Dose-efficient multimodal microscopy of human tissue at a hard X-ray nanoprobe beamline

Abstract

X-ray fluorescence microscopy performed at nanofocusing synchrotron beamlines produces quantitative elemental distribution maps at unprecedented resolution (down to a few tens of nanometres), at the expense of relatively long measuring times and high absorbed doses. In this work, a method was implemented in which fast low-dose in-line holography was used to produce quantitative electron density maps at the mesoscale prior to nanoscale X-ray fluorescence acquisition. These maps ensure more efficient fluorescence scans and the reduction of the total absorbed dose, often relevant for radiation-sensitive (e.g. biological) samples. This multimodal microscopy approach was demonstrated on human sural nerve tissue. The two imaging modes provide complementary information at a comparable resolution, ultimately limited by the focal spot size. The experimental setup presented allows the user to swap between them in a flexible and reproducible fashion, as well as to easily adapt the scanning parameters during an experiment to fine-tune resolution and field of view.

Keywords: X-ray fluorescence emission spectroscopy; X-ray microscopy; in-line holography.

Figure 1

Schematic representation of the multimodal X-ray microscopy experimental setup. KB mirrors focus X-rays down to a focal spot of about 70 nm and with a 1.2 mrad divergence. The sample is mounted on a motor stack enabling xyz translation and θ, φ rotation, i.e. around the x and y axes, respectively. The sample can be translated in and out of the X-ray beam focus via translation along z (total travel range 46 mm), effectively tuning the X-ray beam size. An ion chamber is positioned downstream of the KB chamber to monitor the incoming X-ray photon flux. An optical in-line microscope provides a view onto the sample along the optical z axis. A fluorescence detector is positioned 15 mm away from focus, with a 15° orientation with respect to the focal plane. An in-line area detector is positioned 1.12 m downstream of the focus to collect holograms.

Figure 2

Preliminary reconstructions from a 5 × 5 holography scan covering a potential ROI of a section of an Os-stained human peripheral sural nerve biopsy from a healthy male. These qualitative results were used to delimit a ROI on which to perform a nanoscale X-ray fluorescence scan, as more conveniently annotated in Fig. 3 ▸. Further processing followed. The frame number is annotated on each frame, revealing the scanning sequence.

Figure 3

Quantitative result obtained from processing the images from Fig. 2 ▸. The ROI on which a nanoscale X-ray fluorescence scan was performed is highlighted by the white rectangle. A blood vessel (blue arrow) as well as myelin layers surrounding several axons (red arrows) can be identified. The intensity scale represents relative electron density variations and for convenience was converted into units of Å⁻³.

Figure 4

(a)–(c) Area mass density maps obtained via nanoscale X-ray fluorescence emission spectroscopy for (a) Cl, (b) Fe and (c) Os within a ROI of a section of an Os-stained human peripheral sural nerve biopsy from a healthy male. Linear intensity scales are shown in units of ng mm⁻². (d) RGB representation of the same region including electron density information from the holography scan: red, green and blue channels represent electron density, Fe and Os area mass densities, respectively. A 10 µm scale bar for all images is given in (a).