OSA's Digital Library

Optics Letters

Optics Letters

| RAPID, SHORT PUBLICATIONS ON THE LATEST IN OPTICAL DISCOVERIES

  • Vol. 36, Iss. 7 — Apr. 1, 2011
  • pp: 1269–1271
« Show journal navigation

Hard x-ray Zernike microscopy reaches 30 nm resolution

Yu-Tung Chen, Tsung-Yu Chen, Jaemock Yi, Yong S. Chu, Wah-Keat Lee, Cheng-Liang Wang, Ivan M. Kempson, Y. Hwu, Vincent Gajdosik, and G. Margaritondo  »View Author Affiliations


Optics Letters, Vol. 36, Issue 7, pp. 1269-1271 (2011)
http://dx.doi.org/10.1364/OL.36.001269


View Full Text Article

Acrobat PDF (460 KB)





Browse Journals / Lookup Meetings

Browse by Journal and Year


   


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools

Share
Citations

Abstract

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.

© 2011 Optical Society of America

Conventional x-ray microscopy is negatively affected by weak absorption contrast. Zernike solved the corresponding problem in the visible by introducing a quarter-wavelength shift in the relative phase of specimen-scattered and unscattered waves [1

1. F. Zernike, Science 121, 345 (1955). [CrossRef] [PubMed]

]. This enhances the contrast by small phase differences between waves scattered by different specimen areas.

Recently, absorption microscopy at 8keV reached a 30nm resolution [11

11. Y.-T. Chen, T.-N. Lo, Y. S. Chu, J. Yi, C.-J. Liu, J.-Y. Wang, C.-L. Wang, C.-W. Chiu, T.-E. Hua, Y. Hwu, Q. Shen, G.-C. Yin, K. S. Liang, H.-M. Lin, J. H. Je, and G. Margaritondo, Nanotechnology 19, 395302 (2008). [CrossRef] [PubMed]

], a milestone for biomedical imaging of thick specimens. Could Zernike phase contrast reach this milestone? We present here positive evidence: Fig. 1 shows one of the supporting tests. Figures 1a, 1b are micrographs of a 180nm thick Au star nanopattern without and with Zernike phase shift. The contrast enhancement of Fig. 1b is evident; intensity profiles [e.g., Fig. 1c] quantitatively demonstrate a contrast increase by >3.

As to spatial resolution, the power spectrum analysis (PSA) of Fig. 1d (discussed below) shows similar resolution in Figs. 1b, 1a: 29 and 31nm. Thus, the Zernike contrast enhancement is not at the expense of lateral resolution.

These results were obtained with a newly developed x-ray transmission microscope facility (described in [11

11. Y.-T. Chen, T.-N. Lo, Y. S. Chu, J. Yi, C.-J. Liu, J.-Y. Wang, C.-L. Wang, C.-W. Chiu, T.-E. Hua, Y. Hwu, Q. Shen, G.-C. Yin, K. S. Liang, H.-M. Lin, J. H. Je, and G. Margaritondo, Nanotechnology 19, 395302 (2008). [CrossRef] [PubMed]

]) at the Advanced Photon Source (APS), Argonne National Laboratory. Figure 2a shows its optical layout; the main components are a capillary condenser to focus the x rays on the sample, a pinhole to eliminate unwanted illumination, a precision sample stage, a Fresnel zone plate (FZP) objective, the detector (a thin CsI scintillator and a CCD plate) and a Zernike phase ring [Fig. 2b]. The system is connected to the 32-ID undulator beamline, with a double-bounce Si(111) crystal monochromator.

The facility is equipped with interchangeable objectives acting as phase FZPs, fabricated by the Academia Sinica (Taipei), with a combination of electron-beam writing and electrodeposition [11

11. Y.-T. Chen, T.-N. Lo, Y. S. Chu, J. Yi, C.-J. Liu, J.-Y. Wang, C.-L. Wang, C.-W. Chiu, T.-E. Hua, Y. Hwu, Q. Shen, G.-C. Yin, K. S. Liang, H.-M. Lin, J. H. Je, and G. Margaritondo, Nanotechnology 19, 395302 (2008). [CrossRef] [PubMed]

]. The outer zone width is either 30 or 45nm, and the absorber is 450 or 700nm thick Au; different FZP diameters provide optimal focusing for 812keV photon energies. The space frequency bandwidth reaches 35μm1. Depending on the Au thickness, the FZP efficiency is 1% or 3%, <5% of the theoretical values, a loss attributed to slight pattern imperfections and reduced Au density yielded by electrodeposition. The detector pixel resolution is 5nm.

The Zernike phase ring, located [Fig. 2b] at the condenser conjugate plane (near the back focal plane of the objective zone plate), is made of electroplated Au on a silicon nitride membrane. Its thickness is selected to phase shift the incident radiation beam by 3π/2 for the following reason. For weak scattering, the phase transmittance function is exp(iΩ)1+iΩ and its magnitude is 1. A 3π/2 phase shift of the incident beam changes the phase factor to i+iΩ, magnitude 12Ω, making the object image darker and more visible. This is better than a π/2 shift that would give a phase factor i+iΩ, magnitude 1+2Ω and a brighter object image. In fact, except for a pure phase object, x-ray absorption is also present and works against the effects of the π/2 shift but enhances those of the 3π/2 shift.

Alignment is critical: a “Bertrand lens” [12

12. D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley, 2001).

] (a limited-resolution FZP) is used to position the phase ring with respect to the unscattered annular illumination of the sample. This lens images at the detector the illumination at the condenser conjugate plane. All condensers are optimized for phase contrast. This means that the annular illumination is narrow: the illuminated area is 10% of the FZP area; thus most of the scattered x rays are unaffected by the phase ring and can produce phase contrast.

We assessed these effects by PSA of the images after pixel-by-pixel multiplication by a Hanning window function to avoid edge effects. The two-dimensional Fourier transformation of the image was squared and azimuthally integrated over 2π. The resulting PSA curves show that frequency cutoff is worse for the nonoptimal thicknesses. PSA also revealed aberrations effects caused by the ring diameter. On the other hand, we could improve the resolution by fine tuning the photon energy in 50eV steps: the optimum value was 7.95keV.

Figure 3 shows results for nearly phase objects, polystyrene particles [(C8H8)n], at 8keV, where the refractive index is n=1δiβ with δ=3.88×106 and β=5.58×109. The absorption images, Figs. 3a, 3b, 3c, in focus or slightly out of focus, only show edge-enhanced contrast, whereas the Zernike image [Fig. 3d] reveals contrast enhancement by a factor 2.

Figure 4 shows the importance of <30nm resolution for biology specimens. An absorption image of an EMT (transplantable murine mammary carcinoma) cell is shown together with two phase contrast images obtained with Zernike rings with the same thickness and diameter and different widths. The width-optimized ring (4.5μm) gives the best image, Fig. 4c, whereas the others are more blurred. PSA [Fig. 4d] corroborates this conclusion. Such effects are even stronger in Fig. 5, showing an EMT cell cocultured with Au nanoparticles.

These issues notwithstanding, a 30nm resolution is a remarkable progress for hard x-ray microscopy (for reference see, e.g., [9

9. M. Stampanoni, R. Mokso, F. Marone, J. Vila-Comamala, S. Gorelik, P. Trtik, K. Jefimovs, and C. David, Phys. Rev. B 81, 14105 (2010). [CrossRef]

] and [10

10. H. S. Youn and S.-W. Jung, J. Microsc. 223, 53 (2006). [CrossRef] [PubMed]

]), and Zernike contrast at this resolution opens opportunities not only in biology, medicine, and bioengineering but also in materials science and other areas.

Research was supported by National Science and Technology for Nanoscience and Nanotechnology, the Thematic Project of Academia Sinica (Taiwan), the Fonds National Suisse, and the Center for Biomedical Imaging (CIBM). We used equipment of the Academia Sinica Core Facility for Nanoscience and Nanotechnology and Biomedical NanoImaging. The APS is supported by the United States Department of Energy (DOE) under contract DE-AC02-06CH11357.

Fig. 1 (a), (b) Micrographs of a 180nm thick Au star test pattern obtained with absorption and Zernike phase contrast. (c) Intensity profiles along the red lines in (a) and (b). (d) Power spectra assessing the spatial resolution.
Fig. 2 (a) Layout of the transmission x-ray microscope (TXM). (b) Geometry for the Zernike phase contrast.
Fig. 3 Image of polystyrene particles: (a)–(c) absorption images, (b) in focus and (a), (c) 20μm before or after focus. (d) Zernike phase contrast image with a 100nm particle marked by the arrow.
Fig. 4 Images of an EMT cell by (a) absorption and Zernike contrast by (c) an optimized ring or (b) a slightly narrower ring (the inset shows the illumination leaking). (d) PSA confirms that the optimized ring yields the best results.
Fig. 5 Images of an EMT cell cocultured with Au nano particles, taken (a) without or (b) with a Zernike ring.
1.

F. Zernike, Science 121, 345 (1955). [CrossRef] [PubMed]

2.

D. Rudolph, G. Schmahl, and B. Niemann, in Modern Microscopies, Techniques and Applications, A. Michette and P. Duke, eds. (Plenum, 1990), p. 59–67. [CrossRef]

3.

G. Schmahl, D. Rudolph, P. Guttmann, G. Schneider, J. Thieme, and B. Niemann, Rev. Sci. Instrum. 66, 1282 (1995). [CrossRef]

4.

G. Schneider, Ultramicroscopy 75, 85 (1998). [CrossRef] [PubMed]

5.

U. Neuhäusler, G. Schneider, W. Ludwig, M. A. Meyer, E. Zschech, and D. Hambach, J. Phys. D 36, A79 (2003). [CrossRef]

6.

H. Yokosuka, N. Watanabe, T. Ohigashi, Y. Yoshida, S. Maeda, S. Aoki, Y. Suzuki, A. Takeuchi, and H. Takano, J. Synchrotron Radiat. 9, 179 (2002). [CrossRef] [PubMed]

7.

A. Tkachuk, F. Duewer, H. Cui, M. Feser, S. Wang, and W. Yun, Z. Kristallogr. 222, 650 (2007). [CrossRef]

8.

Y. S. Chu, J. M. Yi, F. De Carlo, Q. Shen, W.-K. Lee, H. J. Wu, C. L. Wang, J. Y. Wang, C. J. Liu, C. H. Wang, S. R. Wu, C. C. Chien, Y. Hwu, A. Tkachuk, W. Yun, M. Feser, K. S. Liang, C. S. Yang, J. H. Je, and G. Margaritondo, Appl. Phys. Lett. 92, 103119 (2008). [CrossRef]

9.

M. Stampanoni, R. Mokso, F. Marone, J. Vila-Comamala, S. Gorelik, P. Trtik, K. Jefimovs, and C. David, Phys. Rev. B 81, 14105 (2010). [CrossRef]

10.

H. S. Youn and S.-W. Jung, J. Microsc. 223, 53 (2006). [CrossRef] [PubMed]

11.

Y.-T. Chen, T.-N. Lo, Y. S. Chu, J. Yi, C.-J. Liu, J.-Y. Wang, C.-L. Wang, C.-W. Chiu, T.-E. Hua, Y. Hwu, Q. Shen, G.-C. Yin, K. S. Liang, H.-M. Lin, J. H. Je, and G. Margaritondo, Nanotechnology 19, 395302 (2008). [CrossRef] [PubMed]

12.

D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley, 2001).

13.

A. G. Michette, Optical Systems for Soft X-rays (Plenum Press, 1986). [CrossRef]

14.

D. L. White, O. R. Wood, J. E. Bjorkholm, S. Spector, A. A. MacDowell, and B. LaFontaine, Rev. Sci. Instrum. 66, 1930 (1995). [CrossRef]

15.

A. H. Bennet, H. Jupnik, H. Osterberder, and O. W. Richards, Phase Microscopy (Wiley, 1951).

OCIS Codes
(340.0340) X-ray optics : X-ray optics
(340.7460) X-ray optics : X-ray microscopy

ToC Category:
Microscopy

History
Original Manuscript: November 29, 2010
Revised Manuscript: February 10, 2011
Manuscript Accepted: March 1, 2011
Published: March 30, 2011

Virtual Issues
Vol. 6, Iss. 5 Virtual Journal for Biomedical Optics

Citation
Yu-Tung Chen, Tsung-Yu Chen, Jaemock Yi, Yong S. Chu, Wah-Keat Lee, Cheng-Liang Wang, Ivan M. Kempson, Y. Hwu, Vincent Gajdosik, and G. Margaritondo, "Hard x-ray Zernike microscopy reaches 30 nm resolution," Opt. Lett. 36, 1269-1271 (2011)
http://www.opticsinfobase.org/ol/abstract.cfm?URI=ol-36-7-1269


Sort:  Author  |  Year  |  Journal  |  Reset  

References

  1. F. Zernike, Science 121, 345 (1955). [CrossRef] [PubMed]
  2. D. Rudolph and G. Schmahl, B. Niemann, in Modern Microscopies, Techniques and Applications, A.Michette and P.Duke, eds. (Plenum, 1990), p. 59–67. [CrossRef]
  3. G. Schmahl, D. Rudolph, P. Guttmann, G. Schneider, J. Thieme, and B. Niemann, Rev. Sci. Instrum. 66, 1282 (1995). [CrossRef]
  4. G. Schneider, Ultramicroscopy 75, 85 (1998). [CrossRef] [PubMed]
  5. U. Neuhäusler, G. Schneider, W. Ludwig, M. A. Meyer, E. Zschech, and D. Hambach, J. Phys. D 36, A79 (2003). [CrossRef]
  6. H. Yokosuka, N. Watanabe, T. Ohigashi, Y. Yoshida, S. Maeda, S. Aoki, Y. Suzuki, A. Takeuchi, and H. Takano, J. Synchrotron Radiat. 9, 179 (2002). [CrossRef] [PubMed]
  7. A. Tkachuk, F. Duewer, H. Cui, M. Feser, S. Wang, and W. Yun, Z. Kristallogr. 222, 650 (2007). [CrossRef]
  8. Y. S. Chu, J. M. Yi, F. De Carlo, Q. Shen, W.-K. Lee, H. J. Wu, C. L. Wang, J. Y. Wang, C. J. Liu, C. H. Wang, S. R. Wu, C. C. Chien, Y. Hwu, A. Tkachuk, W. Yun, M. Feser, K. S. Liang, C. S. Yang, J. H. Je, and G. Margaritondo, Appl. Phys. Lett. 92, 103119 (2008). [CrossRef]
  9. M. Stampanoni, R. Mokso, F. Marone, J. Vila-Comamala, S. Gorelik, P. Trtik, K. Jefimovs, and C. David, Phys. Rev. B 81, 14105 (2010). [CrossRef]
  10. H. S. Youn and S.-W. Jung, J. Microsc. 223, 53 (2006). [CrossRef] [PubMed]
  11. Y.-T. Chen, T.-N. Lo, Y. S. Chu, J. Yi, C.-J. Liu, J.-Y. Wang, C.-L. Wang, C.-W. Chiu, T.-E. Hua, Y. Hwu, Q. Shen, G.-C. Yin, K. S. Liang, H.-M. Lin, J. H. Je, and G. Margaritondo, Nanotechnology 19, 395302 (2008). [CrossRef] [PubMed]
  12. D. B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley, 2001).
  13. A. G. Michette, Optical Systems for Soft X-rays (Plenum Press, 1986). [CrossRef]
  14. D. L. White, O. R. Wood, II, J. E. Bjorkholm, S. Spector, A. A. MacDowell, and B. LaFontaine, Rev. Sci. Instrum. 66, 1930 (1995). [CrossRef]
  15. A. H. Bennet, H. Jupnik, H. Osterberder, and O. W. Richards, Phase Microscopy (Wiley, 1951).

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

Figures

Fig. 1 Fig. 2 Fig. 3
 
Fig. 4 Fig. 5
 

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited