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Abstract
A soft X-ray ptychography system using a Wolter mirror for the illumination optics has been developed. By taking advantage of the achromaticity of the optics, the system is capable of seamlessly imaging at half-period resolution of 50 nm with a broad photon-energy range from 250 eV to 2 keV while maintaining the focal position. Imaging a mammalian cell at various wavelengths was demonstrated, and high-resolution visualization of organelle was achieved. Stereo imaging was also performed with a long working distance of 20 mm. In combination with in-situ/operando and tomographic measurements, this system will be a powerful tool for observing biological and material targets with complex features.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
X-ray microscopy has a wide range of applications in biology and materials science due to its high spatial resolution and analytical techniques. Ptychography, a new X-ray microscopy technique, provides an exceptionally high spatial resolution by reconstructing the sample image using phase recovery calculations from coherent diffraction patterns [1–5].
The combination of ptychography and spectroscopy in the soft X-ray region is promising for investigating samples with light-element-rich heterogeneous structures. For example, using a combination of soft X-ray ptychography and X-ray absorption spectroscopy, mapping the chemical state of magnetic nanoparticles in bacteria [6] and visualization of the cathode degradation mechanism in lithium-ion batteries [7] have been demonstrated.
In conventional ptychography setup, focusing optics are widely used for making an illumination probe [4]. Adequately focused beam for ptychography can improve imaging performance such as field of view and measurement time [8]. For ptychography at multiple X-ray wavelengths, total-reflection mirror optics offer the optimal character of achromaticity and high focusing efficiency. Presently, the achievable focusing size of total-reflection mirrors in soft X-rays is a few hundred nanometers [9,10], much larger than that of zone plates [11,12]. However, the spatial resolution obtained in ptychography is independent of the focusing beam size, which makes total-reflection mirrors a promising candidate. In addition, the long working distance of the mirror optics is suitable for applications in tomographic and in-situ/operando measurements.
In this study, we developed a new total-reflection Wolter mirror optics for soft X-ray ptychography and demonstrated that it could be used to measure cellular samples. The total-reflection Wolter mirror [13–15] was fabricated by modifying our previous precision electroforming process [16,17]. The reflecting surface of the Wolter mirror was covered with Au, which enables seamless ptychographic measurements in a wide soft X-ray energy range from 250 eV to 2 keV. The soft X-ray ptychographic system with a total-reflection Wolter mirror could achieve a half-period resolution of approximately 50 nm in a test chart evaluation. We also showed that it is possible to visualize the microstructure of a mammalian cell without chromatic aberration. Furthermore, taking advantage of the long working distance of the optical system, we attempted stereo imaging with a large rotation angle of the cell sample.
2. Soft X-ray ptychography system with Wolter mirror
The system was constructed at the soft X-ray undulator beamline BL07LSU of SPring-8 [18,19]. The beamline provides a wide photon energy range of soft X-rays from 250 eV to 2 keV with energy resolution higher than 10,000. In X-ray absorption spectroscopy, the range covers the K-edges of light elements from carbon to silicon.
Figure 1(a) shows a schematic illustration of the soft X-ray ptychography system developed in this study. As an illumination system, a total-reflection Wolter mirror with achromaticity and long working distance was employed. Wolter mirrors are ideal illumination optics because of their high imaging property and probe stability. In the hard X-ray region, Wolter mirrors (or advanced Kirkpatrick-Baez mirrors) for microscopic application are constructed by separating the optics into two or more almost flat mirrors [20,21] because of the difficulty in fabricating the cylindrical shape which was originally suggested by H. Wolter in 1952 [13]. However, advanced Kirkpatrick-Baez mirrors do not have sufficient numerical aperture for applications in soft X-rays with longer wavelengths. The complicated alignment of multiple mirrors is also unsuitable for soft X-ray experiments in ultra-high vacuum. Therefore, we applied the cylindrical Wolter mirror for the soft X-ray ptychography system.
Fig. 1. Soft X-ray ptychography system using a Wolter mirror. (a) Schematic illustration of the system. (b) Reflectivity curve of the mirror optics. The dotted line and circles show the theoretical and experimental reflectivity, respectively. (c) Photograph of the Wolter mirror chamber.
The shape of the Wolter mirror is determined by five design variables: working distance, the distance between the light source and the focal point, mirror length, and grazing angles for the ellipsoidal and hyperboloidal parts of the Wolter mirror. A working distance of 20 mm was chosen to allow for tomography and operando measurements. The distance between the light source and the focal point was set to 13.8 m, considering the configuration of the experimental stations of BL07LSU. The grazing angles of the mirror surface were set to about 22.1 mrad (ellipsoidal part) and 42.5 mrad (hyperboloidal part) to obtain a reflectivity of more than 30% in the soft X-ray region below 1000 eV using a mirror with Au as a reflecting surface. The mirror length was set to 200 mm due to the limitations of our current fabrication process. Finally, we designed and fabricated the Wolter mirror with the parameters shown in Table 1.
Table 1. Optical parameters of the Wolter mirror.
We fabricated the Wolter mirror using a precision electroforming process [16,17]. As a reflective layer, 50-nm-thick Au was formed on a 2-mm-thick Cu base material. The Au reflective layer of the mirror was made as an initial electrode surface in the electroforming process. A thin Cr layer was used as an adhesive layer between the Cu and Au layers and prevented them from diffusing and alloying. Figure 1(b) shows the reflectance curve of the mirror. The reflectance curve is seamless since Au has no absorption edge between 250 eV and 2 keV. The measured reflectance was almost the same as that theoretically obtained, considering the grazing angles of the mirror.
In total-reflection mirror optics, which use the specular reflection of X-rays, the working distance and the focal point do not depend on the wavelength. Therefore, the sample position can always be kept the same at any wavelength. The measured focal spot size of the optics was 6.0 µm (horizontal) × 0.5 µm (vertical) at 400 eV. The sample was placed a few hundred micrometers downstream of the mirror’s focus. This defocused positioning increases the illumination area and widens the field of view. At the defocused position, the illumination area was larger than 10 µm in both horizontal and vertical directions.
Figure 1(c) shows a photograph of the system. Three linear stages and one rotation stage are located under the sample to enable sample scanning and tomographic measurement for ptychography. The distance from the sample to the soft X-ray charge-coupled device (CCD) (Princeton Instruments, PIXIS-XO:2048B) is 342.5 mm. The pixel size and sensor size of the CCD detector are 13.5 µm and 27.6 mm, respectively. The full angle subtended by the detector from the sample determines the pixel size of the reconstructed image, which corresponds to the highest half-period resolution. In this configuration, the pixel size of the reconstructed image ranges from 61.4 nm to 7.68 nm at photon energy from 250 eV to 2000 eV.
3. Soft X-ray ptychography results
3.1 Spatial resolution
We evaluated the spatial resolution of the system using a test pattern [2,11,12] fabricated by reactive ion etching of 70-nm-depth trenches on a 200-nm-thick silicon nitride thin film (Fig. 2(a)). Figure 2 shows soft X-ray ptychographic images of the measured test pattern. The line spacing at the innermost edge is 50 nm. Coherent diffraction patterns of 11 × 11 points were measured while scanning the sample with a step size of approximately 500 nm in two directions. The overlap between the adjacent scan positions was over 90% of the defocused X-ray probe. The total measuring time for a single ptychography procedure was approximately eight minutes, mainly due to the readout time of the X-ray CCD. The readout time of the X-ray CCD was almost three seconds per single image. The exposure time of the coherent diffraction pattern itself was 170 ms.
Fig. 2. Assessment of spatial resolution using a test chart. (a) Scanning electron microscope image of the test chart. The dotted rectangle area was observed with soft X-ray ptychography. (b)-(k) Reconstructed absorption (b),(d),(f),(h),(j) and phase (c),(e),(g),(i),(k) images at 400 eV, 800 eV, 1200 eV, 1600 eV, and 2000 eV.
Figures 2(b)-(k) show the absorption and phase images of ptychography results measured at X-ray photon energies from 400 eV to 2000 eV. The reconstructions were performed by the ePIE algorithm [3]. A half-period spatial resolution of about 50 nm was obtained at each energy. The inner 50-nm structure was resolved vertically but not horizontally in each image. Vibrations around the ptychography chamber were measured with an accelerometer and found to be a few hundred nanometers at maximum. The wavelength-independent resolution degradation was mainly due to this vibration.
We observed higher contrast in the phase images than in the absorption images at each energy. Since the real part of the complex refractive index is generally larger than the imaginary part in the X-ray region, phase imaging, which reflects the real part of the complex refractive index, provides more sensitive visualization than absorption imaging, which reflects the imaginary part. This phase imaging is a significant advantage of soft x-ray ptychography over standard scanning transmission X-ray microscopy when measuring high transmittance samples such as biological cells.
3.2 Mammalian cell measurement
To demonstrate the application of this system to microscopy, we measured a mammalian cell sample at multiple X-ray wavelengths. Chinese hamster ovarian cancer cells (CHO-K1) were plated on a 200-nm-thick silicon nitride thin film and fixed with 2% paraformaldehyde/phosphate buffer saline. Figure 3(a) shows a representative CHO-K1 cell image observed with visible light differential interference microscopy. The thickness of the adhered cells was approximately 5 µm, making it difficult to observe the inner microstructures of the cell with electron microscopy. In contrast, soft X-ray ptychography is useful for observing 5-µm -thick specimens in transmission without thinning.
Fig. 3. Soft X-ray ptychography measurement of CHO-K1 cell. (a) Visible light differential interference contrast image. (b)-(k) Reconstructed absorption (b),(d),(f),(h),(j) and phase (c),(e),(g),(i),(k) images at 250 eV, 350 eV, 450 eV, 600 eV, and 1200 eV. The magnified images to the right of the main images are taken from areas corresponding to the dotted rectangles shown in (b).
We measured the cell at photon energies of 250 eV, 350 eV, 450 eV, and 600 eV across the absorption edges of carbon, nitrogen, and oxygen. We also measured the cell at 1200 eV. The sample was kept at the same position in the optical axis direction in these measurements, thanks to the achromatic character of the illumination optics. Figures 3(b)-(k) show absorption and phase images of the reconstruction results at each energy.
We also performed stereo imaging to observe the intercellular structures of the 5-µm-thick cell body, taking advantage of the long working distance of the mirror optics. Figure 4 shows measurements taken by tilting the cell on the silicon nitride membrane by ±45°. By tilting the adhered cells, a raised structure was enhanced in the perinuclear region (see magnified images in Fig. 4). On the other hand, the raised structure in the prenuclear region in a visible light differential interference contrast image was not distinguished from other structures observed in the cytoplasm (Fig. 3(a)). Organelles such as mitochondria, endoplasmic reticulum, and Golgi are clustered in this region. These clusters were also observed in hard X-ray microscopy by mapping phospholipids of these organelle membranes and neutral lipids [22]. The system can be easily extended to soft X-ray tomography to identify these organelle structures in three dimensions.
Fig. 4. Stereo imaging of CHO-K1 cell using the soft X-ray ptychography system. The images were taken by tilting the sample holder by 45° (a), 0° (b), and -45° (c). The magnified images to the right are taken from areas corresponding to the dotted rectangles in the center image of (a). The focused x-ray probe entered the silicon nitride membrane at an incident angle of 7.3° in (b).
4. Conclusion and discussion
We developed a soft X-ray ptychography system with achromatic and long-working-distance Wolter mirror optics at BL07LSU of SPring-8. We confirmed the half-period spatial resolution around 50 nm with test patterns and observed a mammalian cell at various wavelengths across the absorption edges of light elements as a demonstration. The images showed that the intracellular structures in a 5-µm-thick cell body could be visualized at different angles.
We found that the system is highly stable without re-alignment of the mirrors over five days. The vertical half-period resolution was 50 nm at the lowest. The confirmed spatial resolution was limited by the finest structure of the test chart. The horizontal half-period resolution was worse than 50 nm. The chief reason for this is vibrations arising from the ambient environment, including vacuum pumps, which can be significantly suppressed. In this work, we only applied the simple ePIE algorithm for image reconstructions. The multimodal analysis [23] and position-correction algorithm [24] can also improve the spatial resolution.
In this paper, as a demonstration, we observed a CHO-K1 cell at various wavelengths across the absorption edge of light elements. It was shown that the intracellular structure could be observed in transmission for various elements. To understand the details of such cellular structures, it is essential to compare the results with those obtained by visible-light observations, which have already provided many findings. We focused our experiments on soft X-ray imaging, while the total-reflection optical system can also be used for simultaneous imaging with visible light. Since the Wolter mirror is an optical imaging element, it can be used as an objective lens in a visible-light microscope. For example, this Wolter mirror can be used as a visible-light objective lens with a magnification of 200× and a numerical aperture of 0.15. We can construct optical systems such as a fluorescence microscope or Raman microscope with visible-light illumination coaxial with X-rays.
In addition, the system can be used in combination with pump-probe experiments to observe photochemical reactions. The performance of the microscope system is suitable for detailed analysis in biology and materials science to determine complex compositions and three-dimensional structures.
Funding
Japan Society for the Promotion of Science (20H04451, 21K20394); Japan Science and Technology Agency (JPMJPR1772); University of Tokyo (Excellent Young Researcher program).
Acknowledgments
We wish to express our appreciation to Dr. Makina Yabashi of RIKEN for helping in improving the manuscript. This work was carried out as joint research in the Synchrotron Radiation Research Organization and The Institute for Solid State Physics, The University of Tokyo (Proposal Nos. 2021A7402, 2021B7402, and 2022A7402). This work was also supported by “Nanotechnology Platform Japan” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and fabrication was conducted in Takeda Cleanroom with the help of the Nanofabrication Platform Center of School of Engineering, the University of Tokyo, Japan.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
References
1. M. Guizar-Sicairos and J.R. Fienup, “Phase retrieval with transverse translation diversity: a nonlinear optimization approach,” Opt. Express 16(10), 7264–7278 (2008). [CrossRef]
2. P. Thibault, M. Dierolf, A. Menzel, O. Bunk, C. David, and F. Pfeiffer, “High-resolution scanning X-ray diffraction microscopy,” Science 321(5887), 379–382 (2008). [CrossRef]
3. A. M. Maiden and J. M. Rodenburg, “An improved ptychographical phase retrieval algorithm for diffractive imaging,” Ultramicroscopy 109(10), 1256–1262 (2009). [CrossRef]
4. F. Pfeiffer, “X-ray ptychography,” Nat. Photonics 12(1), 9–17 (2018). [CrossRef]
5. M. Hirose, N. Ishiguro, K. Shimomura, D. N. Nguyen, H. Matsui, H. C. Dam, M. Tada, and Y. Takahashi, “Oxygen-diffusion-driven oxidation behavior and tracking areas visualized by X-ray spectro-ptychography with unsupervised learning,” Commun. Chem. 2(1), 50 (2019). [CrossRef]
6. X. Zhu, A. P. Hitchcock, D. A. Bazylinski, P. Denes, J. Joseph, U. Lins, S. Marchesini, H. W. Shiu, T. Tyliszczak, and D. A. Shapiro, “Measuring spectroscopy and magnetism of extracted and intracellular magnetosomes using soft X-ray ptychography,” Proc. Natl. Acad. Sci. U. S. A. 113, E8219–E8227 (2016). [CrossRef]
7. T. Sun, G. Sun, F. Yu, Y. Mao, R. Tai, X. Zhang, G. Shao, Z. Wang, J. Wang, and J. Zhou, “Soft X-ray ptychography chemical imaging of degradation in a composite surface-reconstructed Li-rich cathode,” ACS Nano 15(1), 1475–1485 (2021). [CrossRef]
8. M. Odstrčil, M. Lebugle, M. Guizar-Sicairos, C. David, and M. Holler, “Towards optimized illumination for high-resolution ptychography,” Opt. Express 27(10), 14981–14997 (2019). [CrossRef]
9. Y. Takeo, H. Motoyama, T. Shimamura, T. Kimura, T. Kume, Y. Matsuzawa, T. Saito, Y. Imamura, H. Miyashita, K. Hiraguri, H. Hashizume, Y. Senba, H. Kishimoto, H. Ohashi, and H. Mimura, “A highly efficient nanofocusing system for soft X rays,” Appl. Phys. Lett. 117(15), 151104 (2020). [CrossRef]
10. H. Motoyama, S. Owada, G. Yamaguchi, T. Kume, S. Egawa, K. Tono, Y. Inubushi, T. Koyama, M. Yabashi, H. Ohashi, and H. Mimura, “Intense sub-micrometre focusing of soft X-ray free-electron laser beyond 1016 W cm-2 with an ellipsoidal mirror,” J. Synchrotron Radiat. 26(5), 1406–1411 (2019). [CrossRef]
11. W. Chao, J. Kim, S. Rekawa, P. Fischer, and E. H. Anderson, “Demonstration of 12 nm resolution Fresnel zone plate lens based soft X-ray microscopy,” Opt. Express 17(20), 17669 (2009). [CrossRef]
12. B. Rösner, S. Finizio, F. Koch, et al., “Soft X-ray microscopy with 7 nm resolution,” Optica 7(11), 1602 (2020). [CrossRef]
13. H. Wolter, “Spiegelsysteme streifenden Einfalls als abbildende Optiken für Röntgenstrahlen,” Ann. Phys. 445(1-2), 94–114 (1952). [CrossRef]
14. S. Matsuyama, T. Wakioka, N. Kidani, T. Kimura, H. Mimura, Y. Sano, Y. Nishino, M. Yabashi, K. Tamasaku, T. Ishikawa, and K. Yamauchi, “One-dimensional Wolter optics with a sub-50 nm spatial resolution,” Opt. Lett. 35(21), 3583 (2010). [CrossRef]
15. M. Hoshino and S. Aoki, “Laser plasma soft X-ray microscope with Wolter mirrors for observation of biological specimens in air,” Jpn. J. Appl. Phys. 45(2A), 989–994 (2006). [CrossRef]
16. T. Kume, Y. Takei, S. Egawa, H. Motoyama, Y. Takeo, G. Yamaguchi, and H. Mimura, “Development of electroforming process for soft X-ray ellipsoidal mirror,” Rev. Sci. Instrum. 90(2), 021718 (2019). [CrossRef]
17. G. Yamaguchi, H. Motoyama, S. Owada, Y. Kubota, S. Egawa, T. Kume, Y. Takeo, M. Yabashi, and H. Mimura, “Copper electroforming replication process for soft X-ray mirrors,” Rev. Sci. Instrum. 92(12), 123106 (2021). [CrossRef]
18. S. Yamamoto, Y. Senba, T. Tanaka, et al., “New soft X-ray beamline BL07LSU at SPring-8,” J. Synchrotron Radiat. 21(2), 352–365 (2014). [CrossRef]
19. J. Miyawaki, S. Yamamoto, Y. Hirata, M. Horio, Y. Harada, and I. Matsuda, “Fast and versatile polarization control of X-ray by segmented cross undulator at SPring-8,” AAPPS Bull. 31(1), 25 (2021). [CrossRef]
20. S. Matsuyama, N. Kidani, H. Mimura, Y. Sano, Y. Kohmura, K. Tamasaku, M. Yabashi, T. Ishikawa, and K. Yamauchi, “Hard-X-ray imaging optics based on four aspherical mirrors with 50 nm resolution,” Opt. Express 20(9), 10310–10319 (2012). [CrossRef]
21. S. Matsuyama, Y. Emi, H. Kino, Y. Kohmura, M. Yabashi, T. Ishikawa, and K. Yamauchi, “Achromatic and high-resolution full-field X-ray microscopy based on total-reflection mirrors,” Opt. Express 23(8), 9746–9752 (2015). [CrossRef]
22. M. Shimura, H. Shindou, L. Szyrwiel, S. M. Tokuoka, F. Hamano, S. Matsuyama, M. Okamoto, A. Matsunaga, Y. Kita, Y. Ishizaka, K. Yamauchi, Y. Kohmura, R. Lobinski, I. Shimizu, and T. Shimizu, “Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy,” FASEB J. 30(12), 4149–4158 (2016). [CrossRef]
23. D. J. Batey, D. Claus, and J. M. Rodenburg, “Information multiplexing in ptychography,” Ultramicroscopy 138, 13–21 (2014). [CrossRef]
24. A.M. Maiden, M.J. Humphry, M.C. Sarahan, B. Kraus, and J.M. Rodenburg, “An annealing algorithm to correct positioning errors in ptychography,” Ultramicroscopy 120, 64–72 (2012). [CrossRef]
