly into the object plane 29, 30. The X-ray objective produces a magnified image on a thinned, back-illuminated slow scan CCD-camera. This CCD-detector is nearly optimal for image
Preparation of frozen-hydrated samples
If hydrated biological objects are frozen slowly and at atmospheric pressure, large ice crystals form, which destroy the ultrastructure. However, with cooling rates higher than 10 000 K/s vitreous ice can be formed [33]. In order to obtain these high cooling rates in the specimens the water layer thickness surrounding an object with its limiting thermal conductivity should not exceed about 10 μm. This thickness also guarantees sufficient transparency for radiation of water window wavelengths. In
Experimental results
Up to now amplitude as well as phase contrast X-ray images were obtained at 2.4 nm wavelength mainly from chemically fixed, wet cells at room temperature 34, 35. During the chemical fixation molecules are added to the structures of the samples. Both fixation and radiation damage can change the structures and therefore the image contrast of the initially living sample. However, in cryo samples the image contrast is caused only by the natural element distribution. Amplitude and optimized phase
Prospects of cryo X-ray microscopy
High-resolution X-ray imaging of unstained frozen-hydrated samples is not restricted to the amplitude and phase contrast mode. Due to the unique interactions of X-rays with atoms and molecules additional information can be obtained from the samples. For instance, the X-ray absorption near edge structure (XANES) allows to determine the microscopic distribution of elements and molecules in objects, which has been demonstrated for organic polymers [37] and biomolecules in sperms using a scanning
Conclusion
Frozen-hydrated biological objects were imaged for the first time with a cryo transmission X-ray microscope at 2.4 nm wavelength in amplitude and optimized phase contrast mode. For this purpose a new cryo stage has been developed and implemented at the Göttingen TXM operating at the electron storage ring BESSY in Berlin. In contrast to the cryo stages for electron microscopes, which operate in vacuum, a system was developed where the samples are surrounded by cryogenic nitrogen gas at
Acknowledgements
G. Schmahl and D. Rudolph are gratefully acknowledged for their encouragement and support. The author is indebted to B. Niemann for his important contributions to the development of the cryo technique, P. Guttmann for his help in performing the experiments, T. Schliebe and M. Peuker for the phase contrast objective and the staff of BESSY for excellent working conditions. In addition, the author thanks P. Nieschalk, J. Herbst and H. Düben for technical assistance. The present study was supported
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