Scanning X-Ray Nanodiffraction on Living Eukaryotic Cells in Microfluidic Environments

High-resolution x-ray imaging techniques offer a variety of possibilities for studying the nanoscale structure of biological cells. A challenging task remains the study of cells by x rays in their natural, aqueous environment. Here, we overcome this limitation by presenting scanning x-ray diffraction measurements with beam sizes in the range of a few hundred nm on living and fixed-hydrated eukaryotic cells in microfluidic devices which mimic a native environment. The direct comparison between fixed-hydrated and living cells shows distinct differences in the scattering signal, pointing to structural changes on the order of 30 to 50 nm.

Figure 1

Schematic of the measurement setup at the $P10$ beam line at PETRA III. The x rays are focused onto the sample using two KB mirrors. Microfluidic devices are used as a sample environment for fixed-hydrated and living eukaryotic cells. The scattered intensity is recorded using a Pilatus 300K detector (Dectris Ltd., Baden, Switzerland) (not to scale). The inset shows a detail of the central part of the microfluidic device.

Figure 2

Scanning x-ray nanodiffraction measurements on living cells. (a) Bright-field image of two SK8/18-2 cells recorded directly before attachment of the ${\mathrm{Si}}_3{\mathrm{N}}_4$ membrane window with the cells to the microfluidic device. (b) X-ray dark-field image obtained from a two-dimensional mesh scan with a step size of $1\times 4\mu {\mathrm{m}}^2$ on the marked region in panel (a). (c) Selection of three different regions of interest [blue (dark gray): nucleus, light gray: cell body, red (medium gray): empty region]. (d) Fitting of the radial intensity obtained by azimuthal integration of the averaged scattering patterns from different regions of the cell with a single power law function.

Figure 3

Scanning x-ray nanodiffraction measurements on fixed-hydrated cells in a microfluidic device. (a) Visible light phase contrast image of two SK8/18-2 cells after fixation with formaldehyde. (b) X-ray dark-field image obtained from a two-dimensional mesh scan with a step size of $500\times 500{\mathrm{nm}}^2$ on the same sample region as shown in panel (a). (c) Fitting of the high and low $q_r$-value regions of the background corrected radial intensity profiles from the nuclear region and the cell body with a power law function. The inset indicates the regions used for averaging.

Figure 4

Comparison of power law exponents obtained from fits to the background corrected radial intensities for fixed-hydrated and living SK8/18-2 cells. The error bars indicate the errors obtained from the fits with a power law function.

Figure 5

Averaged radial intensity profiles for living and fixed hydrated cells. (a) Averaged radial intensity profiles of living and fixed hydrated cells for the nucleus and the cell body after background correction. The two sets of data curves are shifted with respect to each other to improve visibility. (b) Difference of the averaged radial intensity profiles for living and fixed hydrated cells (profile for living cells minus profile for fixed cells).