X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam

Abstract

X-ray Fluorescence (XRF) microscopy is a growing approach for imaging the trace element concentration, distribution, and speciation in biological cells at the nanoscale. Moreover, three-dimensional nanotomography provides the added advantage of imaging subcellular structure and chemical identity in three dimensions without the need for staining or sectioning of cells. To date, technical challenges in X-ray optics, sample preparation, and detection sensitivity have limited the use of XRF nanotomography in this area. Here, XRF nanotomography was used to image the elemental distribution in individual E. coli bacterial cells using a sub-15 nm beam at the Hard X-ray Nanoprobe beamline (HXN, 3-ID) at NSLS-II. These measurements were simultaneously combined with ptychography to image structural components of the cells. The cells were embedded in small (3–20 µm) sodium chloride crystals, which provided a non-aqueous matrix to retain the three-dimensional structure of the E. coli while collecting data at room temperature. Results showed a generally uniform distribution of calcium in the cells, but an inhomogeneous zinc distribution, most notably with concentrated regions of zinc at the polar ends of the cells. This work demonstrates that simultaneous two-dimensional ptychography and XRF nanotomography can be performed with a sub-15 nm beam size on unfrozen biological cells to co-localize elemental distribution and nanostructure simultaneously.

Nucleoid remodeling during environmental adaptation is regulated by HU-dependent DNA bundling

“…Although, we observed distinct sub-regions such as nucleoid core and macrodomains, within the nucleoid region in E. coli, their exact composition and protein:nucleic acid ratio cannot be determined with SXT alone. Nevertheless, our observations correlate with previous observations of longitudinal density bundling and radial confinement of E. coli nucleoid 18,38,39 . Similar to histones in eukaryotes, HU that binds non-sequencespecific DNA, is essential for bacterial nucleoid condensation that relates to global gene expression 3,40 .…”

Subcellular Chemical Imaging: New Avenues in Cell Biology

“…This has been recently performed on a freeze-dried human phagocytic cell [16] and human red blood cells infected with the malaria parasite Plasmodium falciparum [70]. An alternative to phase contrast imaging is ptychography [71], which can be combined with XRF tomography to obtain the 3D localization of elements in cells, such as bacteria in [72].…”

Spectroscopic imaging with single acquisition ptychography and a hyperspectral detector

“…Unlike other methods such as fluorescence imaging with nano-probes 40,41 , or spectral ptychography through energy scans 10–14 , the proposed technique gives access to spectral information at resolutions beyond the beam size from a single two dimensional scan. The focus of this study was on the spectral capabilities of the technique and the chosen sample is not sufficient for a meaningful evaluation of the spatial resolution.…”