Imaging biomineralizing bacteria in their native-state with X-ray fluorescence microscopy

Abstract

Understanding the interactions between metal-based nanoparticles and biological systems in complex environments (e.g., the human body, soils, and marine settings) remains challenging, especially at single-cell and nanoscale levels. Capturing the dynamics of these interactions, such as metal distribution, nanoparticle growth, or degradation, in their native state (in vivo) is particularly difficult. Here, we demonstrate the direct measurement of iron content in hydrated, magnetite-biomineralizing magnetotactic bacteria using synchrotron-based nanobeam-scanning X-ray fluorescence microscopy combined with a liquid cell environment. In addition to X-ray fluorescence imaging, we collected iron chemical speciation information from individual bacteria in liquid using X-ray absorption spectroscopy. To follow biomineralization in situ, we developed a microfluidic device to track magnetite nanoparticle formation over several hours under the X-ray beam. This approach highlights the potential of X-ray fluorescence microscopy in liquid cell setups to provide elemental and chemical insights into biological processes at the single-cell level. Combining X-ray nanobeam techniques with liquid cell devices will enable more "on-chip" experiments on metals in biological contexts to be conducted at the synchrotron.

Fig. 1. Nanobeam-scanning X-ray fluorescence imaging (nano-XRF) of MSR-1 dried and in a liquid cell. (A) Schematic of a SiN-“sandwich” liquid cell device with immobilized magnetotactic bacteria for nano-XRF measurement (top) and image of the assembled liquid cell with the optical microscopy image of the SiN window region (bottom). MSR-1 measured under dried conditions (B and C) at 100 nm scanning resolution and the liquid cell (D and E) at 200 nm scanning resolution with Fe Kα XRF (B and D) maps and phase gradient (PG) contrast imaging (C and E). The XRF intensity scale (in counts (cts)) in (B) applies to all XRF maps. Arrows in (E) identify weak phase contrast from individual bacteria. Scale bars 2 μm.

Fig. 2. (A) Fe K-edge X-ray absorption near-edge structure (XANES) spectra of MSR-1 bacteria under hydrated conditions (liquid cell) and dried conditions with a magnetite (Fe₃O₄) reference. Zoomed in regions for (B) pre-edge (promotion of 1s electrons to unoccupied 3d valence levels) and (C) white-line (promotion of 1s electrons to unoccupied 4p valence levels) features.

Fig. 3. Representative (A and B) transmission electron microscopy images and (C and D) Fe Kα XRF maps of MSR-1 bacteria show a decrease in intracellular magnetite content in response to iron concentration in the culture medium (dried conditions, 50 μM Fe-citrate, left; 10 μM Fe-citrate, right). (E) Extracted total XRF counts per cells and maximum XRF counts per pixel for dried and liquid cell conditions. The XRF intensity scale (in counts (cts)) applies to all XRF maps. The dotted rectangle in (D) shows a typical integration area of 2 μm² for a single cell. Scale bars are 500 nm (A and B) and 1 μm (C and D).

Fig. 4. Customized microfluidic device for nanobeam-scanning X-ray fluorescence microscopy (nano-XRF). (A) Layer design for soft lithography preparation of a microfluidic layer in PDMS. (B) Assembled microfluidic device with pins and tubing connected and microchannel design overlaid (inset, side view depiction of the device). (C) In-line optical microscope image of the microfluidic device mounted on a nano-scanning stage at the beamline with squares indicating XRF mapped regions in (D) (smaller square) and (E). Fe Kα XRF mapping of MSR-1 in the customized microfluidic device at (D) 150 nm and (E) 200 nm step size. The XRF intensity scale (in counts (cts)) applies to all XRF maps. Scale bars 100 μm (C), 3 μm (D) and 5 μm (E).

Fig. 5. Live–dead fluorescence assay conducted post-X-ray measurement. Epifluorescence microscopy images of (A) X-ray measured channel (top, channel 1 – position a) and (B) distant channel (bottom, channel 6 – position a) (DAPI and PI fluorescence signals are blue and red, respectively). (C) Live–dead assay results corresponding to the position in the microfluidic layer (“X” corresponds to the irradiated region). Scale bars 20 μm.

Fig. 6. In situ magnetite biomineralization experiment conducted in the custom SiN-PDMS microfluidic device. (A) Schematic of the magnetosome induction experiment conducted in the microfluidic device. (B) Fe Kα XRF maps at selected time points from 1 to 8 h over the course of magnetosome formation. (C) Average Fe Kα XRF total counts (top) and maximum count values (bottom) per bacterium for all collected time points. Dashed horizontal lines in (C) refer to the average total Fe Kα XRF counts (top) and maximum counts (bottom) for MSR-1 grown with 10 μM Fe-citrate. The XRF intensity scale applies to all XRF maps. Scale bars 3 μm.