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Mask-based digital soft X-ray Fourier transform holography using coherent synchrotron radiation is applied to image diatoms and freeze-dried 3T3 fibroblast cells. At soft X-ray wavelengths between 4 nm and 8 nm we achieve spatial resolutions down to 450 nm over a 33.5 μm field of view. As holography records amplitude and phase of the scattered wavefield, images with different contrast mechanisms can be generated a posteriori by numerical processing of the holographic data (“omni-microscopy”). We present Zernike phase contrast, Schlieren phase contrast, differential interference and gradient contrast images based on amplitude and phase information. We discuss the potential of this approach for destructive single-shot imaging beyond dose limitations of biological samples at coherent femtosecond-pulsed X-ray sources.
©2009 Optical Society of America
1. Introduction
For the understanding of structure and function of cells it is essential to have access to imaging tools powerful enough to resolve and identify different intracellular structures. As the resolution in far-field light-based microscopy is limited by the photon wavelength, X-rays are used for high-resolution imaging. Different approaches have been successfully advanced in recent years, including scanning and full field transmission microscopy [1, 2, 3], holography [4], coherent diffraction imaging [5, 6, 7] and interferometry [8] at X-ray wavelengths. Beyond the capability for high spatial resolution, X-rays facilitate the use of special contrast mechanisms such as atomic, chemical or magnetic sample contrast via resonant scattering. Apart from such contrast based on the intrinsic scattering processes in the sample, the optical system can add specific contrast modes such as Zernike phase contrast or differential interference contrast [9, 10]. The widespread use of such techniques in light microscopy particularly for biological and medical specimens underlines their usefulness in visualizing and interpreting structure in the images.
In microscopy, the means of generating e. g. Zernike phase contrast is “hard-wired” into the experimental setup by the introduction of appropriate optical elements, such as a phase ring in the back focal plane. Different optical configurations have to be used to generate the desired contrast. In holography, on the other hand, the complete wavefield information consisting of amplitude and phase is recorded. As a result, the analog of any optical element behind the sample can be simulated by an appropriate transfer function. In consequence, any desired optics-related contrast can be generated numerically by virtual optics, i. e. the computer becomes an integral part of the imaging system. This approach has been named omni-microscopy and was demonstrated at hard X-ray wavelengths by Paganin et al. [11, 12].
Holograms of biological objects have been recorded in in-line geometry in the past. Utilizing visible or UV laser sources, holography of biological objects with spatial resolutions around one micrometer below have been achieved in conjunction with short exposure times allowing to track motion of the specimen on the millisecond timescale [13, 14]. For in-line holography with X-rays, the spatial resolution on the 2D X-ray detector recording the hologram is typically a limiting factor for the achievable spatial resolution and thus it has been challenging to make significant resolution advancements into the sub-micrometer realm. High-resolution of approximately 50 nm was achieved using photoresists to record the hologram in conjunction with an atomic force microscope to read out the holographic information [4, 15]. Fully digital soft X-ray in-line holography of biological objects at approximately 800 nm resolution has recently been reported by Rosenhahn et al. using a charged coupled device (CCD) camera [16, 17].
In Fourier transform holography (FTH), a small reference structure in the object plane creates the reference wave needed to encode a hologram of the sample. The resolution is directly limited by the radius of the reference wave source [18, 19]. High-resolution imaging with 50 nm resolution has been achieved with a Fresnel zone plate focus to generate a reference wave [20] and with a mask-based approach which locks the specimen and the reference object rigidly together [21]. The latter approach has recently been employed for high-resolution imaging of magnetic materials [21, 22, 23, 24, 25, 26], including magnetic phase contrast imaging [27]. The integrated mask-sample design has inherent stability permitting very long exposures and allowing for multiplexed experiments with multiple samples and/or multiple reference beams [28, 29].
In this work, we present soft X-ray omni-microscopy of biological objects to explore the potential of FTH-based imaging for biological specimens. For the initial experiments, we designed our experiment to balance sub-μm image resolution with a large field of view (FOV). As we will discuss below, with the same experimental setup it is straightforward to obtain ten times higher resolution by simply shrinking the field of view, the reference hole size and the object-reference separation correspondingly. Furthermore, the approach is ideally suited for high-resolution imaging below 20 nm via destructive single-shot imaging at free electron lasers.
Two prototype specimens have been investigated: (i) diatom skeletons consisting of nano-and microstructured silica and (ii) fixed embryonic mouse fibroblasts from cell culture (3T3 cells).
2. Methods
In FTH the object and the reference wave originate from the same plane which is perpendicular to the incoming beam. When working with X-rays, it is convenient to implement FTH in a lensless configuration [18]. Here, the reference wave is created by a small pinhole next to the object which needs to be suitably transversely confined. Object and reference have to be illuminated coherently and the overall geometry and coherence lengths have to be such that object and reference beam can interfere on the detector.
We realize a suitable FTH geometry for soft X-rays by a mask approach [21] as sketched in Fig.1. An X-ray transparent silicon nitride (Si3N4) membrane is covered by a 1 μm thick X-ray opaque gold film. In this film, a comparatively large object hole (33.5 μm diameter) has been defined by UV lithography in a lift-off process. This aperture defines the field of view for the holographic image formation. The Si3N4 membrane below the object hole remains intact.
The specimens to be imaged are positioned on the Si3N4 side of this structure. 3T3 cells were cultivated in Dulbeccos’ modified eagles medium (+10 % fetal bovine serum + 1 % 200 mM L-glutamin) [30] on the membrane. In order to ensure a proper adhesion of the cells, the membrane was covered with fibronectin before cultivation. The cell density was chosen to create non-confluent cells. After growth, the cells were fixed with a solution of 3.7 % paraformaldehyde in water and then freeze-dried. Diatoms are prepared by depositing a drop of a diatom suspension on the membrane followed by simple evaporation of the water. After the sample deposition, reference apertures are milled through the gold layer and the membrane by a focused ion beam (FIB, Zeiss Crossbeam 1540 EsB). For image formation, we use reference apertures with a radius of 450 nm at a distance of 65 μm from the center of the object in the diatom case and with a radius of 340 nm at a distance of 72 μm in the 3T3 cell case.
Fig. 1. Schematic of the Fourier transform holography setup and the omni microscopy procedure. The object’s scattered wave interferes with a reference wave to form the hologram on the detector. The phase relation between the reference and the object is transferred into an intensity modulation in the hologram. An inverse Fourier transform reconstructs a complex image of the object containing the amplitude and phase information and a spatially separated twin image. Virtual optics allows to compute images with different contrast a posteriori.
The experiments were performed at the BESSY II synchrotron source in Berlin, Germany, at the UE52-SGM undulator beamline. Soft X-rays with a photon energy of 150 eV (λ=8.27 nm), 225 eV (λ=5.51 nm), and 300 eV (λ =4.13 nm) were used to record holograms. For uniform and coherent illumination the sample-mask structure is placed 30 cm downstream of the beamline focus, in a normal incidence transmission geometry. The non-opaque areas thus define a small angular acceptance, ensuring transversely coherent illumination. Upstream of the sample, the X-ray beam is monochromatized with an energy resolution E/ΔE of approximately 5000. As the maximum optical path length difference (for hypothetical scattering under π/2) is smaller than 100 μm, this ensures sufficient longitudinal coherence for interference at the detector even for the shortest wavelength illumination at this beamline. The hologram is detected by a CCD camera (Princeton Instruments PI-MTE, 2048×2048 pixels, pixel size 13.5 μm) located 1.51 m downstream of the sample. For this particular detector pixel geometry, the distance between detector and specimen is necessitated by the large FOV in order to resolve the hologram modulation caused by the interference between the reference and the object wave.
Due to the limited dynamic range of the CCD, the intense central part of the hologram around zero momentum transfer was blocked using a central beamstop with 1.4 mm diameter. A 2D fast Fourier transform (FFT) of this dataset after suitable edge-windowing and zero-padding is calculated. The complex FFT yields two separate matrices containing the amplitude and the phase image of the sample. We demonstrate the use of virtual optics by further applying suitable operators as outlined below.
To compare the resolution and information content of the reconstructed images with conventional imaging methods we record images of our sample with a scanning electron microscope (Zeiss Crossbeam 1540 EsB) and light microscope in Zernike phase contrast (Leica DMI 6000B).
3. Results and discussion
As an example for the data quality (signal-to-noise ratio and modulation visibility as a function of momentum transfer function), holograms of the diatoms and 3T3 cells recorded with a photon energy of 150 eV are presented in Fig. 2. The intensity modulations in the holograms are clearly visible indicating adequate coherence of the X-ray beam for the experiment. The accumulated exposure time is 1800 seconds for the diatoms and 1600 seconds for the 3T3 cells. The Poisson signal to noise ratio for the highest detected scattering angles at the rim of the CCD is 12.4 for the diatomic sample and 7.8 for the 3T3 cells, i. e. the signal is clearly detectable. Consequently, the maximum momentum transfer measured in the holograms is limited by the solid angle covered by the detector in conjunction with the X-ray wavelength used.
Fig. 2. Holograms of the diatom sample (left) and 3T3 cells (right) at a photon energy of 150 eV on a logarithmic intensity grey scale (counts/pixel). In the inset of the diatomic specimen the hologram modulation is clearly visible (inset not shown for the hologram of the 3T3 cells). The accumulated exposure time amounts to 1800 s for the diatoms and 1600 s for the 3T3 cells. The central part of the holograms is blocked by a beamstop.
Based on such holograms we reconstruct images of the specimen. In Fig. 3, reconstructions of a diatom skeleton sample are presented. Panels (a) through (c) depict amplitude reconstructions of the specimen from holograms recorded with increasing photon energy from 150 eV to 300 eV. Diatom skeleton structures are clearly visible in the entire FOV. Only in panel (c), a modulation in the background deteriorates the contrast in the upper half of the image. This effect is caused by limited transverse coherence over the 82 μm object-reference distance at 300 eV, resulting in decreased image signal to background noise ratio. The same specimen is imaged by SEM and light microscopy; micrographs are presented in panels (d) and (e), respectively. To characterize the spatial resolution, panels (ai) to (ci) highlight holographic images of a fan-shaped structure containing holes of approximately 300 nm diameter, as evident from the SEM micrograph shown in panel (di). As expected, the spatial resolution is increasing with shorter X-ray wavelength; highest resolution is obtained at 300 eV photon energy. Convolving the SEM image with an Airy disk of variable width we determine the Rayleigh resolution in the reconstructed image shown in panel ci to be 450±30 nm. As expected, this resolution is limited by the reference hole diameter of 450 nm. The momentum transfer limited in-plane Rayleigh resolution resolution at the CCD edge would amount to approximately 300 nm.
In Fig. 4 (a) and (b), we present amplitude and phase contrast images of the 3T3 cell sample, respectively. The hologram was recorded at 150 eV photon energy. In both images, the cell is clearly visible and inner-cell compartments, e. g. the nucleus, can be identified. Both image types show significant differences. In the phase image, the contrast between cytoplasm and nucleus is much stronger and small structures within the nucleus, probably the nucleolus, are unveiled. By comparing the FTH images of the 3T3 cells with an SEM image in panel (i) the resolution can be estimated to be 600 nm, i. e. diffraction limited at 8.27 nm wavelength in the present geometry. For comparison, we also show a light microscopy image in Zernike phase contrast in panel (j).
Fig. 3. Reconstructed images of the diatom sample recorded at different photon energies in comparison to (d) electron and (e) light micrographs. (a) E = 150 eV. (b) E = 225 eV. (c) E = 300 eV. Insets are shown for (ai) E = 150 eV, (bi) E = 225 eV and (ci) E = 300 eV to compare structures directly to an (di) electron micrograph. The scale bar for (a) – (e) is shown in (d) and corresponds to 4 μm. The scale bar for (ai) – (di) is shown in (ai) and corresponds to 1 μm.
The spatial resolution obtained in the reconstructions of our Fourier transform holograms is better than previously reported resolutions for digital soft X-ray holography, which was performed in in-line geometry [16, 17]. We reconstruct images over a 33.5 μm field of view, which necessitates an even larger object-reference distance and a correspondingly large sample-detector distance to record the hologram fringes. Together with the number of pixels per axis on our detector and the transverse reference hole size, this limits our spatial resolution. On the other hand, the spatial resolution achieved is about a factor of 10 lower than in state-of-the-art X-ray microscopy of biological samples [1, 2], coherent diffraction imaging [5, 6, 7, 31] or non-digital holographic approaches based on atomic force microscopy readout of holograms recorded in photoresists [4]. The FTH approach is not detector pixel limited to achieve highest spatial resolution if the entire mask structure is correspondingly reduced in its transverse size. Consequently, it will be straight forward to improve the resolution by a factor of ten, as already demonstrated in mask-based digital FTH imaging of non-biological objects [21, 25]. While the achievable resolution in holography at 3rd generation synchrotron sources can be competitive with state-of-the-art X-ray microscopy, the imaging efficiency is significantly lower than in full field transmission microscopy based on Fresnel zone plates as evidenced by the comparatively long exposure time due to the modulation depth in the high spatial frequency domains. While multi-reference approaches can increase the efficiency [28, 31], we stipulate that the main potential for the holographic omni-microscopy approach in bio-imaging lies in the use of intense, femtosecond-pulsed, coherent X-ray beams from free electron lasers. Here, ultimate dose limitations for the achievable spatial resolution [32] can be circumvented by fs-snapshot imaging, where the sample is destroyed after the collection of the diffraction pattern or hologram [33, 34, 35]. First imaging experiments of a biological sample at the FLASH free electron laser reached a spatial resolution of 75 nm [31].
Fig. 4. Reconstructed 3T3 cell images of (a) the amplitude and (b) the phase recorded at 150 eV photon energy in comparison to (i) electron and (j) light micrographs. Further images based on virtual optics are calculated as described in the text: Zernike phase contrast with (c) ψ = +ψ/2 and (d) ψ = -π/2; differential interference contrast of (e) the phase and (g) applied to the amplitude; (f) Schlieren phase contrast; (h) gradient phase contrast. The scale bar in (i) corresponds to 4 μm and applies to all panels.
In single-shot imaging, it is useful to collect as much information as possible in the single, destructive shot. This pertains not only to highest possible spatial resolution, but also to the maximum information on the spatial variation of the complex refractive index in the sample [36]. In particular, for many biological samples it would be useful to be able to compare a single-shot absorption image to e. g. a Zernike phase contrast image or a differential interference contrast image produced in the very same shot. In fact, due to the destructive, single-shot nature of such experiments, it would be helpful to have all this information on the sample accessible. In light microscopy of biological objects, the possibility to use different contrast mechanisms in order to enhance specific features is very useful and thus common practice - in a single shot experiment this has to be done a posteriori.
For phase contrast to be visible in conventional, lens-based full-field microscopy, the phase information must be transferred into a detectable image intensity variation by the optical system. The optical elements creating the phase contrast manipulate the scattered wavefield and make phase information visible in this way. For lens-based, single-shot full-field microscopy this implies that only one type of contrast can give rise to the image, depending on the optical configuration chosen. In holography, on the other hand, the contrast generation on the basis of the spatial variations of the complex refractive index can be emulated a posteriori by virtual optics as both amplitude and phase information of the scattered wavefield were measured, i. e. the complete optical information has been captured over a certain momentum-transfer range. Virtual optics generated by suitable operators allow to numerically calculate several images with the desired contrast mechanism from one holographic dataset, as demonstrated by Paganin et al. [11, 12]. In the following, we demonstrate this omni-microscopy concept in conjunction with X-ray Fourier transform holography, which is well suited for high-resolution single-shot imaging with X-rays [21, 37].
We use the amplitude A(x,y) and phase Φ(x,y) information of the real space object directly obtained via FTH to calculate several types of images I(x,y) of the 3T3 cell sample. Amplitude and phase information alone are presented in panels (a) and (b) of Fig. 4, respectively. As virtual optics, the following operators are employed [12]. Here, the 2D spatial Fourier transform connecting real (x,y) and reciprocal (kx,ky) space is denoted by ℱ{}(kx,ky):
- Zernike phase contrast; shown in Fig. 4 (c) and (d):
In conventional optics, the transmitted light is phase-shifted by a phase ring centered in the back focal plane such that it can interfere with the diffracted wave from the sample.
We use
with the phase shift φ = ψ 0 ≠ 0 applied within the phase ring radius ∣k∣ ≤ k 0 and φ = 0 otherwise. Strongest phase contrast is achieved for φ 0 = ±π/2.
- Differential interference contrast (DIC), shown in Figs. 4 (e) and (g):
In classical DIC, the sample is additionally illuminated by a slightly transversely displaced copy of the original incident illumination beam. Both rays are brought to overlap and interfere behind the sample. In light microscopy this is typically realized using Wollanski prisms as a beam splitters and polarizers. For DIC of the phase presented in Fig. 4 (e), we use
where Δr is the in plane displacement vector and φ = φ 0 ≠ 0 the phase shift between both beams. In contrast to the typical optical situation, we also calculate DIC of the amplitude information by replacing Φ(x,y) with A(x,y) in the formula above (Fig. 4 (g)).
- Gradient contrast, shown in Fig. 4 (h):
We highlight phase gradients in any in-plane direction within in the sample by using
This is an example of a contrast without a direct optical analog. It bears some resemblance to dark field microscopy.
- Schlieren phase contrast, shown in Fig. 4 (f):
In conventional microscopy, Schlieren phase contrast is generated by a knife-edge in the back focal plane blocking half the diffraction pattern. We emulate this effect by
with
Here, α defines the angular position of the knife-edge.
As evident in Fig. 4, the use of virtual optics allows to highlight different sample features. As expected, the Zernike phase contrast image with φ = +π/2. (Fig. 4 (c)) is very similar to the direct holographic phase image. A shift to φ = −π/2 inverts the contrast (Fig. 4 (d)). In the DIC image (Fig. 4 (e)) the typical edge enhancement perpendicular to a selected in-plane direction is visible. As amplitude and phase information are separately available for numerical processing, one can generate contrast methods which are not realized in optical full-field microscopy such as DIC for the amplitude image or gradient contrast in panels (g) and (h), respectively. While emulations of existing and established microscopy contrast modes may be initially most helpful, new types of contrast can be devised for a given scientific question.
Here, we do not want to discuss any particular cellular features in the images of the 3T3 cells, as we suspect the presence of artifacts due to the fixation and freeze-dry procedure. For the purpose of this paper it is sufficient to note that from a single X-ray Fourier transform hologram, images with various contrast mechanisms can be generated via virtual optics. We would like to point out that the omni-microscopy approach is also accessible after successful iterative phase retrieval in coherent diffraction imaging or in combined holography/phase retrieval approaches [37].
4. Conclusion
Mask-based digital Fourier transform holography with soft X-rays is employed to image fixed 3T3 cells and diatom silica skeletons at spatial resolutions down to 450 nm. We demonstrate that the amplitude and phase information stored in the hologram can be accessed via virtual optics to generate images with different contrast mechanisms such as Zernike Phase contrast or differential interference contrast. The omni-microscopy approach will be particularly useful for destructive single-shot imaging at intense, coherent short-wavelength radiation sources such as UV-lasers [38] or X-ray free electron lasers [39] where highest spatial resolution beyond dose limitations for biological objects can be expected below 20 nm to be achieved via destructive single-shot imaging. For this approach, omni-microscopy based on Fourier transform holography will be able to extract a maximum of information on the specimen out of a single shot.
Acknowledgments
We wish to thank the group of Axel Rosenhahn for the introduction into the preparation of cells on silicon nitride membranes. We thank John Banhart and Nelia Wanderka for the opportunity to use the FIB and Helmut Zabel and his group for the possibility to use the ALICE scattering chamber for the experiments. We thank Christian Weniger for the fabrication and lift-off patterning of silicon nitride membranes, and we thank Peter Guttmann for providing the diatoms and Gerd Schneider for discussions.
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