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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 2, Iss. 11 — Nov. 26, 2007
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Fresnel-propagated submicrometer x-ray imaging of water-immersed tooth dentin

Simon Zabler, Peter Cloetens, and Paul Zaslansky  »View Author Affiliations


Optics Letters, Vol. 32, Issue 20, pp. 2987-2989 (2007)
http://dx.doi.org/10.1364/OL.32.002987


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Abstract

Structural investigations of materials in diverse fields such as biomimetics, materials engineering, and medicine have much to benefit from 3D nondestructive microscopy of representative samples of wet tissues. Phase contrast appearing in tomograms produced by Fresnel propagation of partially coherent x-ray fields is useful for visualizing submicrometer features within water-immersed samples. However, spurious contributions such as those due to randomly appearing bubbles lead to distorted images. By improving the statistics during image acquisition and reconstruction, submicrometer-sized tubules in human tooth dentin are observed. This type of wet imaging is directly applicable to the study of many mineralized tissues.

© 2007 Optical Society of America

X-ray absorption and phase-contrast tomography are important tools for the study of biomaterials. Hard x rays are often used, where biological materials appear partially transparent due to their low absorption so that the distribution of attenuation coefficients and corresponding constituent densities are visualized nondestructively [1

1. O. Prymak, H. Tiemann, I. Soetje, J. C. Marxen, A. Klocke, B. Kahl-Nieke, F. Beckmann, T. Donath, and M. Epple, JBIC, J. Biol. Inorg. Chem. 10, 688 (2005). [CrossRef]

]. Specifically for mineralized tissues such as tooth or bone, representative millimeter-sized samples require the use of energies of 2050keV (λ=0.20.6Å) to obtain reasonable radiographs. With phase enhancement [2

2. D. M. Paganin, Coherent X-Ray Optics (Oxford U. Press, 2006). [CrossRef]

], coherent imaging allows visualization of micrometer-sized features, which characterize most skeletal tissues.

A substantial increase in detail visibility occurs when phase-shift effects are recorded. This is particularly useful for visualizing interfaces and inhomogeneities in the microstructure of biomaterials, where precise attenuation coefficients are not needed. With monochromatic partially coherent sources, phase enhancement may be induced by various methods [2

2. D. M. Paganin, Coherent X-Ray Optics (Oxford U. Press, 2006). [CrossRef]

], the simplest of which suggests increasing D [6

6. J.-P. Guigay, Optik (Stuttgart) 49, 121 (1977).

], as shown recently in seeds or teeth [7

7. P. Cloetens, R. Mache, M. Schlenker, and S. Lerbs-Mache, Proc. Natl. Acad. Sci. U.S.A. 103, 14626 (2006). [CrossRef] [PubMed]

, 8

8. S. Zabler, P. Zaslansky, H. Riesemeier, and P. Fratzl, Opt. Express 14, 8584 (2006). [CrossRef] [PubMed]

]. Yet under high-flux conditions (>1010photonssmm2), x-ray interactions with the sample environment result in degraded images. Specifically, in water-immersed samples, bubbles appear, probably due to the dissociation of H2O molecules, as shown, for example, for ice [9

9. W. L. Mao, H. Mao, Y. Meng, P. J. Eng, M. Y. Hu, P. Chow, Y. Q. Cai, J. Shu, and R. J. Hemley, Science 314, 636 (2006). [CrossRef] [PubMed]

]. The resulting free radicals form bubbles that continuously expand, move, and float up to the surface. Silhouettes of such bubbles are often observed in phase-enhanced images and are also seen with other liquids (e.g., ethanol).

We consider here various experimental aspects of tomography of water-immersed samples of tooth dentin and propose a method for reliable acquisition and reconstruction of high-resolution 3D images of the microstructure. Our results indicate this to be directly applicable to x-ray imaging of many wet biomaterials at submicrometer resolution.

The experimentally recorded radiographs are conventionally normalized by subtraction of the CCD dark current IDC(x,y) and division by bright-field images (IBF(x,y)=uinc(x,y)2) so that normalized intensity ratios ID(corr)(x,y,φ) for each projection angle φ are obtained
ID(corr)(x,y,φ)=ID(x,y,φ)IDC(x,y)IBF(x,y)IDC(x,y).
(3)
This normalization is vital for the removal of inhomogeneities in the beam profile and in the detector response. A 3D virtual volume is then assembled by calculating a stack of y planes of the microstructural feature distribution ρ(x,z)=f[δ(x,z),β(x,z)], from lines of log[ID(corr)(x,φ)] in the projection images. Inversion of the Radon transform (R) [11

11. J. Radon, Ber. Verh. Saechs. Akad. Wiss. Leipzig, Math.-Phys. Kl. 69, 262 (1917).

],
log(ID(corr))=Rρ(x,φ)=sampledvdwδ(vsin(φ)+wcos(φ)x)ρ(v,w).
(4)
[(v,w) coordinates of each rotating sample slice] is calculated by standard filtered backprojection so that approximations of ρ(x,z) are obtained. As shown elsewhere [12

12. P. Cloetens, M. Pateyron-Salome, J. Y. Buffiere, G. Peix, J. Baruchel, F. Peyrin, and M. Schlenker, J. Appl. Phys. 81, 5878 (1997). [CrossRef]

] the phase retardation is incorporated into ρ(x,z) and corresponds to the microstructure densities and constitution. Large contributions from stray intensities of log[ID(corr)(x,φ)] must thus be minimized. Despite appearing in only a limited interval of angular projections, TV,TB variations dominate over intensity differences due to TS. Discontinuities [13

13. T. J. Davis, T. E. Gureyev, D. Gao, A. W. Stevenson, and S. W. Wilkins, Phys. Rev. Lett. 74, 3173 (1995). [CrossRef] [PubMed]

], most notably bubble edges, increase TB and extreme dark or bright contributions result in severe perturbations of the 3D image, because they are integrated into many ρ(x,z) points.

To reduce the spurious intensities of the moving bubbles TB we exploit the fact that they vary randomly over time. In a sequence of repeated tomographic acquisitions of a given sample recorded at different times, the overlap between extreme TB contributions is quasi negligible. With a set of N (preferably odd) identical projections from different acquisition runs, each offset by a small vertical sample shift Δyj but recorded at the same projection angle φ, one obtains a series of images ID(j)(x,yΔyj,φ). After normalization, the images j=2N are aligned with ID(1) and time-median images are produced, whereby each projection image point contains only median values. By choosing sufficiently long time intervals Δt=tj+1tj, improved estimates of TS may be obtained, while removing the destructive effects of TB.

The above approach was used to image micrometer-sized tubules in small samples of tooth crown dentin (1mm×1mm×4mm). Samples were immersed in water within poly(methyl methacrylate) (PMMA) vials. The samples, from several different teeth, were arranged such that remaining small fragments of the enamel (which normally coats the external surface of teeth) were situated on the upper edge, assisting in orientation. Each sample and vial were placed at the center of a high-precision rotation stage of the partially coherent high-flux x-ray imaging beam line ID19 in the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The x-ray detector (17μm thick LAG:Eu scintillator, 10×0.40 Olympus lens, 2× eyepiece, and FRELON 2K CCD camera) was positioned at one of several distances (D=10200mm) behind the sample (Fig. 1).

In uncorrected reconstructed slices of the water-immersed dentin [Fig. 2a, D=100mm, x-ray energy E=22keV] white streaks affecting large regions in the 3D reconstructed image are seen, caused by stray intensities induced by bubbles [inset Fig. 2b]. Improved reconstructions [Fig. 2c] were produced from time-median images [Fig. 2d], created from the median values of matching radiograph points from three sequential sets and recorded at identical sample projection angles (Δt=20min). Artifacts [Fig. 3a ] are significantly reduced in the improved reconstruction [Fig. 3b], revealing the outlines of the tubules and variations in the density and 3D orientations. In particular, differences in δ due to variations in thickness and projected electron density ρel of the 0.54μm mineral sheaths surrounding the tubules are seen (note that δλ2ρel [4

4. A. Guinier, X-ray diffraction in crystals, imperfect crystals and amorphous bodies (Freeman & Co., 1963).

]). With increased propagation distance [D=180mm, inset Fig. 3c] fringes around the tubules appear broader, yet the tubule center-to-center distances remain unchanged [8

8. S. Zabler, P. Zaslansky, H. Riesemeier, and P. Fratzl, Opt. Express 14, 8584 (2006). [CrossRef] [PubMed]

]. To the best of our knowledge, no other form of nondestructive (whole tissue) microscopy reveals such 3D structural detail in similar biomaterials.

We conclude that most time-dependent stray intensity contributions can be removed from projection images of water-immersed biomineralized samples. Stray contributions, attributed mainly to the formation of gas bubbles degrade the visibility of micrometer-sized features that characterize skeletal tissues. During such experiments, wet samples do not undergo dehydration, and therefore it is possible to acquire large numbers of radiographs repeatedly. When additionally small deliberate shifts to the sample stage are induced, artifacts caused by detector components are reduced, leading to additional improvement in the visibility of features in the microstructure. In samples of water-immersed dentin, improved visibility of interference fringes resulted in detailed representation of the 0.51μm sized tubules and peritubular sheaths in the tomograms. Additional structural insights may arise by exchanging water for other liquids (ethanol, saltwater) such that diffusion effects may be tracked. It is thus believed that this form of nondestructive liquid-immersed 3D microscopy has a wide application, far beyond tooth structure imaging.

The authors thank John Currey and Peter Fratzl for discussions and support.

Fig. 1 Component setup for Fresnel-propagated microtomography: images of the liquid immersed sample are acquired at several detector positions.
Fig. 2 Projection radiographs and tomographic slices of wet dentin samples: (a) Reconstructed slice of Fresnel-propagated radiographs: note white streaks, which are due to stray intensity contributions originating from bubbles in the water. (b) Radiograph of water-immersed dentin (dense enamel on upper area appears dark) contains high-contrast bubble silhouettes. (c) Slice in a 3D time-median reconstruction with fewer stray intensities. Structural detail is observed (see Fig. 3). (d) Typical time-median radiograph.
Fig. 3 Microstructural detail in dentin (a) high- and low-density regions of mineral in the dentin are obscured by image noise in slices of tomograms produced from conventional radiographs. The combination of ring artifacts and stray intensity contributions makes visualization and analysis difficult. (b) Improved reconstructed images, produced from three sequential scans, result in high-quality virtual microscopy images of interference rings corresponding to wet 1μm thick tubules (D=100mm). Images obtained for larger propagation distances (c) (D=180mm) show enlarged tubules, yet the center-to-center distances (810μm) remain constant.
1.

O. Prymak, H. Tiemann, I. Soetje, J. C. Marxen, A. Klocke, B. Kahl-Nieke, F. Beckmann, T. Donath, and M. Epple, JBIC, J. Biol. Inorg. Chem. 10, 688 (2005). [CrossRef]

2.

D. M. Paganin, Coherent X-Ray Optics (Oxford U. Press, 2006). [CrossRef]

3.

H. A. Lowenstam and S. Weiner, On Biomineralization (Oxford U. Press, 1989).

4.

A. Guinier, X-ray diffraction in crystals, imperfect crystals and amorphous bodies (Freeman & Co., 1963).

5.

J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1968).

6.

J.-P. Guigay, Optik (Stuttgart) 49, 121 (1977).

7.

P. Cloetens, R. Mache, M. Schlenker, and S. Lerbs-Mache, Proc. Natl. Acad. Sci. U.S.A. 103, 14626 (2006). [CrossRef] [PubMed]

8.

S. Zabler, P. Zaslansky, H. Riesemeier, and P. Fratzl, Opt. Express 14, 8584 (2006). [CrossRef] [PubMed]

9.

W. L. Mao, H. Mao, Y. Meng, P. J. Eng, M. Y. Hu, P. Chow, Y. Q. Cai, J. Shu, and R. J. Hemley, Science 314, 636 (2006). [CrossRef] [PubMed]

10.

S. Zabler, P. Cloetens, J.-P. Guigay, J. Baruchel, and M. Schlenker, Rev. Sci. Instrum. 76, 073705 (2005). [CrossRef]

11.

J. Radon, Ber. Verh. Saechs. Akad. Wiss. Leipzig, Math.-Phys. Kl. 69, 262 (1917).

12.

P. Cloetens, M. Pateyron-Salome, J. Y. Buffiere, G. Peix, J. Baruchel, F. Peyrin, and M. Schlenker, J. Appl. Phys. 81, 5878 (1997). [CrossRef]

13.

T. J. Davis, T. E. Gureyev, D. Gao, A. W. Stevenson, and S. W. Wilkins, Phys. Rev. Lett. 74, 3173 (1995). [CrossRef] [PubMed]

OCIS Codes
(110.7440) Imaging systems : X-ray imaging
(170.1850) Medical optics and biotechnology : Dentistry
(170.3660) Medical optics and biotechnology : Light propagation in tissues
(170.6960) Medical optics and biotechnology : Tomography
(340.6720) X-ray optics : Synchrotron radiation

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: June 25, 2007
Revised Manuscript: August 16, 2007
Manuscript Accepted: August 29, 2007
Published: October 8, 2007

Virtual Issues
Vol. 2, Iss. 11 Virtual Journal for Biomedical Optics

Citation
Simon Zabler, Peter Cloetens, and Paul Zaslansky, "Fresnel-propagated submicrometer x-ray imaging of water-immersed tooth dentin," Opt. Lett. 32, 2987-2989 (2007)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=ol-32-20-2987


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References

  1. O. Prymak, H. Tiemann, I. Soetje, J. C. Marxen, A. Klocke, B. Kahl-Nieke, F. Beckmann, T. Donath, and M. Epple, JBIC, J. Biol. Inorg. Chem. 10, 688 (2005). [CrossRef]
  2. D. M. Paganin, Coherent X-Ray Optics (Oxford U. Press, 2006). [CrossRef]
  3. H. A. Lowenstam and S. Weiner, On Biomineralization (Oxford U. Press, 1989).
  4. A. Guinier, X-ray diffraction in crystals, imperfect crystals and amorphous bodies (Freeman & Co., 1963).
  5. J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1968).
  6. J.-P. Guigay, Optik (Stuttgart) 49, 121 (1977).
  7. P. Cloetens, R. Mache, M. Schlenker, and S. Lerbs-Mache, Proc. Natl. Acad. Sci. U.S.A. 103, 14626 (2006). [CrossRef] [PubMed]
  8. S. Zabler, P. Zaslansky, H. Riesemeier, and P. Fratzl, Opt. Express 14, 8584 (2006). [CrossRef] [PubMed]
  9. W. L. Mao, H. Mao, Y. Meng, P. J. Eng, M. Y. Hu, P. Chow, Y. Q. Cai, J. Shu, and R. J. Hemley, Science 314, 636 (2006). [CrossRef] [PubMed]
  10. S. Zabler, P. Cloetens, J.-P. Guigay, J. Baruchel, and M. Schlenker, Rev. Sci. Instrum. 76, 073705 (2005). [CrossRef]
  11. J. Radon, Ber. Verh. Saechs. Akad. Wiss. Leipzig, Math.-Phys. Kl. 69, 262 (1917).
  12. P. Cloetens, M. Pateyron-Salome, J. Y. Buffiere, G. Peix, J. Baruchel, F. Peyrin, and M. Schlenker, J. Appl. Phys. 81, 5878 (1997). [CrossRef]
  13. T. J. Davis, T. E. Gureyev, D. Gao, A. W. Stevenson, and S. W. Wilkins, Phys. Rev. Lett. 74, 3173 (1995). [CrossRef] [PubMed]

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