Claus Flachenecker

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.

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.

The Bionanoprobe: Synchrotron-based Hard X-ray Fluorescence Microscopy for 2D/3D Trace Element Mapping.

Trace elements, particularly metals, play an important role in a large variety of cellular processes in a biological system. In the context of biological organisms and tissues, the term trace element means that over the entire organism an element is present at only trace levels, say 100 ppm or lower. Trace element distribution and content can be analyzed using several techniques, for example, visible light optical fluorescence imaging, energy-dispersive x-ray spectroscopy on an electron microscope, synchrotron-based x-ray fluorescence (XRF) imaging, secondary-ion mass spectrometry, and laser ablation inductively coupled with mass spectrometry. Comprehensive reviews on these techniques are given by Lobinski et al. [1] and McRae et al. [2]. Among these techniques, synchrotron-based XRF microscopy, particularly utilizing third-generation x-ray sources and advanced x-ray focusing optics, offers the most suitable capabilities to perform trace element studies of biological samples: The penetrating power and non-destructive nature of x-rays allows one to image many-micron-thick biological samples such as biological whole cells in a way that visible light or electron microscopes cannot; the sensitivity of x-ray-induced XRF is down to parts per million, several orders of magnitude better than standard electron-based techniques due to the absence of bremsstrahlung background in x-ray-induced x-ray emission. The capability of imaging frozen samples in both 2D and 3D with sub-50 nm resolution in various x-ray modes has greatly advanced a broad range of scientific studies. This article describes how this technique can be used to track the incorporation of nanocomposites into cancer cells.

2D/3D cryo X-ray fluorescence imaging at the bionanoprobe at the advanced photon source

Trace elements, particularly metals, play very important roles in biological systems. Synchrotron-based hard X-ray fluorescence microscopy offers the most suitable capabilities to quantitatively study trace metals in thick biological samples, such as whole cells and tissues. In this manuscript, we have demonstrated X-ray fluorescence imaging of frozen-hydrated whole cells using the recent developed Bionanoprobe (BNP). The BNP provides spatial resolution down to 30 nm and cryogenic capabilities. Frozen-hydrated biological cells have been directly examined on a sub-cellular level at liquid nitrogen temperatures with minimal sample preparation.

Fluorescence Micro-tomography of Frozen-hydrated Whole Cells using the Bionanoprobe

1 Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439, USA 2 Carl Zeiss, Pleasanton, CA 94588, USA 3 Department of Radiation Oncology, Northwestern University, Chicago, IL 60611, USA 4 Applied Physics, Northwestern University, Evanston, IL 60208, USA 5 Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA 6 Synchrotron Research Center, Northwestern University, Argonne, IL 60439, USA 7 Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA

Sub-100-nm 3D-elemental mapping of frozen-hydrated cells using the bionanoprobe

Hard X-ray fluorescence microscopy is one of the most sensitive techniques to perform trace elemental analysis of unsectioned biological samples, such as cells and tissues. As the spatial resolution increases beyond sub-micron scale, conventional sample preparation method, which involves dehydration, may not be sufficient for preserving subcellular structures in the context of radiation-induced artifacts. Imaging of frozen-hydrated samples under cryogenic conditions is the only reliable way to fully preserve the three dimensional structures of the samples while minimizing the loss of diffusible ions. To allow imaging under this hydrated “natural-state” condition, we have developed the Bionanoprobe (BNP), a hard X-ray fluorescence nanoprobe with cryogenic capabilities, dedicated to studying trace elements in frozen-hydrated biological systems. The BNP is installed at an undulator beamline at Life Sciences Collaboration Access Team at the Advanced Photon Source. It provides a spatial resolution of 30 nm for fluorescence imaging by using Fresnel zone plates as nanofocusing optics. Differential phase contrast imaging is carried out in parallel to fluorescence imaging by using a quadrant photodiode mounted downstream of the sample. By employing a liquid-nitrogen-cooled sample stage and cryo specimen transfer mechanism, the samples are well maintained below 110 K during both transfer and X-ray imaging. The BNP is capable for automated tomographic dataset collection, which enables visualization of internal structures and composition of samples in a nondestructive manner. In this presentation, we will describe the instrument design principles, quantify instrument performance, and report the early results that were obtained from frozen-hydrated whole cells.