Multimodality hard-x-ray imaging of a chromosome with nanoscale spatial resolution

Abstract

We developed a scanning hard x-ray microscope using a new class of x-ray nano-focusing optic called a multilayer Laue lens and imaged a chromosome with nanoscale spatial resolution. The combination of the hard x-ray's superior penetration power, high sensitivity to elemental composition, high spatial-resolution and quantitative analysis creates a unique tool with capabilities that other microscopy techniques cannot provide. Using this microscope, we simultaneously obtained absorption-, phase-, and fluorescence-contrast images of Pt-stained human chromosome samples. The high spatial-resolution of the microscope and its multi-modality imaging capabilities enabled us to observe the internal ultra-structures of a thick chromosome without sectioning it.

Figure 1. Schematic of the experimental setup.

A bright and coherent x-ray beam is generated from an undulator source. A monochromatic beam is then selected by the double-crystal monochromator and further shaped by a set of slits. The x-ray beam is focused down to a 13 × 33 nm² spot by two MLLs placed in a cross geometry. In this study because the sample was well isolated, an order-sorting-aperture (OSA) was not used to maximize the working distance. A far-field pixel-array detector and an energy-dispersive detector are used to collect different types of signals as the sample is raster scanned through the focus, thereby yielding absorption-, phase- and fluorescence-contrast images simultaneously.

Figure 2

(a) scanning electron micrograph (SEM) of the examined chromosome (performed after the x-ray measurement). Many globules can be seen on the surface. (b,c) are the x-ray phase-gradient images in horizontal and vertical directions, respectively. They provide enhanced contrast to small features. (d) is the transmission image showing the absorption contrast. Maximum absorption is about 5%, seen around the center of the image. (e) is the phase image, reconstructed from (b,c). Maximum phase retard is seen around the same position in (d) where a high concentration of Ag (f) is present. (f–i) are the concentration maps of Ag, Pt, Ba and Cl, respectively. The units are μg/cm². Note the measured concentration is a projection through the entire thickness. In order to show the weak signal in (g), log scale is used. (j) depicts the β/δ ratio variation. It indicates the composition change. The Pt particle along the top edge can be seen clearly in this map. The scan-step size in (b–j) is 50 nm.

Figure 3. Magnified x-ray images of the enclosed area in Fig. 2a,d,e.

(a) is the SEM image. (b,c) are phase-gradient images in horizontal and vertical directions, respectively. Small globules can be seen. (b) shows more fine features than (c), indicating a better resolution in the horizontal direction. (d) is the absorption-contrast image. Fibrous ultrastructures can be observed. (e) is the phase image (unit in radian). Globules are not as apparent as in (b,c) due to the weak contrast and worse resolution. (f–h) are the distributions of Ag, Pt and Cl, respectively. Units are μg/cm2. (i) is the map. Different types of structures can be seen in the centromere and chromatins. The scan-step is 12 nm.