Scanning X-ray microprobes operating in the multi-keV region are excellent tools for the quantitative mapping of trace elements by X-ray fluorescence (XRF), often combined with X-ray absorption near-edge structure (XANES) measurements to provide localized information about chemical speciation (see e.g.
Paunesku et al.
; Fahrni, 2007
). However, these techniques often do not image the ultrastructure of biological specimens well owing to the low photoelectric absorption and low fluorescent yield of light elements in this energy range. This can make it difficult to put trace elements into their structural context.
Phase contrast has received increased recognition as a complementary contrast mechanism in recent years (Momose, 2005
). At lower photon energies, it can significantly reduce the radiation dose imposed on the specimen compared with absorption imaging (Schmahl et al.
; Schneider, 1998
). At higher energies, phase contrast dominates over absorption contrast particularly for light elements (see Fig. 1) and provides a means to image weakly absorbing specimens such as biological tissue.
Figure 1 Number of photons required to see a 50 nm-thick protein structure in either air or 50 nm of water (Hornberger et al., 2006 ). The calculation is based on a simple projection (for absorption) and refraction [for differential (more ...)
In an X-ray microprobe (which at lower photon energies is typically called a scanning transmission X-ray microscope, or STXM) an image is formed by raster-scanning the specimen through a focused X-ray beam (see Fig. 2). A fluorescence spectrum can be collected at each scan position using an energy-dispersive detector, and the total transmitted intensity and therefore specimen absorption is commonly measured with an area-integrating detector downstream of the specimen. Phase variations in the specimen do not affect the total transmitted intensity, but refract and diffract the beam, leading to a redistribution of intensity in the detector plane (for a mathematical description, see, for example, Hornberger et al.
). This redistribution can be measured with an appropriately configured detector.
Figure 2 Schematic of a scanning X-ray microprobe. An optic, in our case a Fresnel zone plate (Michette, 1986 ), produces an X-ray focus, through which the specimen is raster-scanned. The image is acquired by recording the detector signal(s) at each scan (more ...)
The most flexible configuration is a fully pixelated detector such as a CCD (Chapman et al.
; Gianoncelli et al.
), whose response function can be modified arbitrarily in software after data acquisition. However, the pixel dwell time for transmission images in modern microprobes can be less than a millisecond; such short readout times are difficult to achieve with currently available pixel detectors. Moreover, to obtain a statistically meaningful signal in a large number of detector pixels a high dose to the specimen is required. Therefore, we have pursued the approach of a detector with fewer (eight to ten) segments, which has the advantages of fast readout (about 10 µs) and far reduced storage and data processing requirements compared with acquiring a full CCD frame at each image pixel. With such a detector, the difference signal of opposing segments provides differential phase contrast (Dekkers & Lang, 1974
; Palmer & Morrison, 1991
), which is a measure of the phase gradient of the specimen. The sum of all segments provides the same absorption signal as a single large-area detector. More elaborate analysis techniques allow the quantitative reconstruction of the specimen phase shift (see §3
We have recently reported on the development of a segmented detector for use with a soft X-ray STXM at the National Synchrotron Light Source (NSLS) (Feser et al.
). Now we describe the adaptation of that detector for use with harder X-rays at a third-generation synchrotron such as the Advanced Photon Source (APS). The detector consists of a segmented silicon photodiode chip and a set of charge integrating electronics, providing excellent performance even at the highest flux rates. The signal can be collected in parallel with the fluorescence spectrum for simultaneous absorption, phase and fluorescence imaging with intrinsic registration of all images. We present differential phase contrast examples which demonstrate greatly improved contrast over absorption imaging in the multi-keV range.