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.