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Vacuum-ultraviolet Gabor holography with synchrotron radiation
ARTICLE IN PRESS
Ultramicroscopy 107 (2007) 1171–1177 www.elsevier.com/locate/ultramic
Vacuum-ultraviolet Gabor holography with synchrotron radiation A. Rosenhahna,, R. Bartha, X. Caoa, M. Schu¨rmanna, M. Grunzea, S. Eisebittb a
Angewandte Physikalische Chemie, Universita¨t Heidelberg, INF 253, 69120 Heidelberg, Germany b BESSY m.b.H., Albert-Einstein-Str. 15, 12489 Berlin, Germany
Received 16 October 2006; received in revised form 12 January 2007; accepted 23 January 2007
Abstract We present the realization of high-resolution holographic microscopy using the original Gabor geometry and imaging with radiation in the vacuum-ultraviolet (VUV) spectral region. Synchrotron VUV radiation with a wavelength of 13.8 nm was focused on a small pinhole generating a highly divergent light cone suitable for digital in-line holography. Objects of different thickness and materials have been used to test the imaging properties of holographic microscopy in the VUV wavelength range. The effective numerical aperture was limited by the illuminated area of the detector, yielding a theoretical resolution below 1 mm and an experimental one of approximately 1 mm. r 2007 Elsevier B.V. All rights reserved. Keywords: Gabor; Holography; Digital in-line holography; Holographic microscopy; Synchrotron radiation; VUV radiation; Reconstruction; Kirchhoff–Helmholtz; Coherent scattering; Polystyrene; Iron oxide; Fibroblast
1. Introduction Since its invention by Gabor in the year 1948 [1], holography has been proven to be a powerful and inspiring technique. By now, a variety of imaging and measurement techniques are based on this coherent scattering concept, ranging from everyday applications like document protection or encryption to new measurement devices that help to solve scientific questions [2]. In Gabor’s original idea of a holographic microscope [1], a coherent wave is scattered from an object and interferes with the unscattered wave that passes the object undisturbed (the ‘‘reference wave’’). As sketched in Fig. 1, the result is an interference pattern known as hologram. The diverging wave creates a projection of the sample, thus magnifying the object and working as a coherent microscope. From such a hologram, real space information can either optically or digitally be retrieved (‘‘reconstruction’’) [3,4]. As both amplitude and phase are recorded up to a maximum angle given by the numerical aperture of the detection system, three-dimensional information is present in a hologram. Therefore, not only a single Corresponding author. Tel.: +49 6221 545065; fax: +49 6221 545060.
E-mail address: [email protected] (A. Rosenhahn). 0304-3991/$ - see front matter r 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2007.01.010
real space plane but also a whole stack of image planes can be reconstructed from a single scattering pattern. Holography was first conceived by Gabor for electron waves and this has by now been realized [5–7]. Apart from this, neutrons [8,9] or gamma radiation [10] was also used to obtain holographic images. Photon based holography is especially suited for the liquid environment due to high penetration depths and the high phase contrast caused by biological objects. In the visible range, the three-dimensional imaging of the marine organisms Ditylum brightwellii, the head of a fruit fly [3,4] and of human epithelial cells [11] are striking examples of the potential of digital in-line holography. Especially with the latest developments in immersion holography [12], it is clear that holography is rapidly approaching the performance of modern, state-of-the-art optical microscopes. As the resolution of a holographic microscope is determined by the wavelength of the photons and the numerical aperture of the detection system, it is straightforward to consider an increase of the photon energy in order to enhance the performance of the technique. As demonstrated by X-ray fluorescence holography, the short wavelength of X-rays allows to determine atomic positions in ordered solids with angstrom accuracy [13–16]. We pursue a decrease of the wavelength for digital in-line
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A. Rosenhahn et al. / Ultramicroscopy 107 (2007) 1171–1177
Fig. 1. Schematic geometry of a digital in-line holography experiment. The hologram is formed by interference of the undisturbed primary wave front and the scattered, secondary wave front.
holography into the vacuum-ultraviolet (VUV) range. The general possibility to use VUV radiation to produce in-line holograms was earlier demonstrated with a selenium X-ray laser at a wavelength of 21 nm with an obtained resolution of 5 mm [17]. Recently, in higher harmonic generation experiments coherent photons with a wavelength of 30 nm were generated and coherent scattering experiments were conducted [18]. Exploiting the natural, small divergence of the VUV beam thus resulted in a magnification of 3.8 at a resolution of 6.8 mm with suggestions for improving this to 500 nm [18]. Soft X-rays allow for much higher resolution holographic images, especially in the water window which is frequently used in X-ray microscopy of biological specimen [19,20]. With a wavelength of 1.89 nm, fibroblast fibers were imaged with a resolution of 40–50 nm using a photo resist as recording medium and thus overcoming detector resolution limitations at these short wavelengths [21]. Hard X-rays have been exploited to image holographically [22,23] and via coherent scattering and iterative phase retrieval [24–26], but without the availability of an X-ray laser, experiments with shorter wavelengths generally suffer from a dramatic decrease of coherent photon flux obtainable from noncoherent sources. The number of photons extractable from an incoherent source into a coherence volume scales as 1/l3. At short wavelengths, Fourier transform setups are advantageous in order to achieve high spatial resolution [27,28]. Here, we will focus exclusively on VUV radiation, also known as extreme ultraviolet (EUV). As 13.4 nm wavelength radiation is used in the next generation lithography for microelectronics, new lab sources may become available [29] for holographic imaging in this spectral range. 2. Experiment and samples All experiments have been carried out at the synchrotron light source BESSY II in Berlin at the undulator beamline
UE52-SGM. The focus of the beamline is 18 18 mm2 fullwidth at half-maximum (FWHM), enabling a high flux per area, required for the experiments. The photon energy was set to 90 eV, corresponding to a wavelength of 13.8 nm, which is used in all the results presented here. The pinhole–detector distance of 1000 mm was dictated by the available experimental setup in the ultra high vacuum chamber. The CCD camera used in the experiments was a Roper Scientific PI-SX-2048, using the back-illuminated Marconi CCD42-40 chip with 2048 2048 pixels and a pixel size of 13.5 13.5 mm2. For the given photon energy, the quantum efficiency of the CCD chip is about 45%. The pinholes used in the experiments were purchased from National Aperture, Inc. with a nominal size of 1 mm. A quantitative analysis of the Airy pattern obtained revealed a real diameter of 1.4 mm. The pinhole size is large compared to the wavelength of the photons used in the experiment. In an ideal holography experiment, one would use smaller pinholes, but we are limited by the incident coherent photon flux. The chosen pinhole represents a good compromise between hologram intensity and spatial resolution. The effective numerical aperture of the setup is not limited by the acceptance angle of the CCD chip (NA ¼ 0.014) but by the radius of the first Airy minimum at a value of NA ¼ 0.012. The theoretical resolution of the described experimental geometry is 725 nm as given by the wavelength and the effective numerical aperture. As the Airy pattern provides a quasihomogeneous illumination within the first minimum with only a small fraction of light directly penetrating the pinhole, no beam block had to be used in these scattering experiments. To reconstruct the images we applied the Kreuzer-implementation of the Kirchhoff–Helmholtz transformation, allowing the direct reconstruction of different planes between the pinhole and the detector without further phase assumptions or iterative refinement [4] Z n 2 ~ KðrÞ ¼ d xIðnÞ exp ik r . (1) jnj screen The integration extends over the two-dimensional surface of the screen with coordinates x ¼ ðX ; Y ; LÞ, where L is the distance from the source (pinhole) to the center of the screen (CCD chip) and I~ðnÞ is the measured hologram. The wave front K(r) can be reconstructed on a number of planes at various distances from the source in the vicinity of the object until a plane in focus is found. For the numerical implementation of the transform a fast algorithm has been developed that evaluates K(r) without any approximations. A more detailed description about reconstruction and resolution of digital in-line holographic microscopy can be found in Ref. [30]. Particle mixtures, lithographic structures and fibroblast cells are used as objects to evaluate the contrast and resolution properties of digital in-line holographic VUV microscopy. All are prepared on 100 nm thin Si3N4membrane windows with a window diameter of 1 mm
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(Silson Ltd., Northampton). For the preparation of particle mixtures on the surface, first a droplet of magnetic pigment (Magnetpigment 025 BASF, average particle size: 4–6 mm) was suspended in distilled water and dispersed onto the membrane. After evaporation of the water, a second droplet with polystyrene beads (Polystyrene micro spheres, Polysciences, size 6 mm, 10% variance) was added and the sample subsequently air-dried. For electron beam lithography, SU8-25 negative tone photo resist by Micro Chem was spin coated in a BalTec Spin Coater at 4000 rpm to achieve a film thickness of 700 nm. In order to evaporate the solvent and compact the film, the substrate was first soft baked on a level hot plate at 65 and 95 1C for 1 min at each temperature. Structures with different line width were written with a scanning electron microscope (SEM, Leo1530 with a Raith Elphy Quantum lithography unit) at an excessive dose of 25 mC/cm2. Subsequently, the sample was post exposure baked on a hot plate to selectively cross-link the exposed film at 65 and 95 1C for 2 min before developing it by immersion for 1.5 min in SU-8 developer by Micro Chem. REF 52WT fibroblast cells were cultivated on fibronectin coated membranes for 24 h in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), all purchased at Gibco. After fixation in glutaraldehyde, the cell water was slowly exchanged against ethanol by six different ethanol/water concentrations and the cells were finally critical point dried (Bal-Tec CPD 030). For comparison, all samples were additionally characterized by optical microscopy (Zeiss Axioplan 2, Zeiss 40 Neofluar, numerical aperture 0.75, AxioCam MRm) and/ or scanning electron microscopy. For electron microscopy a Leo1530 has been used and, if necessary, the samples were coated with a 2 nm thick carbon layer prior to imaging. 3. Results and discussion Fig. 2(a) shows a hologram of an arrangement of mixed polystyrene and iron oxide particles prepared by the abovedescribed protocol. A mixture of particles was used in order to probe possible material specific contrast differences for organic and metal oxide materials. The hologram shows pronounced interference fringes, indicating both a sufficient divergence of the scattered light, as well as a sufficient coherence of the synchrotron radiation. In the corners of the hologram, the first minimum of the Airy disk can barely be seen. Fig. 2(b) and the corresponding image panels at the right hand side show the reconstruction of the hologram in (a). For comparison, the same region was also imaged by optical microscopy (c) and electron microscopy (d). Especially when comparing the optical microscopy picture (c) and the reconstruction (b) it becomes obvious that the iron oxide particles (darker in (c)) and the polystyrene particles (brighter in (c)) exhibit nearly the same contrast properties in digital in-line holography with VUV radiation. As the objective used in optical microscopy
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(Zeiss 40 Neofluar objective) had a numerical aperture of NA ¼ 0.75, the theoretical resolution was about 450 nm. The linescans in Fig. 2(e) through two small iron oxide particles show a FWHM according to the SEM image of 330 nm (particle A) and 650 nm (particle B). As this size is smaller than the resolution in the VUV holography experiments and also smaller than the theoretical resolution of the optical microscope, the FWHM of the profiles can serve directly to characterize the resolution by the measured broadening. For optical microscopy the obtained FWHM was 1.01 mm (particle A) and 1.18 mm (particle B). Digital in-line holography results in images with a FWHM of 1.05 mm (particle A) and 1.19 mm (particle B). This result comes close to the theoretical prediction for the holography experiment of 725 nm and the resolution limit given by the pinhole radius. A possible reason for the broadening might be vibrations and/or thermal drift of the sample with respect to the CCD camera, which could not entirely be avoided as the samples were mounted onto a manipulator of 30 cm length. To elucidate contrast sensitivity in greater detail, thin lithographic structures were prepared. The thickness of the SU-8 photo resist was adjusted by the spin coating speed to 700 nm and the structure width at the broadest position within the lines were chosen to be 1.5 mm. Thus, the thickness of this object is only 19th of the diameter of the aforementioned particles. The structures were designed to have dots and lines, some of the latter tapered towards one end to check the sensitivity of the technique towards structural sizes below the resolution limit. The hologram obtained from such a structure is shown in Fig. 3(a). Again, the interference fringes are well visible and resolved. The apparent smearing of the lower right structure is real due to a resist drop edge across the lithographic structure. Comparing the reconstruction (b) with the electron microscopy picture (c) and the optical microscopy picture (d) reveals that especially the structures on the left part of the image are well resolved. This demonstrates that the photo resist with a thickness of 700 nm provides enough material to cause significant scattering of VUV radiation, leading to a good reconstruction. As third objects of interest, fibroblast cells adhered to silicon nitride windows were used. The main question in this case is the capability of VUV in-line holography to image biological samples and the sensitivity of the technique to resolve small structures inside these extended objects. As shown in Fig. 4(a), the hologram again shows pronounced and well resolved interference fringes which are necessary for the retrieval of high quality real space images. The reconstruction in Fig. 4(b) shows the fibroblast cell with remarkable intracellular details. The comparison with the surface sensitive electron microscopy picture in (c) and the optical microscopy picture in (d) shows the high contrast of holography for both the outside borders of the object (compared to (c)) and the internal structures of the cell (compared to (d)). The reason why a good contrast is observed is partly due to the wavelength range
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Fig. 2. Mixture of polystyrene and iron oxide particles dispersed on a silicon nitride membrane. (a) Hologram, l ¼ 13.8 nm, NA ¼ 0.014, NAeff ¼ 0.012; (b) numerical reconstruction of (a), image size 190 190 mm2; (c) optical microscopy picture, Zeiss Axioplan 2, Zeiss 40 Neofluar objective, NA ¼ 0.75, image size 190 140 mm2; (d) electron microscopy picture, Leo 1530, magnification 402 , electron energy 10 keV, secondary electron detector, image size 190 160 mm2. Magnified image sections: image size 90 55 mm2. (e) Linescans through the beads indicated by the arrows in the magnified insets of (b) and (d).
used. In general, the mass absorption coefficient for example, for carbon containing material is relative high at 90 eV (104 cm2/g [31]) thus providing a high contrast in
holography. In our opinion, the strong contrast especially of filament arrangements in cells in conjunction with an improved resolution has significant potential for cell
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Fig. 3. Electron lithographically written structure in SU-8 photo resist on silicon nitride membrane. (a) Hologram, l ¼ 13.8 nm, NA ¼ 0.014, NAeff ¼ 0.012; (b) numerical reconstruction of (a), image size 150 150 mm2; (c) scanning electron microscopy picture, Leo 1530, magnification 673 , electron energy 3 keV, secondary electron detector, image size 150 120 mm2; (d) optical microscopy picture, Zeiss Axioplan 2, Zeiss 40 Neofluar objective, NA ¼ 0.75, image size 150 120 mm2. Magnified image sections: image size 45 35 mm2.
biological applications in the future. The fact that internal structures are well visible is interesting for a second, more fundamental reason. Especially with extended objects like cells, the background wave front which is essential to produce a distinct scattering pattern is disturbed and the question arises whether the remaining primary wave outside the object provides enough reference photons for a reasonable reconstruction of the hologram. As internal structures are well resolved in (b), either the reference wave present outside the object is sufficient, or thinner parts within the cell allow sufficient transmission for the
reference wave. Two more points deserve to be pointed out: first, the nucleus of the cell is not clearly resolved. We assume that the material within the nucleus is either too dense to allow significant transmission at 90 eV or the contrast variation at this energy is too low to generate sufficient scattering. The second point is a noticeable background noise in the reconstructed images. Summarizing, especially as the setup of the technique is relatively simple, the high sensitivity for biological material such as filaments and membrane suggests VUV digital in-line holography as useful microscopy technique in cell biology.
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synchrotron radiation available at third generation synchrotrons like BESSY are sufficient to perform these types of coherent scattering experiments. Nevertheless, the coherent photon flux is a critical and limiting factor for such experiments in order to obtain higher spatial resolution. We show that transverse coherence filter pinholes with a diameter significantly larger than the wavelength are able to produce a suitably coherent and divergent beam which can serve as a homogeneous illumination source in Gabor holography. For different samples, material contrast and the obtainable resolution was characterized and discussed. In addition to organic and inorganic test samples, biological samples were investigated as well. For all structures, digital in-line holography with VUV radiation shows good contrast with a resolution limited by the effective numerical aperture. Even in the case of relatively large objects like adherent fibroblast cells, internal structures such as filaments are well resolved and exhibit remarkable contrast. This indicates that despite the large object the reference wave reaching the detector is suitable for the formation of interpretable holograms. Especially the observed contrast behavior suggests potential applications in biology and biophysics. The presented work shows the feasibility to use digital in-line holography with VUV radiation and suggests the use of EUV lithography lab sources for DIXH. In order to further enhance the technique for applications in biology and material sciences, more sophisticated geometries, shorter wavelength and smaller pinholes in conjunction with higher numerical aperture detection will be pursued. The development of short wavelength sources with increased coherent photon flux, such as the free electron laser facilities presently in construction and already in operation, will be pivotal in this respect. While not addressed in this article, we expect the intrinsic three dimensionality of holographic imaging to be of use for biological imaging in the future. Acknowledgments
Fig. 4. REF 52WT cells cultivated on a silicon nitride membrane. (a) Hologram, l ¼ 13.8 nm, NA ¼ 0.014, NAeff ¼ 0.012; (b) numerical reconstruction of (a), image size 210 100 mm2; (c) scanning electron microscopy picture, Leo 1530, magnification 363 , electron energy 5 keV, secondary electron detector, image size 300 100 mm2; (d) optical microscopy picture, Zeiss Axioplan 2, Zeiss 40 Neofluar objective, NA ¼ 0.75, image size 290 100 mm2.
4. Conclusion We have presented data on digital VUV in-line holography in the classical Gabor geometry. The reconstructions provide high quality microscopic images of different test objects. The longitudinal and transverse coherence as well as the coherent photon flux of
A.R. acknowledges the Fonds der Chemischen Industrie for a Liebig research grant and the Landesstiftung BadenWu¨rttemberg for support within the Elitefo¨rderprogramm. This work was funded by the BMBF 05KS4VH1/5 within the programme ‘‘Erforschung kondensierter Materie mit GroXgera¨ten’’ and partially supported by the EU STREP ‘‘Nanocues’’. We are indebted to H.J. Kreuzer for valuable discussions and advices. The support of the cell culture team and access to the microscopy facility of J.P. Spatz is gratefully acknowledged. We thank H. Zabel and his group for the possibility to use the ALICE scattering chamber for these experiments. References [1] D. Gabor, Nature 161 (1948) 777. [2] U. Schnars, W. Juepner, Digital Holography, Springer, Berlin, 2004.
ARTICLE IN PRESS A. Rosenhahn et al. / Ultramicroscopy 107 (2007) 1171–1177 [3] W. Xu, M.H. Jericho, I.A. Meinertzhagen, H.J. Kreuzer, PNAS 98 (20) (2001) 11301. [4] W. Xu, M.H. Jericho, I.A. Meinertzhagen, H.J. Kreuzer, Appl. Opt. 41 (2002) 5367. [5] C.S. Fadley, in: R.Z. Bachrach (Ed.), Advances in Surface and Interface Science, vol. 1: Techniques, Plenum Press, New York, 1992. [6] M. Zharnikov, H.-P. Steinru¨ck, J. Phys.: Condens. Matter 13 (2001) 10533. [7] A. Go¨lzha¨user, B. Vo¨lkel, M. Grunze, H.J. Kreuzer, Micron 33 (2002) 241. [8] B. Sur, R.B. Rogge, R.P. Hammond, V.N.P. Anghel, J. Katsaras, Nature 414 (2001) 525. [9] L. Cser, G. To¨ro¨k, G. Krexner, I. Sharkov, B. Farago´, Phys. Rev. Lett. 89 (2002) 17. [10] P. Korecki, J. Korecki, T. Slezak, Phys. Rev. Lett. 79 (1997) 18. [11] H.J. Kreuzer, N. Pomerleau, K. Blagrave, M.H. Jericho, SPIE 3744 (1999) 65. [12] J. Garcia-Sucerquia, W. Xu, M.H. Jericho, H.J. Kreuzer, Opt. Lett. 31 (9) (2006) 1211. [13] M. Tegze, G. Faigel, Europhys. Lett. 16 (1991) 41. [14] M. Tegze, G. Faigel, Nature 380 (1996) 49. [15] M. Tegze, G. Faigel, S. Marchesini, M. Belakhovski, O. Ulrich, Nature 407 (2000) 38. [16] S. Omori, L. Zhao, S. Marchesini, M.A. Van Hove, C.S. Fadley, Phys. Rev. B 65 (2001) 141061-1. [17] J.E. Trebes, S.B. Brown, E.M. Campbell, D.L. Matthews, D.G. Nilson, G.F. Stone, D.A. Whelan, Science 238 (1987) 517.
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[18] R.A. Bartels, A. Paul, H. Green, H.C. Kapteyn, M.M. Murnane, S. Backus, I.P. Christov, Y. Liu, D. Attwood, C. Jacobsen, Science 297 (2002) 376. [19] S. Vogt, G. Schneider, A. Steuernagel, J. Lucchesi, E. Schulze, D. Rudolph, G. Schmahl, J. Struct. Biol. 132 (2) (2000) 123. [20] W. Meyer-Ilse, D. Hamamoto, A. Nair, S.A. Lelivie`vre, G. Denbeaux, L. Johnson, A.L. Pearson, D. Yager, M.A. Legros, C.A. Larabell, J. Microsc. 201 (3) (2001) 395. [21] S. Lindaas, M. Howells, C. Jacobsen, A. Kalinovsky, J. Opt. Soc. Am. A 13 (9) (1996) 1788. [22] J.E. Trebes, S.B. Brown, E.M. Campbell, D.L. Matthews, D.G. Nilson, G.F. Stone, D.A. Whelan, Science 238 (1987) 517. [23] P. Spanne, C. Raven, I. Snigireva, A. Snigirev, Phys. Med. Biol. 44 (1999) 741. [24] J. Miao, K.O. Hodgson, T. Ishikawa, C.A. Larabell, M.A. LeGros, Y. Nishino, PNAS 100 (1) (2003) 110. [25] J. Miao, T. Ishikawa, E.H. Anderson, K.O. Hodgson, Phys. Rev. B 67 (2003) 174104. [26] S. Marchesini, H. He, H.N. Chapman, S.P. Hau-Riege, A. Noy, M.R. Howells, U. Weierstall, J.C.H. Spence, Phys. Rev. B 68 (2003) 140101(R). [27] I. McNulty, J. Kirz, C. Jacobsen, E.H. Anderson, M.R. Howells, D.P. Kern, Science 256 (1992) 1009. [28] S. Eisebitt, J. Lu¨ning, W.F. Schlotter, M. Lo¨rgen, O. Hellwig, W. Eberhardt, J. Sto¨hr, Nature 432 (2004) 886. [29] P. Jaegle´, Coherent Sources of XUV Radiation, Springer Series in Optical Sciences, Springer, New York, 2006. [30] J. Garcia-Sucerquia, W. Xu, S.K. Jericho, M.H. Jericho, P. Klages, H.J. Kreuzer, Appl. Opt. 45 (2006) 836. [31] X-ray Data Booklet, Center for X-ray Optics and Advanced Light Source, LBNL, January 2001.
Ultramicroscopy 107 (2007) 1171–1177 www.elsevier.com/locate/ultramic
Vacuum-ultraviolet Gabor holography with synchrotron radiation A. Rosenhahna,, R. Bartha, X. Caoa, M. Schu¨rmanna, M. Grunzea, S. Eisebittb a
Angewandte Physikalische Chemie, Universita¨t Heidelberg, INF 253, 69120 Heidelberg, Germany b BESSY m.b.H., Albert-Einstein-Str. 15, 12489 Berlin, Germany
Received 16 October 2006; received in revised form 12 January 2007; accepted 23 January 2007
Abstract We present the realization of high-resolution holographic microscopy using the original Gabor geometry and imaging with radiation in the vacuum-ultraviolet (VUV) spectral region. Synchrotron VUV radiation with a wavelength of 13.8 nm was focused on a small pinhole generating a highly divergent light cone suitable for digital in-line holography. Objects of different thickness and materials have been used to test the imaging properties of holographic microscopy in the VUV wavelength range. The effective numerical aperture was limited by the illuminated area of the detector, yielding a theoretical resolution below 1 mm and an experimental one of approximately 1 mm. r 2007 Elsevier B.V. All rights reserved. Keywords: Gabor; Holography; Digital in-line holography; Holographic microscopy; Synchrotron radiation; VUV radiation; Reconstruction; Kirchhoff–Helmholtz; Coherent scattering; Polystyrene; Iron oxide; Fibroblast
1. Introduction Since its invention by Gabor in the year 1948 [1], holography has been proven to be a powerful and inspiring technique. By now, a variety of imaging and measurement techniques are based on this coherent scattering concept, ranging from everyday applications like document protection or encryption to new measurement devices that help to solve scientific questions [2]. In Gabor’s original idea of a holographic microscope [1], a coherent wave is scattered from an object and interferes with the unscattered wave that passes the object undisturbed (the ‘‘reference wave’’). As sketched in Fig. 1, the result is an interference pattern known as hologram. The diverging wave creates a projection of the sample, thus magnifying the object and working as a coherent microscope. From such a hologram, real space information can either optically or digitally be retrieved (‘‘reconstruction’’) [3,4]. As both amplitude and phase are recorded up to a maximum angle given by the numerical aperture of the detection system, three-dimensional information is present in a hologram. Therefore, not only a single Corresponding author. Tel.: +49 6221 545065; fax: +49 6221 545060.
E-mail address: [email protected] (A. Rosenhahn). 0304-3991/$ - see front matter r 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2007.01.010
real space plane but also a whole stack of image planes can be reconstructed from a single scattering pattern. Holography was first conceived by Gabor for electron waves and this has by now been realized [5–7]. Apart from this, neutrons [8,9] or gamma radiation [10] was also used to obtain holographic images. Photon based holography is especially suited for the liquid environment due to high penetration depths and the high phase contrast caused by biological objects. In the visible range, the three-dimensional imaging of the marine organisms Ditylum brightwellii, the head of a fruit fly [3,4] and of human epithelial cells [11] are striking examples of the potential of digital in-line holography. Especially with the latest developments in immersion holography [12], it is clear that holography is rapidly approaching the performance of modern, state-of-the-art optical microscopes. As the resolution of a holographic microscope is determined by the wavelength of the photons and the numerical aperture of the detection system, it is straightforward to consider an increase of the photon energy in order to enhance the performance of the technique. As demonstrated by X-ray fluorescence holography, the short wavelength of X-rays allows to determine atomic positions in ordered solids with angstrom accuracy [13–16]. We pursue a decrease of the wavelength for digital in-line
ARTICLE IN PRESS 1172
A. Rosenhahn et al. / Ultramicroscopy 107 (2007) 1171–1177
Fig. 1. Schematic geometry of a digital in-line holography experiment. The hologram is formed by interference of the undisturbed primary wave front and the scattered, secondary wave front.
holography into the vacuum-ultraviolet (VUV) range. The general possibility to use VUV radiation to produce in-line holograms was earlier demonstrated with a selenium X-ray laser at a wavelength of 21 nm with an obtained resolution of 5 mm [17]. Recently, in higher harmonic generation experiments coherent photons with a wavelength of 30 nm were generated and coherent scattering experiments were conducted [18]. Exploiting the natural, small divergence of the VUV beam thus resulted in a magnification of 3.8 at a resolution of 6.8 mm with suggestions for improving this to 500 nm [18]. Soft X-rays allow for much higher resolution holographic images, especially in the water window which is frequently used in X-ray microscopy of biological specimen [19,20]. With a wavelength of 1.89 nm, fibroblast fibers were imaged with a resolution of 40–50 nm using a photo resist as recording medium and thus overcoming detector resolution limitations at these short wavelengths [21]. Hard X-rays have been exploited to image holographically [22,23] and via coherent scattering and iterative phase retrieval [24–26], but without the availability of an X-ray laser, experiments with shorter wavelengths generally suffer from a dramatic decrease of coherent photon flux obtainable from noncoherent sources. The number of photons extractable from an incoherent source into a coherence volume scales as 1/l3. At short wavelengths, Fourier transform setups are advantageous in order to achieve high spatial resolution [27,28]. Here, we will focus exclusively on VUV radiation, also known as extreme ultraviolet (EUV). As 13.4 nm wavelength radiation is used in the next generation lithography for microelectronics, new lab sources may become available [29] for holographic imaging in this spectral range. 2. Experiment and samples All experiments have been carried out at the synchrotron light source BESSY II in Berlin at the undulator beamline
UE52-SGM. The focus of the beamline is 18 18 mm2 fullwidth at half-maximum (FWHM), enabling a high flux per area, required for the experiments. The photon energy was set to 90 eV, corresponding to a wavelength of 13.8 nm, which is used in all the results presented here. The pinhole–detector distance of 1000 mm was dictated by the available experimental setup in the ultra high vacuum chamber. The CCD camera used in the experiments was a Roper Scientific PI-SX-2048, using the back-illuminated Marconi CCD42-40 chip with 2048 2048 pixels and a pixel size of 13.5 13.5 mm2. For the given photon energy, the quantum efficiency of the CCD chip is about 45%. The pinholes used in the experiments were purchased from National Aperture, Inc. with a nominal size of 1 mm. A quantitative analysis of the Airy pattern obtained revealed a real diameter of 1.4 mm. The pinhole size is large compared to the wavelength of the photons used in the experiment. In an ideal holography experiment, one would use smaller pinholes, but we are limited by the incident coherent photon flux. The chosen pinhole represents a good compromise between hologram intensity and spatial resolution. The effective numerical aperture of the setup is not limited by the acceptance angle of the CCD chip (NA ¼ 0.014) but by the radius of the first Airy minimum at a value of NA ¼ 0.012. The theoretical resolution of the described experimental geometry is 725 nm as given by the wavelength and the effective numerical aperture. As the Airy pattern provides a quasihomogeneous illumination within the first minimum with only a small fraction of light directly penetrating the pinhole, no beam block had to be used in these scattering experiments. To reconstruct the images we applied the Kreuzer-implementation of the Kirchhoff–Helmholtz transformation, allowing the direct reconstruction of different planes between the pinhole and the detector without further phase assumptions or iterative refinement [4] Z n 2 ~ KðrÞ ¼ d xIðnÞ exp ik r . (1) jnj screen The integration extends over the two-dimensional surface of the screen with coordinates x ¼ ðX ; Y ; LÞ, where L is the distance from the source (pinhole) to the center of the screen (CCD chip) and I~ðnÞ is the measured hologram. The wave front K(r) can be reconstructed on a number of planes at various distances from the source in the vicinity of the object until a plane in focus is found. For the numerical implementation of the transform a fast algorithm has been developed that evaluates K(r) without any approximations. A more detailed description about reconstruction and resolution of digital in-line holographic microscopy can be found in Ref. [30]. Particle mixtures, lithographic structures and fibroblast cells are used as objects to evaluate the contrast and resolution properties of digital in-line holographic VUV microscopy. All are prepared on 100 nm thin Si3N4membrane windows with a window diameter of 1 mm
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(Silson Ltd., Northampton). For the preparation of particle mixtures on the surface, first a droplet of magnetic pigment (Magnetpigment 025 BASF, average particle size: 4–6 mm) was suspended in distilled water and dispersed onto the membrane. After evaporation of the water, a second droplet with polystyrene beads (Polystyrene micro spheres, Polysciences, size 6 mm, 10% variance) was added and the sample subsequently air-dried. For electron beam lithography, SU8-25 negative tone photo resist by Micro Chem was spin coated in a BalTec Spin Coater at 4000 rpm to achieve a film thickness of 700 nm. In order to evaporate the solvent and compact the film, the substrate was first soft baked on a level hot plate at 65 and 95 1C for 1 min at each temperature. Structures with different line width were written with a scanning electron microscope (SEM, Leo1530 with a Raith Elphy Quantum lithography unit) at an excessive dose of 25 mC/cm2. Subsequently, the sample was post exposure baked on a hot plate to selectively cross-link the exposed film at 65 and 95 1C for 2 min before developing it by immersion for 1.5 min in SU-8 developer by Micro Chem. REF 52WT fibroblast cells were cultivated on fibronectin coated membranes for 24 h in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), all purchased at Gibco. After fixation in glutaraldehyde, the cell water was slowly exchanged against ethanol by six different ethanol/water concentrations and the cells were finally critical point dried (Bal-Tec CPD 030). For comparison, all samples were additionally characterized by optical microscopy (Zeiss Axioplan 2, Zeiss 40 Neofluar, numerical aperture 0.75, AxioCam MRm) and/ or scanning electron microscopy. For electron microscopy a Leo1530 has been used and, if necessary, the samples were coated with a 2 nm thick carbon layer prior to imaging. 3. Results and discussion Fig. 2(a) shows a hologram of an arrangement of mixed polystyrene and iron oxide particles prepared by the abovedescribed protocol. A mixture of particles was used in order to probe possible material specific contrast differences for organic and metal oxide materials. The hologram shows pronounced interference fringes, indicating both a sufficient divergence of the scattered light, as well as a sufficient coherence of the synchrotron radiation. In the corners of the hologram, the first minimum of the Airy disk can barely be seen. Fig. 2(b) and the corresponding image panels at the right hand side show the reconstruction of the hologram in (a). For comparison, the same region was also imaged by optical microscopy (c) and electron microscopy (d). Especially when comparing the optical microscopy picture (c) and the reconstruction (b) it becomes obvious that the iron oxide particles (darker in (c)) and the polystyrene particles (brighter in (c)) exhibit nearly the same contrast properties in digital in-line holography with VUV radiation. As the objective used in optical microscopy
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(Zeiss 40 Neofluar objective) had a numerical aperture of NA ¼ 0.75, the theoretical resolution was about 450 nm. The linescans in Fig. 2(e) through two small iron oxide particles show a FWHM according to the SEM image of 330 nm (particle A) and 650 nm (particle B). As this size is smaller than the resolution in the VUV holography experiments and also smaller than the theoretical resolution of the optical microscope, the FWHM of the profiles can serve directly to characterize the resolution by the measured broadening. For optical microscopy the obtained FWHM was 1.01 mm (particle A) and 1.18 mm (particle B). Digital in-line holography results in images with a FWHM of 1.05 mm (particle A) and 1.19 mm (particle B). This result comes close to the theoretical prediction for the holography experiment of 725 nm and the resolution limit given by the pinhole radius. A possible reason for the broadening might be vibrations and/or thermal drift of the sample with respect to the CCD camera, which could not entirely be avoided as the samples were mounted onto a manipulator of 30 cm length. To elucidate contrast sensitivity in greater detail, thin lithographic structures were prepared. The thickness of the SU-8 photo resist was adjusted by the spin coating speed to 700 nm and the structure width at the broadest position within the lines were chosen to be 1.5 mm. Thus, the thickness of this object is only 19th of the diameter of the aforementioned particles. The structures were designed to have dots and lines, some of the latter tapered towards one end to check the sensitivity of the technique towards structural sizes below the resolution limit. The hologram obtained from such a structure is shown in Fig. 3(a). Again, the interference fringes are well visible and resolved. The apparent smearing of the lower right structure is real due to a resist drop edge across the lithographic structure. Comparing the reconstruction (b) with the electron microscopy picture (c) and the optical microscopy picture (d) reveals that especially the structures on the left part of the image are well resolved. This demonstrates that the photo resist with a thickness of 700 nm provides enough material to cause significant scattering of VUV radiation, leading to a good reconstruction. As third objects of interest, fibroblast cells adhered to silicon nitride windows were used. The main question in this case is the capability of VUV in-line holography to image biological samples and the sensitivity of the technique to resolve small structures inside these extended objects. As shown in Fig. 4(a), the hologram again shows pronounced and well resolved interference fringes which are necessary for the retrieval of high quality real space images. The reconstruction in Fig. 4(b) shows the fibroblast cell with remarkable intracellular details. The comparison with the surface sensitive electron microscopy picture in (c) and the optical microscopy picture in (d) shows the high contrast of holography for both the outside borders of the object (compared to (c)) and the internal structures of the cell (compared to (d)). The reason why a good contrast is observed is partly due to the wavelength range
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Fig. 2. Mixture of polystyrene and iron oxide particles dispersed on a silicon nitride membrane. (a) Hologram, l ¼ 13.8 nm, NA ¼ 0.014, NAeff ¼ 0.012; (b) numerical reconstruction of (a), image size 190 190 mm2; (c) optical microscopy picture, Zeiss Axioplan 2, Zeiss 40 Neofluar objective, NA ¼ 0.75, image size 190 140 mm2; (d) electron microscopy picture, Leo 1530, magnification 402 , electron energy 10 keV, secondary electron detector, image size 190 160 mm2. Magnified image sections: image size 90 55 mm2. (e) Linescans through the beads indicated by the arrows in the magnified insets of (b) and (d).
used. In general, the mass absorption coefficient for example, for carbon containing material is relative high at 90 eV (104 cm2/g [31]) thus providing a high contrast in
holography. In our opinion, the strong contrast especially of filament arrangements in cells in conjunction with an improved resolution has significant potential for cell
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Fig. 3. Electron lithographically written structure in SU-8 photo resist on silicon nitride membrane. (a) Hologram, l ¼ 13.8 nm, NA ¼ 0.014, NAeff ¼ 0.012; (b) numerical reconstruction of (a), image size 150 150 mm2; (c) scanning electron microscopy picture, Leo 1530, magnification 673 , electron energy 3 keV, secondary electron detector, image size 150 120 mm2; (d) optical microscopy picture, Zeiss Axioplan 2, Zeiss 40 Neofluar objective, NA ¼ 0.75, image size 150 120 mm2. Magnified image sections: image size 45 35 mm2.
biological applications in the future. The fact that internal structures are well visible is interesting for a second, more fundamental reason. Especially with extended objects like cells, the background wave front which is essential to produce a distinct scattering pattern is disturbed and the question arises whether the remaining primary wave outside the object provides enough reference photons for a reasonable reconstruction of the hologram. As internal structures are well resolved in (b), either the reference wave present outside the object is sufficient, or thinner parts within the cell allow sufficient transmission for the
reference wave. Two more points deserve to be pointed out: first, the nucleus of the cell is not clearly resolved. We assume that the material within the nucleus is either too dense to allow significant transmission at 90 eV or the contrast variation at this energy is too low to generate sufficient scattering. The second point is a noticeable background noise in the reconstructed images. Summarizing, especially as the setup of the technique is relatively simple, the high sensitivity for biological material such as filaments and membrane suggests VUV digital in-line holography as useful microscopy technique in cell biology.
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synchrotron radiation available at third generation synchrotrons like BESSY are sufficient to perform these types of coherent scattering experiments. Nevertheless, the coherent photon flux is a critical and limiting factor for such experiments in order to obtain higher spatial resolution. We show that transverse coherence filter pinholes with a diameter significantly larger than the wavelength are able to produce a suitably coherent and divergent beam which can serve as a homogeneous illumination source in Gabor holography. For different samples, material contrast and the obtainable resolution was characterized and discussed. In addition to organic and inorganic test samples, biological samples were investigated as well. For all structures, digital in-line holography with VUV radiation shows good contrast with a resolution limited by the effective numerical aperture. Even in the case of relatively large objects like adherent fibroblast cells, internal structures such as filaments are well resolved and exhibit remarkable contrast. This indicates that despite the large object the reference wave reaching the detector is suitable for the formation of interpretable holograms. Especially the observed contrast behavior suggests potential applications in biology and biophysics. The presented work shows the feasibility to use digital in-line holography with VUV radiation and suggests the use of EUV lithography lab sources for DIXH. In order to further enhance the technique for applications in biology and material sciences, more sophisticated geometries, shorter wavelength and smaller pinholes in conjunction with higher numerical aperture detection will be pursued. The development of short wavelength sources with increased coherent photon flux, such as the free electron laser facilities presently in construction and already in operation, will be pivotal in this respect. While not addressed in this article, we expect the intrinsic three dimensionality of holographic imaging to be of use for biological imaging in the future. Acknowledgments
Fig. 4. REF 52WT cells cultivated on a silicon nitride membrane. (a) Hologram, l ¼ 13.8 nm, NA ¼ 0.014, NAeff ¼ 0.012; (b) numerical reconstruction of (a), image size 210 100 mm2; (c) scanning electron microscopy picture, Leo 1530, magnification 363 , electron energy 5 keV, secondary electron detector, image size 300 100 mm2; (d) optical microscopy picture, Zeiss Axioplan 2, Zeiss 40 Neofluar objective, NA ¼ 0.75, image size 290 100 mm2.
4. Conclusion We have presented data on digital VUV in-line holography in the classical Gabor geometry. The reconstructions provide high quality microscopic images of different test objects. The longitudinal and transverse coherence as well as the coherent photon flux of
A.R. acknowledges the Fonds der Chemischen Industrie for a Liebig research grant and the Landesstiftung BadenWu¨rttemberg for support within the Elitefo¨rderprogramm. This work was funded by the BMBF 05KS4VH1/5 within the programme ‘‘Erforschung kondensierter Materie mit GroXgera¨ten’’ and partially supported by the EU STREP ‘‘Nanocues’’. We are indebted to H.J. Kreuzer for valuable discussions and advices. The support of the cell culture team and access to the microscopy facility of J.P. Spatz is gratefully acknowledged. We thank H. Zabel and his group for the possibility to use the ALICE scattering chamber for these experiments. References [1] D. Gabor, Nature 161 (1948) 777. [2] U. Schnars, W. Juepner, Digital Holography, Springer, Berlin, 2004.
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