The Bionanoprobe
Figure 3 : Components of the Bionanoprobe: (a) liquid nitrogen dewar attached to the vacuum chamber. Liquid nitrogen provides conductive cooling to components holding the specimen inside the chamber. A laser interfer- ometer system is used for precise stage positioning. (b) Robot used for sample exchange inside the chamber. Reproduced with permission of the International Union of Crystallography from [ 3 ].
cell (NIH/3T3 originally purchased from ATCC, USA). Quan- titative information on the elemental concentration is obtained by comparing the fl uorescence intensities with a calibration curve derived from measurements of a thin-fi lm XRF standard (RF8-200-S2453, AXO DRESDEN GmbH, Germany).
Bionanoprobe . Challenges arise in both instrumentation and sample preparation: How can an incident X-ray beam be produced suffi ciently small and stable to probe individual organelles? How to preserve both the structure and chemistry of samples as in their natural states? How to prevent damage of a sample under intense X rays and repeated imaging? To overcome these challenges, we developed the Bionanoprobe (BNP), an X-ray microscope with a sub-50 nm X-ray probe size, tomography cap- abilities, and a cryogenic sample environment.
Figure 4 : A survey scan acquired using an 850 nm step size of a frozen-hydrated HeLa cell sample treated with Fe 3 O 4 @TiO 2 nanocomposites for 30 min. Cells were identifi ed using the S fl uorescence signal. The white box indicates the cell examined in Figures 5 and 6 . The yellow box shows where the background signal was acquired. Reproduced with permission of the International Union of Crystallography from [ 3 ].
Figure 5 : X-ray fl uorescence spectra, from the cell and a background region of Figure 4 . The elements S, Ca, Ti, and Fe exhibit signals above background within the cell.
2015 May •
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T e BNP is housed at an undulator beamline of the Life Sciences Collaboration Access Team at the Advanced Photon Source (APS) of Argonne National Laboratory, where the incident X-ray energy (E) is in the range of 4.5–35 keV with an energy resolution (Δ E/E) of 2×10 -4 . T e BNP is dedicated to the studies of trace elements within frozen biological samples and other materials at sub-50 nm spatial resolution. It operates under high vacuum (10 -7 -10 -8 torr) and cryogenic (<110 K) conditions ( Figure 3 ). Samples are conductively cooled using liquid nitrogen. T e vacuum condition protects frozen samples from frosting and minimizes air absorption of low-energy fl uorescence X rays. T e motion of the scanning stages is precisely controlled using laser interferometer systems. Using the BNP at an incident photon energy of 10 keV, we have observed 25 nm features on a resolution test pattern [ 3 ]. T e practical spatial resolution of analysis is about an order of magnitude better than the other existing XRF microscopes at the APS. Very recently we have demonstrated ptychographic imaging at the same time of XRF imaging, providing phase contrast images with a spatial resolution theoret- ically only limited by the wavelength of the incident X rays [ 4 ]. Biological samples have been studied in the frozen- hydrated state at a temperature below 110 K. A robotic sample loading mechanism is used to transfer samples onto the sample stage under cryogenic conditions. T e ability to perform cryogenic experiments has greatly advanced X-ray fl uorescence studies, particularly for organic samples, such as biological cells and tissues. Biological cells typically contain more than 90% of water. By fast freezing, cellular water is retained as amorphous ice, and the cellular structure and ionic distributions are more faithfully preserved in their natural states compared to samples prepared by dehydration methods. Methods for cryogenic sample preparation have been investigated such as discussed by Jin et al. [ 5 ]. In addition, the radiation resistance of organic materials is improved at low temperatures [ 6 ]. Tomography . However, difficulties arise in accurately interpreting 2D images, particularly those collected from thick samples, such as cryogenically fixed biological whole cells because of the lack of information at various depths. X-ray fluorescence tomography is highly desired in this case. The BNP employs a rotation sample stage with a vertical rotation axis, enabling a total rotation of 180 °. Typically, about sixty 2D projections are collected with an estimated accumulated dose of 10 10 Gy. No radiation
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