X-ray phase-contrast microscopy and microtomography

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X-ray phase-contrast microscopy and microtomography
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  X-ray phase-contrast microscopy and microtomography S.C. Mayo, T.J. Davis, T.E. Gureyev, P.R. Miller, D. Paganin, A. Pogany, A.W. Stevenson, S.W. Wilkins CSIRO Manufacturing and Infrastructure Technology, Private Bag 33,Clayton South, VIC 3169, Australia Sherry.Mayo@csiro.au Abstract:  In-line phase contrast enables weakly absorbing specimens to be imaged successfully with x-rays, and greatly enhances the visibility of fine scale structure in more strongly absorbing specimens. This type of phase contrast requires a spatially coherent beam, a condition that can be met by a microfocus x-ray source. We have developed an x-ray microscope, based on such a source, which is capable of high resolution phase-contrast imaging and tomography. Phase retrieval enables quantitative information to be recovered from phase-contrast microscope images of homogeneous samples of known composition and density, and improves the quality of tomographic reconstructions. ©2003 Optical Society of America OCIS codes:  (340.7460) X-ray microscopy; (100.5070) Phase retrieval References and links 1. U. Bonse and M. Hart, “An x-ray interferometer,” Appl. Phys. Lett. 6 , 155-157 (1965). 2. M. Ando and S. Hosoya, “An attempt at x-ray phase-contrast microscopy,” in Proc. 6 th  Intern. Conf. On X-ray Optics and Microanalysis, G. Shinoda, K. Kohra and T. Ichinokawa Eds. (Univ. of Tokyo Press, Tokyo, 1972) pp. 63-68. 3. A. Momose, T.Takeda and Y. Itai, “Phase-contrast x-ray computed tomography for observing biological specimens and organic materials,” Rev. Sci. Instr. 66 , 1434-1436 (1995). 4. Y. Kohmura, H. Takano, Y. Suzuki and T. Ishikawa, “Shearing x-ray interferometer with an X-ray prism and its improvement,”, Proc. 7 th  Intern. Conf. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp. 571-574. 5. C. David, B. Nöhammer, H.H. Solak and E. Ziegler, “Hard x-ray shearing interferometer,” Proc. 7 th  Intern. Conf. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp 595-598. 6. T. Wilhein et al, “Differential interference contrast x-ray microscopy with twin zone plates at ESRF beamline ID21,” Proc. 7 th  Intern. Conf. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp 535-541. 7. G. Schmahl, D. Rudolph, G. Schneider, P. Guttmann and B. Niemann, “Phase contrast x-ray microscopy studies,” Optik, 97, 181-182 (1994). 8. Y. Kohmura, A. Takeuchi, H. Takano, Y. Suzuki and T. Ishikawa, “Zernike phase-contrast x-ray microscope with an x-ray refractive lens,” Proc. 7 th  Intern. Con.f. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp 603-606. 9. J. R. Palmer and G. R. Morrison, “Differential phase-contrast imaging in the scanning transmission x-ray microscope,” in OSA Proc. On Short Wavelength Coherent Radiation: Generation and Applications, P.H. Buckbaum and N.M. Ceglio, eds., Vol. 11 of OSA Proceedings Series (Optical Society of America, Washington, D.C., 1991), pp. 141-145. 10. M. Feser, C. Jacobsen, P. Rehak and G. DeGeronimo, “Scanning transmission x-ray microscopy with a segmented detector,” Proc. 7 th  Intern. Con.f. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp 529-534. 11. R.W. Gerchberg and W.O. Saxton, “A practical algorithm for the determination of phase from images and diffraction plane pictures,”   Optik, 35 , 237-246 (1972). 12. J. Miao, P. Charalambous, J. Kirz and D. Sayre, “Extending the methodology of x-ray crystallography to allow imaging of micrometre-sized non-crystalline specimens,” Nature, 400 , 342-344 (1992). (C) 2003 OSA22 September 2003 / Vol. 11, No. 19 / OPTICS EXPRESS 2289 #2790 - $15.00 USReceived July 24, 2003; Revised September 10, 2003  13. M.R. Howells, et al. , “X-ray microscopy by phase-retrieval methods at the Advanced Light Source,” Proc. 7 th  Intern. Conf. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp 557-561. 14. Gabor, D., “A new microscopic principle,” Nature 161 , 777-778 (1948) 15. C. Jacobsen, M. Howells, J. Kirz and S. Rothman, “X-ray holographic microscopy using photoresists,” J. Opt. Soc. Am.   A  7 , 1847-1861 (1990). 16. N. Watanabe et al ., “Optical Holography in the hard x-ray domain,” Proc. 7 th  Intern. Con.f. on X-ray Microscopy, J. Susini, D. Joyeux, F. Polack, Eds. (EDP Sciences, Les Ulis) pp 551-556. 17. A. Snigirev, I. Snigireva, V. Kohn, S. Kuznetsov and I. Schelokov, “On the possibilities of x-ray phase contrast microimaging by coherent high-energy synchrotron radiation,” Rev.Sci.Inst. 66, 5486-5492 (1995) 18. K.A. Nugent, T.E. Gureyev, D.J. Cookson, D. Paganin, and Z. Barnea, “Quantitative phase imaging using hard X rays,” Phys. Rev. Lett. 77 , 2961-2964 (1996) 19. S.W. Wilkins, T.E. Gureyev, D. Gao, A. Pogany and A.W. Stevenson, “Phase-contrast imaging using polychromatic hard x-rays,” Nature 384 , 335-338 (1996) 20. P. Cloetens, R. Barrett, J. Baruchel, J.-P. Guigay, and M. Schlenker, “Phase objects in synchrotron radiation hard X-ray imaging,” J.Phys. D: Appl. Phys. 29 , 133-146 (1996) 21. P. Cloetens, W. Ludwig, J. Baruchel, D. Van Dyck, J. Van Landuyt, J.-P. Guigay, and M. Schlenker, “Holotomography: Quantitative phase tomography with micrometre resolution using hard synchrotron radiation X-rays,” Appl. Phys. Lett. 75 , 2912-2914 (1999) 22. H. Yoshimura, D. Shoutsu, T. Horikoshi, H. Chiba, S. Kumagai, K. Takahashi and T. Mitsui, “Application of SEM-modified x-ray microscope to entomology and histology, and effects of x-ray coherence in imaging,” J. Elect. Micros .   49 , 621-628 (2000) 23. V.E. Cosslett and W.C. Nixon, W,  X-ray Microscopy , (Cambridge Univ. Press, London,1960) 24. S.C. Mayo et al.,  “Quantitative x-ray projection microscopy: phase-contrast and multi-spectral imaging,” J. Microscopy 207, 79-96 (2002). 25. J.M. Cowley,  Diffraction Physics , 3 rd  revised edition, (North-Holland, Amsterdam, 1995). 26. J.C.H. Spence,  Experimental High-resolution Electron Microscopy . 2 nd  edition, (Oxford Univ. Press: New York, 1988). 27. T.E. Gureyev, S. Mayo, S.W. Wilkins, D. Paganin, and A.W. Stevenson, “Quantitative in-line phase-contrast imaging with multienergy X-rays,” Phys. Rev. Lett. 86  (25), 5827-5830 (2001) 28. D Paganin, S. C Mayo, T. E Gureyev, P. R Miller and S. W Wilkins, “Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object,” J. Microscopy, 206 , 33-40 (2002) 29. T.E. Gureyev, “Composite techniques for phase retrieval in the Fresnel region,” Opt. Commun. 220 , 49-58 (2003). 30. L.A. Feldkamp, L.C. Davis and J.W. Kress, “Practical cone-beam algorithm,” J. Opt. Soc. Am. A  1 , 612-619 (1984). 31. A.V. Bronnikov, “Reconstruction formulas in phase-contrast tomography,” Opt. Commun. 171 , 239-244 (1999) 1. Introduction Recent years have seen a growing interest in various forms of x-ray phase-contrast imaging. Conventional x-ray imaging relies on absorption contrast which is only effective in absorbing samples. Absorption contrast is also poor at revealing small high spatial frequency features where they occur in the presence of larger lower frequency features. By comparison, phase-contrast is often particularly sensitive to high spatial frequency features, giving an alternative view of the sample. It also enables weakly absorbing or non-absorbing samples to be imaged effectively. Phase shifts imposed on an x-ray wavefront by a sample can be transformed into contrast, and hence measured or reconstructed indirectly by a number of methods. One of the older methods uses crystal interferometers of the type developed by Bonse and Hart [1]. The use of this method for phase contrast imaging was pioneered by Ando and coworkers [2] and has produced excellent phase imaging and tomography results [3]. More recently other interferometric methods have been developed including shearing interferometers [4,5] and twin zone plates [6]. Zernike methods, analogous to those used in light optics have also been developed with both diffractive [7] and refractive optics [8]. (C) 2003 OSA22 September 2003 / Vol. 11, No. 19 / OPTICS EXPRESS 2290 #2790 - $15.00 USReceived July 24, 2003; Revised September 10, 2003  Scanning x-ray microscopes can be used with a segmented detector to generate phase (and other) contrast images from observed deflections in the beam. This was first explored by Palmer and Morrison [9] and subsequently by Feser and coworkers [10]. A major class of methods for extracting both phase and absorption information employ mathematical phase-retrieval techniques. Typically these extract such information from holographic or diffraction images of the sample. For high resolution imaging, methods have been developed based on oversampling of far-field diffraction images combined with Gerchberg-Saxton phase retrieval [11,12,13]. Alternative approaches, more suited to larger samples, are based on Gabor [14] holography. These include synchrotron-based methods using a pinhole or a Fresnel zone plate focus as a secondary source[15,16]. A simpler method, requiring no additional optics, is in-line phase-contrast imaging where the wave is allowed to propagate beyond the sample sufficiently for Fresnel diffraction to occur. This was proposed in the mid 90’s [17] and is used at synchrotrons and with laboratory microfocus sources [18-22]. At its simplest, the in-line phase contrast approach can be used to produce edge-enhanced images of the sample which are often very useful in themselves. However, to obtain quantitative information about the sample, or to exploit the greater contrast available in the holographic imaging regime, the ability to perform phase retrieval on the images becomes increasingly important. Phase retrieval enables the phase shift imposed on the wave by the sample (e.g. in the object plane) to be recovered from the diffracted intensity distribution in the image plane or planes. In recent years we have been exploring the potential of inline phase contrast imaging using laboratory sources. This paper describes one such instrument, dubbed the x-ray ultra-microscope (XuM), which uses a point-projection geometry for x-ray imaging. The XuM is an attachment to an electron microscope, exploiting the fine focus of the microscope to generate a microfocus x-ray source. The use of an SEM as an x-ray source has a history going back over 50 years to early work by Cosslett and Nixon [23]. Here, however, we exploit the small source that can be produced with a modern SEM, to produce a spatially coherent x-ray beam suitable for generating phase-contrast images. This enhances the information content of images, and, together with phase retrieval, enables the extraction of quantitative information. When combined with a modern CCD detector system the result is a practical and versatile instrument well suited to imaging a wide range of samples. The ease of data acquisition, together with the imaging geometry also lend themselves to tomographic data collection. The use of phase-retrieval to improve the results of tomographic reconstruction will be described. 2. The X-ray Ultra-Microscope (XuM) This instrument is hosted on an FEI XL-30 SFEG SEM. The warm-FEG source has high brightness enabling a high beam current to be focused into a small spot on a metal target to generate a submicron source of x-rays. The primary components of the XuM are a direct detection CCD camera, mounted of the right side of the SEM chamber, and a target positioner, mounted on the right side. The arrangement of the components is shown in Fig 1. The target positioner enables the target – typically a metal foil – to be positioned a few millimeters below the pole piece where the electron beam is focused onto it to produce a microfocus x-ray source. The sample is mounted on the SEM sample stage which enables x, y and z translation and rotation about the vertical axis. Figure 2 shows how the point projection geometry results in magnification of the image on the detector. For a target-sample distance R 1  and a sample-detector distance R 2 , the magnification, M, is determined by the ratio of the target-detector distance, (R 1 +R 2 ) to the target-sample distance R 1 , such that; M=(R 1 +R 2 )/R 1 . Since the target-detector distance is 250mm and the target-sample distance can be varied between 0 and 25 mm the minimum magnification is 10x and the maximum is limited only by the practicalities of operating with the sample very close to the target. In practice a typical upper limit is around 3000x. (C) 2003 OSA22 September 2003 / Vol. 11, No. 19 / OPTICS EXPRESS 2291 #2790 - $15.00 USReceived July 24, 2003; Revised September 10, 2003  Fig. 1. Diagram showing the main components of the XuM. Object X-ray source Phase-contrast image z = R 2   contact image z = 0    Fig. 2. Sketch of the point-projection microscope geometry, indicating R 1  the source-sample distance, and R 2  the sample-detector distance. The x-ray range generated in the XuM is determined by the target material and the accelerating voltage. The spectrum is typically dominated by a characteristic line (or lines) of the target material, with a significant amount of bremsstrahlung also present. The energy range that can be used to form an image is further limited by the sensitivity of the detectors. The main detector used is a deep depletion CCD with a beryllium window, sensitive to around 2-10 keV. A back illuminated windowless CCD is also used when soft-x-ray sensitivity (800eV – 8keV) is required. The SEM accelerating voltage goes up to a maximum of 30 kV, more than sufficient to excite characteristic lines within the detector sensitivity range. Selected targets with different characteristic lines can be used, together with changes to accelerating voltage to modify the x-ray energy spectrum and to tailor it to particular features of interest in the sample. The imaging resolution is ultimately limited by the x-ray source size, which depends on the target geometry and composition, however resolution down to 60nm can be achieved. Images typically take a few minutes to acquire with the camera described above. More recently a short R 2  camera has been developed which enables similar images to be acquired in a minute or so. With binning and a fast readout speed, this camera can also be used for real time navigation, albeit with noisier image quality. Sample stage (mounted on SEM chamber door) Target positioner Detector Module Sample Secondary Electron detector (for SEM) SEM pole piece SEM sample chamber SEM column X-rays Electron beam Target Vacuum Pump Computer for detector control and data acquisition R 1  R 2   (C) 2003 OSA22 September 2003 / Vol. 11, No. 19 / OPTICS EXPRESS 2292 #2790 - $15.00 USReceived July 24, 2003; Revised September 10, 2003  3. Phase contrast, phase retrieval and image processing In its simplest form, in-line phase-contrast imaging provides images of samples in which edges and boundaries are enhanced by near-field Fresnel diffraction. For a sufficiently small source and a high resolution detector this type of contrast is in fact unavoidable, although the nature of the contrast is changed substantially by modifying the imaging conditions. This is in contrast to lens based x-ray microscopy systems which produce pure absorption images unless specifically modified to give phase contrast [7,8]. Such edge-enhanced images may often be sufficient in themselves for observing features of interest in the sample. If we consider a wavefront propagating along z, then for a pure phase object the near field image is (to a good approximation) the Laplacian (in x and y) of the phase shift imposed on the wavefront by the sample. For samples which also show absorption contrast the situation is a little more complicated, although the high spatial frequency features are still much enhanced by phase contrast. Subtle features involving small but abrupt changes in the sample’s projected density are typically very clearly visible, making the technique ideal for detecting cracks, voids and surface texture. Figure 3 shows examples where the raw phase contrast image gives useful information about the sample. The first is of a dust mite, and clearly shows the grooves on its back despite the small size of these features compared to the overall thickness of the mite. These features would be very difficult to see in an image relying on conventional absorption contrast. The second image is of a 1mm diameter multilayer sphere composed of concentric shells of different materials. Phase contrast makes a crack in the outermost 30 µ m thick shell clearly visible. Fig. 3. Left: Phase contrast images of a dust mite. The grooved texture of the mite’s back is clearly visible due to phase contrast, despite the small size of these grooves compared to the overall thickness of the mite (10 min. exposure, R 1 =1.9mm, R 1 +R 2 =250mm). Right: Part of a 1mm diameter multilayer composed of concentric shells of different thicknesses. showing a fine crack in the outer shell (10 min exposure, R 1 =3.6mm, R 1 +R 2  = 250mm). 3.1 Contrast formation While such near field images are often useful in their raw form, recovering quantitative information from phase contrast data requires a fuller understanding of the image formation process. Initially we consider the case of a plane wave, U 0 , of wavelength λ,  propagating along the z direction and which encounters the sample at z = 0. The wave is modified by the sample such that the wave, U(x,y), on exiting the sample is given by (C) 2003 OSA22 September 2003 / Vol. 11, No. 19 / OPTICS EXPRESS 2293 #2790 - $15.00 USReceived July 24, 2003; Revised September 10, 2003
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