Quantitative X-ray projection microscopy: phase-contrast and multi-spectral imaging

We outline a new approach to X‐ray projection microscopy in a scanning electron microscope (SEM), which exploits phase contrast to boost the quality and information content of images. These developments have been made possible by the combination of a high‐brightness field‐emission gun (FEG)‐based SEM, direct detection CCD technology and new phase retrieval algorithms. Using this approach we have been able to obtain spatial resolution of < 0.2 µm and have demonstrated novel features such as: (i) phase‐contrast enhanced visibility of high spatial frequency image features (e.g. edges and boundaries) over a wide energy range; (ii) energy‐resolved imaging to simultaneously produce multiple quasi‐monochromatic images using broad‐band polychromatic illumination; (iii) easy implementation of microtomography; (iv) rapid and robust phase/amplitude‐retrieval algorithms to enable new real‐time and quantitative modes of microscopic imaging. These algorithms can also be applied successfully to recover object–plane information from intermediate‐field images, unlocking the potentially greater contrast and resolution of the intermediate‐field regime. Widespread applications are envisaged for fields such as materials science, biological and biomedical research and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to projection microscopy using other sources of radiation, such as visible light and electrons.     

Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object

We demonstrate simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object. Subject to the assumptions explicitly stated in the derivation, the algorithm solves the twin‐image problem of in‐line holography and is capable of analysing data obtained using X‐ray microscopy, electron microscopy, neutron microscopy or visible‐light microscopy, especially as they relate to defocus and point projection methods. Our simple, robust, non‐iterative and computationally efficient method is applied to data obtained using an X‐ray phase contrast ultramicroscope.     

Scanning transmission X-ray microscopy with a fast framing pixel detector

Scanning transmission X-ray microscopy (STXM) is a powerful imaging technique, in which a small X-ray probe is raster scanned across a specimen. Complete knowledge of the complex-valued transmission function of the specimen can be gained using detection schemes whose every-day use, however, is often hindered by the need of specialized configured detectors or by slow or noisy readout of area detectors. We report on sub-50 nm-resolution STXM studies in the hard X-ray regime using the PILATUS, a fully pixelated fast framing detector operated in single-photon counting mode. We demonstrate a range of imaging modes, including phase contrast and dark-field imaging.     

Quantitative phase-amplitude microscopy. III. The effects of noise

We explore the effect of noise on images obtained using quantitative phase‐amplitude microscopy – a new microscopy technique based on the determination of phase from the intensity evolution of propagating radiation. We compare the predictions with experimental results and also propose an approach that allows good‐quality quantitative phase retrieval to be obtained even for very noisy data.     

Desktop X-ray microscopy and microtomography

Recent developments in X-ray microtomography have made it possible to miniaturize a CT scanner into a versatile and cost-effective desktop system that fits into any laboratory environment. The possibilities of the technique are demonstrated for a range of applications. It is also shown how an existing scanning electron microscope with an X-ray detector can, with a specially developed attachment, be transformed into an X-ray microscope and microtomograph.     

Hard X-ray microscopy with Zernike phase contrast

Zernike phase contrast has been added to a full‐field X‐ray microscope with Fresnel zone plates that was in operation at 6.95 keV. The spatial resolution has also been improved by increasing the magnification of the microscope objective looking at the CsI(Tl) scintillation crystal. Cu no. 2000 meshes and a zone plate have been imaged to see the contrast as well as the spatial resolution. A Halo effect coming from the Zernike phase contrast was clearly visible on the images of meshes.     

X-ray microscopy using computerized axial tomography

Computerized axial tomography with MoK_a X-radiation has been used to study a 0.8times0.8 mm column of human femoral bone at a resolution of 15 μm. This non-destructive X-ray microscopic technique allowed ‘sections' to be made with 25 μm separation and the distribution of linear absorption coefficient in each section to be determined.     

Linear phase imaging using differential interference contrast microscopy

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.     

Soft X-ray contact microscopy with nanosecond exposure times

High resolution (better than 20 nm) contact micrographs have been produced with exposure times of about a nanosecond. The illuminating source was a short-lived carbon plasma produced by focusing a single short (～1 ns) 100 J pulse from the Vulcan laser at the Rutherford Appleton Laboratory (RAL) to a 300 μm spot on a graphite target. This plasma emits strongly in the soft X-ray region, particularly at the CVI (3.37 nm) and CV (4.03 nm) lines. The specimens were behind a 100 nm thick Si₃N₄ window, at atmospheric pressure in an environmental cell. The images of diatoms recorded on X-ray resist showed features down to the limit of resolution of the SEM used to view the developed resist, which was about 20 nm.     

An environmental sample chamber for X-ray microscopy

The manufacture, construction and performance of a special specimen holder for biological samples suspended in aqueous solution is described. The holder was designed for use in a scanning transmission X-ray microscope. Using it, we have successfully obtained high-resolution images of fresh cellular and subcellular specimens in suspension and have been able to vary the sample environment during viewing in the microscope.     

Size-selective colloidal-gold localization in transmission X-ray microscopy

Colloidal gold is a useful marker for functional‐imaging experiments in transmission X‐ray microscopy. Due to the low contrast of gold particles with small diameters it is necessary to develop a powerful algorithm to localize the single gold particles. The presented image‐analysis algorithm for identifying colloidal gold particles is based on the combination of a threshold with respect to the local absorption and shape discrimination, realized by fitting a Gaussian profile to the identified regions of interest. The shape discrimination provides the possibility of size‐selective identification and localization of single colloidal gold particles down to a diameter of 50 nm. The image‐analysis algorithm, therefore, has potential for localization studies of several proteins simultaneously and for localization of fiducial markers in X‐ray tomography.     

Wavelet-based image restoration for compact X-ray microscopy

Compact water‐window X‐ray microscopy with short exposure times will always be limited on photons owing to sources of limited power in combination with low‐efficency X‐ray optics. Thus, it is important to investigate methods for improving the signal‐to‐noise ratio in the images. We show that a wavelet‐based denoising procedure significantly improves the quality and contrast in compact X‐ray microscopy images. A non‐decimated, discrete wavelet transform (DWT) is applied to original, noisy images. After applying a thresholding procedure to the finest scales of the DWT, by setting to zero all wavelet coefficients of magnitude below a prescribed value, the inverse DWT to the thresholded DWT produces denoised images. It is concluded that the denoising procedure has potential to reduce the exposure time by a factor of 2 without loss of relevant image information.     

Scanning luminescence X-ray microscopy: Imaging fluorescence dyes at suboptical resolution

Scanning luminescence X-ray microscopy is based on the use of the very small focused probe of a scanning X-ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X-ray source and a Fresnel zone plate to produce a focused X-ray probe, images of P31 phosphor grains with a resolution of 50–75 nm have been obtained, and luminescence from polystyrene spheres loaded with 50–100 μmol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X-ray probe (the microscope has imaged features at 36-nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye-tagged sites of biochemical activity at the resolution of the X-ray microscope in wet, unsectioned, and unfixed cells, especially with soft X-ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye-tagged features should be 2–22 times lower than it is in transmission X-ray microscopy. A possible extension of the technique for three-dimensional imaging at the transverse resolution of the X-ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.     

High resolution protein localization using soft X-ray microscopy

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive 'water window', where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.     

Holographic measurement of the wave aberration of an electron microscope by means of the phases in the Fourier spectrum

The resolution limit achievable by holographic correction of the aberrations of an electron microscope depends critically on the information available about the microscope parameters when the hologram was taken. The measuring technique based on symmetry relations of the phases in the Fourier spectrum of the reconstructed electron wave is outlined and experimentally tested.     

Flash X-ray microscopy with a gas jet plasma source

A novel flash X-ray source, the gas jet plasma source, has been used for contact X-ray microscopy. Using a wavelength range of 2–7 nm a resolution of the order of 30 nm can be obtained. The gas jet plasma source provides a new and unique tool which should allow future imaging of wet live cells.     

Advanced thin film technology for ultrahigh resolution X-ray microscopy

Further progress in the spatial resolution of X-ray microscopes is currently impaired by fundamental limitations in the production of X-ray diffractive lenses. Here, we demonstrate how advanced thin film technologies can be applied to boost the fabrication and characterization of ultrahigh resolution X-ray optics. Specifically, Fresnel zone plates were fabricated by combining electron-beam lithography with atomic layer deposition and focused ion beam induced deposition. They were tested in a scanning transmission X-ray microscope at 1.2 keV photon energy using line pair structures of a sample prepared by metalorganic vapor phase epitaxy. For the first time in X-ray microscopy, features below 10 nm in width were resolved.     

A vacuum-compatible wet-specimen chamber for compact X-ray microscopy

Soft X‐ray microscopy is a powerful tool for investigations of, for example, polymers or soils in their natural liquid environment. This requires a wet‐specimen chamber. Compact X‐ray microscopy allows the horizontal mounting of such samples, thereby reducing the influence of gravitational forces. We have developed a wet‐specimen chamber for such compact X‐ray microscope. The chamber is vacuum compatible, which reduces the exposure time. The vacuum sealing is achieved by a combination of mechanical sealing and sealing by bio‐compatible glue. With the wet‐specimen chamber the specimens can be kept in an aqueous environment in a vacuum of 10⁻⁴ mbar for several hours. Imaging of lipid droplets in water demonstrates the function of the wet‐specimen chamber.     

Ultrastructural characterization of isolated rat Kupffer cells by transmission X-ray microscopy

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X-ray microscopy as well as transmission electron microscopy. X-ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent-shaped vacuoles that were half X-ray lucent, half X-ray dense; others were uniformly dense. The number of crescent-shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent-shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X-ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent-shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent-shaped versus uniform vacuoles — might be due to different three-dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron-lucent and electron-dense. Based on the observation of only one type of vacuole by transmission X-ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent-shaped vacuole. From the present data it can be concluded that transmission X-ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.     

Reconstruction of the complex reflectance function in acoustic microscopy

We describe the theory and practical implementation of an iterative computer algorithm (an extension of the Gerchberg Saxton algorithm, originally developed for electron microscopy) for enabling an estimate of the complex reflectance function of a material to be reconstructed from measured values of only the magnitude of the response of an acoustic microscope (i.e. without the phase of the transducer voltage). Results are presented for eight materials measured with a spherical lens (at 320 MHz) and five with a cylindrical line-focus lens (at 210 and 228 MHz).