Subcellular Imaging of Trace Metal Distribution and Chemistry by X-Ray Microfluorescence*
Barry Lai
Advanced Photon Source, Argonne National Laboratory
X-ray fluorescence microscopy is ideally suited for studying trace-metal distribution due to its inherent elemental sensitivity of ~ 0.01-10 part per million (ppm). It enables studies of inter- and intra-cellular distributions of elements from phosphorus to heavy metals, with simple yet accurate quantification. Because a finely focused x-ray beam is used to excite the atomic emission, the total metal concentration can be measured directly without the need of labeling with fluorescent sensors. This provides a complementary technique to conventional optical fluorescence microscopy, which mainly detects chelatable metals.
Currently a spatial resolution of ~ 200 nm is achieved routinely at the 2-ID-D station of the APS, with the minimum detection limit as low as 3 attograms for zinc (3x10-18 gm, or 2.7x104 atoms) within one second of data acquisition time. Typically, images of as much as 10-15 different elements are acquired simultaneously, thus ensuring complete alignment between the images. The large penetration depth of x-rays allows the study of whole cells without sectioning, tissue sections of > 10-mm thickness, and hydrated/frozen samples. In addition, the possibility of performing micro-XANES analysis at discrete locations enables the oxidation state for the element of interest to be determined. These unique capabilities had been employed in single cells and bacteria studies of environmental toxins (As, Hg, Pb, U), carcinogens (Cr), therapeutic agents (Pt, Sb), nanobiocomposites (Ti), and metalloproteins (Fe, Cu, Zn).
With the proposed CHESS ERL source and the advance of x-ray nanofocusing optics, it will be possible to produce a 1-nm x-ray spot with > 1012 ph/s/0.01%BW. This will allow the detection of even a few metal atoms with nanometer spatial resolution, which is sufficient to locate most metalloproteins and metal-containing macromolecules individually within a cell. This unprecedented capability however must be harvested against the challenge of radiation damage which will be particularly severe for any nano-spectroscopy measurement. We will discuss instrumentation and methods that have been implemented, demonstrate their application in ongoing studies, and delineate the future prospects.
Figure: Thin section of a melanoma cell MNT-1 treated with cisplatin CDDP. Top row: elemental image of Pt, Cu, and P. Bottom row: overlay image of the three elements, and the corresponding electron micrograph (courtesy of Richard Leapman, NIH).
* Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-ENG-38.
