Synchrotron X-ray chemical nano-imaging reveals iron sub-cellular distribution.

<p>The synchrotron X-ray fluorescence nanoprobe end-station installed at ESRF was designed to provide a high flux hard X-ray beam of less than 90 nm size (FWHM, full width at half maximum). The intensity distribution in the focal plane is shown in (A); dopamine producing cells were exposed <i>in vitro</i> to 300 µM FeSO₄ during 24 h (B). Chemical element distributions, here potassium and iron, were recorded on distinct cellular areas such as cell bodies (C), neurite outgrowths, and distal ends (D). Iron was found in 200 nm structures in the cytosol, neurite outgrowths, and distal ends, but not in the nucleus. Iron rich structures are not always resolved by the beam and clusters of larger dimension are also observed. Min-max range bar units are arbitrary. Scale bars = 1 µm.</p

Cellular iron and zinc concentrations (µg/g dry mass; mean±SD; n = 6), obtained by PIXE quantitative micro-analysis.

<p>The data are the mean of six independent analyses performed on areas containing several hundred of cells for each condition of culture. The inhibition of dopamine synthesis (AMT, and AMT+Fe) results in a decrease of total iron concentration, while zinc concentration is not changed, suggesting a specific role of dopamine in iron homeostasis.</p

Iron is localized within dopamine neurovesicles.

<p>Visible light microscopy of freeze-dried cells (A), and epifluorescence microscopy of the same freeze-dried cells (B) enable the identification of dopamine distribution, while synchrotron X-ray fluorescence nano-imaging reveals the distribution of iron (C, D). Panels C and D represent comparison of the same region imaged in a fluorescent mode to visualize dopamine and with X-ray fluorescence to localize iron. Dopamine and iron are co-located within 200 nm structures characteristic of dopamine neurovesicles as identified by epifluorescence microscopy. A large number of iron and dopamine neurovesicles are found in neurite outgrowths (C) and distal ends (D). Min-max range bar units are arbitrary. Scale bars = 1 µm.</p

Nano-imaging of potassium, iron, and zinc in distal ends.

<p>Each series of images are representative of the entire cell population for each condition (control, 1mM AMT and/or 300 µM FeSO₄). The scanned area (left images, red squares) is shown on a bright field microscopy view of the freeze dried cell. Iron is located within dopamine vesicles of 200 nm size or more (Control, and Fe conditions). Iron concentration is close to the limit of detection in distal ends of AMT, and AMT+Fe cells; only a basal level of Fe is observed. Min-max range bar units are arbitrary for potassium and zinc distributions. For iron distribution the maximum threshold values in micrograms per squared centimeter are shown for each color scale. Scale bars = 1 µm.</p

Retrieved information in the reconstructed images.

<p>[<b>a</b>] Transverse, [<b>b</b>] frontal and [<b>c</b>] sagittal slices through the images reconstructed from phase data. Grayscale is proportional to local density. Osteocyte lacunae [Lc] and canaliculi [Ca] can clearly be seen. The heterogeneous organization of the matrix by mineralized collagen fibers can also be distinguished [box]. In this sample, a continuous change in collagen orientation can be seen between adjacent lamellae. The cement line [Cm], separating osteonal [On] and interstitial [It] tissue, can clearly be distinguished as more mineralized than the surrounding matrix. Tissue close to osteocyte lacunae is also hypermineralized. [<b>d</b>] Zoom on the boxed area in C. Matrix orientation is clearly visible and canaliculi are seen as black dots. [<b>e</b>] Mass density histograms in the three tissue types. [<b>f</b>] Samples were extracted from the mid diaphysis of a human femur. [<b>g</b>] The blue cylinder shows the imaged region inside the sample. [<b>h</b>] Schematic of a transverse section showing the organization of lamellar bone in osteons, interstitial tissue and cement lines. Blue circle shows the positioning of A. [<b>i</b>] Rendering of the electron density in the sample [blue] and porosity [yellow]. Structures such as osteocyte lacunae [Lc] and canaliculi [Ca], the cement line [Cm] and collagen fibers are revealed.</p

Experimental setup and image reconstruction.

<p>[<b>a</b>] Schematic of experimental setup. The X-ray beam [X] is monochromatized and focused into a focal spot [F] by X-ray reflective optics [KB]. The sample [S] is positioned on a translation-rotation stage downstream of the focus and imaged onto a stationary detector. Due to the resulting divergent beam, different spot-sample distances [D1] and different free space propagation distances [D2] imply different magnification factors on the detector. [<b>b</b>] Images were recorded at four focus-to-sample distances over a complete turn of the sample at 2999 projection angles. The images were used to reconstruct the phase shift at each angle [phase retrieval PR], which was used as input to a tomographic reconstruction algorithm to reconstruct the 3D local mass density.</p

3D renderings of ultrastructural bone features.

<p>[<b>a</b>] Rendering of osteocyte lacunae and canaliculi in the whole imaged volume overlayed over the bottom slice shown in grayscale. Colors correspond to connected components and grayscale to mass density. Note the difference in structure in the interstitia and osteon: the connected cells are all in the osteonal tissue, the others in the interstitial. The canaliculi are considerably reduced in the interstitia. [<b>b</b>] Zoom on the highlighted lacuna in A showing the interaction between the canaliculi [pink] and the cement line [green], and branching of the canaliculi.</p

Compressive Residual Strains in Mineral Nanoparticles as a Possible Origin of Enhanced Crack Resistance in Human Tooth Dentin

The tough bulk of dentin in teeth supports enamel, creating cutting and grinding biostructures with superior failure resistance that is not fully understood. Synchrotron-based diffraction methods, utilizing micro- and nanofocused X-ray beams, reveal that the nm-sized mineral particles aligned with collagen are precompressed and that the residual strains vanish upon mild annealing. We show the link between the mineral nanoparticles and known damage propagation trajectories in dentin, suggesting a previously overlooked compression-mediated toughening mechanism

Deciphering the Resistance-Counteracting Functions of Ferroquine in <i>Plasmodium falciparum</i>-Infected Erythrocytes

The aminoquinoline chloroquine (CQ) has been widely used for treating malaria since World War II. Resistance to CQ began to spread around 1957 and is now found in all malarious areas of the world. CQ resistance is caused by multiple mutations in the <i>Plasmodium falciparum</i> chloroquine resistance transporter (PfCRT). These mutations result in an increased efflux of CQ from the acidic digestive vacuole (DV) to the cytosol of the parasite. This year, we proposed a strategy to locate and quantify the aminoquinolines in situ within infected red blood cells (iRBCs) using synchrotron based X-ray nanoprobe fluorescence. Direct measurements of unlabeled CQ and ferroquine (FQ) (a ferrocene-CQ conjugate, extremely active against CQ-resistant strains) enabled us to evidence fundamentally different transport mechanisms from the cytosol to the DV between CQ and FQ in the CQ-susceptible strain HB3. These results inspired the present study of the localization of CQ and FQ in the CQ-resistant strain W2. The introduction of the ferrocene core in the lateral side chain of CQ has an important consequence: the transporter is unable to efflux FQ from the DV. We also found that resistant parasites treated by FQ accumulate a sulfur-containing compound, credibly glutathion, in their DV

Imaging of carbon nanotube contamination in lung tissue.

<p>(A) Slice from a low resolution electron density volume. (B) Low resolution 2D fluorescence image of the specimen showing iron, phosphorus and sulfur distributions. The circle indicates the ROI that was chosen for higher resolution imaging. The scanned area was about 0.2×0.3 mm² and the step size was 1.8 µm. (C) A schematic representation of the imaging geometry, showing the ROI inside the sample and the two slices that were chosen for further analysis. Phase contrast slice with 60 nm pixel size (D), Fe fluorescence slice with 500 nm pixel size (E) and correlative image of Fe and phase contrast (F) for slice #1 in the lung specimen. Numbers 1, 2, and 3 indicate positions of alveolar macrophages. Phase contrast (G), Fe fluorescence signal (H) and correlative image of Fe and phase contrast (I) for slice #2 in the lung specimen. Number 4 indicates the position of a type 1 pneumocyte, while number 5 indicates the position of an alveolar macrophage. Notice the factor of 10 difference in the relative fluorescence signal between parts (E, F) and (H, I).</p