The Bionanoprobe has been developed to study trace elements in frozen-hydrated biological systems with sub-100 nm spatial resolution. Here its performance is demonstrated and first results reported.

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolution beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ∼90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context. ptychography | X-ray fluorescence microscopy | cryogenic biological samples X -ray fluorescence microscopy (XFM) offers unparalleled sensitivity for quantitative mapping of elements, especially trace metals which play a critical role in many biological processes (1-3). It is complementary to light microscopy, which can study some elemental content in live cells (with superresolution techniques possible) but which is more difficult to quantitate because it depends on the binding affinities of fluorophores. However, XFM does not usually show much cellular ultrastructure, because the light elements (such as H, C, N, and O, which are the main constituents of biological materials) have low fluorescence yield (4). At the multi-keV X-ray energies needed to excite most X-ray fluorescence lines of interest, these light elements show little absorption contrast, but phase contrast can be used to image cellular structure (5, 6) and this can be combined with scanned-beam XFM (7-11).One can also acquire phase-contrast X-ray images with a resolution beyond X-ray lens limits by recording the diffraction pattern from a coherently illuminated, noncrystalline sample in an approach called coherent diffraction imaging (CDI) (12). This approach has been used to image isolated dried cells (13-15), and 3-nm resolution has been achieved when imaging silver nanocubes (16). The traditional CDI approach requires that samples meet a so-called "finite support" (17) requirement with no observable scattering outside of a defined region; although some limited success has been obtained (18,19), this finite support condition has proven difficult to achieve with single cells surrounded by ice layers. Ptychography (20-22) is a recently realized CDI method [with an older history (23)] that circumvents this isolated cell requirement by instead scanning a limitedsize coherent illumination spot across the sample. Ptychography has been used to image freeze-dried diatoms at 30-nm resolution (24) and bacter...

Sequestration within the cytoplasm often limits the efficacy of therapeutic nanoparticles that have specific subcellular targets. To allow for both cellular and subcellular nanoparticle delivery we have created Epidermal Growth Factor Receptor (EGFR) targeted Fe3O4@TiO2 nanoparticles that use the native intracellular trafficking of EGFR to improve internalization and nuclear translocation in EGFR-expressing HeLa cells. While bound to EGFR these nanoparticles do not interfere with the interaction between EGFR and karyopherin-β, a protein that is critical for the translocation of ligand-bound EGFR to the nucleus. Thus, a portion of the EGFR targeted nanoparticles taken up by the cells also reaches cell nuclei. We were able to track nanoparticle accumulation in cells by flow cytometry and nanoparticle subcellular distribution by confocal fluorescent microscopy indirectly, using fluorescently labeled nanoparticles. More importantly, we imaged and quantified intracellular nanoparticles directly, by their elemental signatures, using X-ray fluorescence microscopy at the Bionanoprobe, the first instrument of its kind in the world. The Bionanoprobe can focus hard X-rays down to a 30 nm spot size to map the positions of chemical elements tomographically within whole frozen-hydrated cells. Finally, we show that photoactivation of targeted nanoparticles in cell nuclei, dependent on successful EGFR nuclear accumulation, induces significantly more double-stranded DNA breaks then photoactivation of nanoparticles that remain exclusively in the cytoplasm.

X-ray ptychography and fluorescence imaging reveal 3D elemental composition and ultrastructure in frozen-hydrated green algae.

SummaryTrace metals play important roles in biological function, and x‐ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side‐by‐side comparison shows that plunge‐freezing‐based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with fresh media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. When chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x‐ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.