Junjing Deng

The Bionanoprobe: Hard X-ray Fluorescence Nanoprobe with Cryogenic Capabilities

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.

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Significance X-ray fluorescence microscopy provides unparalleled sensitivity for measuring the distribution of trace elements in many-micrometer-thick specimens, whereas ptychography offers a path to the imaging of weakly fluorescing biological ultrastructure at beyond-focusing-optic resolution. We demonstrate here for the first time, to our knowledge, the combination of fluorescence and ptychography for imaging frozen-hydrated specimens at cryogenic temperatures, with excellent structural and chemical preservation. This combined approach will have significant impact on studies of the intracellular localization of nanocomposites with attached therapeutic or diagnostic agents, help elucidate the roles of trace metals in cell development, and further the study of diseases where trace metal misregulation is suspected (including neurodegenerative diseases). 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.

Epidermal Growth Factor Receptor Targeted Nuclear Delivery and High Resolution Whole Cell X-Ray Imaging of Fe3O4@TiO2 Nanoparticles in Cancer Cells

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 than photoactivation of nanoparticles that remain exclusively in the cytoplasm.

Parallel ptychographic reconstruction

Ptychography is an imaging method whereby a coherent beam is scanned across an object, and an image is obtained by iterative phasing of the set of diffraction patterns. It is able to be used to image extended objects at a resolution limited by scattering strength of the object and detector geometry, rather than at an optics-imposed limit. As technical advances allow larger fields to be imaged, computational challenges arise for reconstructing the correspondingly larger data volumes, yet at the same time there is also a need to deliver reconstructed images immediately so that one can evaluate the next steps to take in an experiment. Here we present a parallel method for real-time ptychographic phase retrieval. It uses a hybrid parallel strategy to divide the computation between multiple graphics processing units (GPUs) and then employs novel techniques to merge sub-datasets into a single complex phase and amplitude image. Results are shown on a simulated specimen and a real dataset from an X-ray experiment conducted at a synchrotron light source.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. By working with cells that have been rapidly frozen without the use of chemical fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.

Ultraviolet Germicidal Irradiation and Its Effects on Elemental Distributions in Mouse Embryonic Fibroblast Cells in X-Ray Fluorescence Microanalysis

Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.

Nanoscale x-ray imaging of circuit features without wafer etching

Modern integrated circuits (ICs) employ a myriad of materials organized at nanoscale dimensions, and certain critical tolerances must be met for them to function. To understand departures from intended functionality, it is essential to examine ICs as manufactured so as to adjust design rules, ideally in a non-destructive way so that imaged structures can be correlated with electrical performance. Electron microscopes can do this on thin regions, or on exposed surfaces, but the required processing alters or even destroys functionality. Microscopy with multi-keV x-rays provides an alternative approach with greater penetration, but the spatial resolution of x-ray imaging lenses has not allowed one to see the required detail in the latest generation of ICs. X-ray ptychography provides a way to obtain images of ICs without lens-imposed resolution limits, with past work delivering 20-40 nm resolution on thinned ICs. We describe a simple model for estimating the required exposure, and use it to estimate the future potential for this technique. Here we show for the first time that this approach can be used to image circuit detail through an unprocessed 300 μm thick silicon wafer, with sub-20 nm detail clearly resolved after mechanical polishing to 240 μm thickness was used to eliminate image contrast caused by Si wafer surface scratches. By using continuous x-ray scanning, massively parallel computation, and a new generation of synchrotron light sources, this should enable entire non-etched ICs to be imaged to 10 nm resolution or better while maintaining their ability to function in electrical tests.

Opportunities and limitations for combined fly-scan ptychography and fluorescence microscopy

X-ray fluorescence offers unparalleled sensitivity for imaging the nanoscale distribution of trace elements in micrometer thick samples, while x-ray ptychography offers an approach to image weakly fluorescing lighter elements at a resolution beyond that of the x-ray lens used. These methods can be used in combination, and in continuous scan mode for rapid data acquisition when using multiple probe mode reconstruction methods. We discuss here the opportunities and limitations of making use of additional information provided by ptychography to improve x-ray fluorescence images in two ways: by using position-error-correction algorithms to correct for scan distortions in fluorescence scans, and by considering the signal-to-noise limits on previously-demonstrated ptychographic probe deconvolution methods. This highlights the advantages of using a combined approach.

Strategies for high-throughput focused-beam ptychography

X-ray ptychography is being utilized for a wide range of imaging experiments with a resolution beyond the limit of the X-ray optics used. Here, strategies for data sampling and for increasing imaging throughput when the specimen is at the focus of an X-ray beam are discussed, and the tradeoffs between large and small illumination spots are examined.

Simultaneous x-ray nano-ptychographic and fluorescence microscopy at the bionanoprobe

Hard X-ray fluorescence (XRF) microscopy offers unparalleled sensitivity for quantitative analysis of most of the trace elements in biological samples, such as Fe, Cu, and Zn. These trace elements play critical roles in many biological processes. With the advanced nano-focusing optics, nowadays hard X-rays can be focused down to 30 nm or below and can probe trace elements within subcellular compartments. However, XRF imaging does not usually reveal much information on ultrastructure, because the main constituents of biomaterials, i.e. H, C, N, and O, have low fluorescence yield and little absorption contrast at multi-keV X-ray energies. An alternative technique for imaging ultrastructure is ptychography. One can record far-field diffraction patterns from a coherently illuminated sample, and then reconstruct the complex transmission function of the sample. In theory the spatial resolution of ptychography can reach the wavelength limit. In this manuscript, we will describe the implementation of ptychography at the Bionanoprobe (a recently developed hard XRF nanoprobe at the Advanced Photon Source) and demonstrate simultaneous ptychographic and XRF imaging of frozen-hydrated biological whole cells. This method allows locating trace elements within the subcellular structures of biological samples with high spatial resolution. Additionally, both ptychographic and XRF imaging are compatible with tomographic approach for 3D visualization.