X-ray fluorescence microscopy (XFM) offers high sensitivity for quantitative mapping of elements in biological samples. However, a majority of biological structures are not easily visualized in XFM since XFM is relatively blind to the light elements (such as H, C, N, O) which are the main constituents of biological materials. Ptychography, a more recent method of coherent diffraction imaging (CDI) that delivers quantitative phase contrast and high spatial resolution, provides a good solution to visualize biological ultrastructure. While the spatial resolution of ptychography can in theory reach the wavelength limit, radiation damage to biological samples affects the obtainable resolution. A good solution towards this problem is to work with frozen-hydrated biological specimens under cryogenic conditions. We describe here the combination of ptychography and fluorescence for imaging frozen-hydrated specimens at cryogenic temperature on the Bionanoprobe [1]. By this combination, we can obtain simultaneous views of ultrastructure and elemental compositions of specimens at high resolution [2]. To address the bottlenecks for fast ptychography imaging, we develop fly-scan ptychography [3] so that the data acquisition time in this combined technique can be greatly reduced. The challenges in data analysis of this combined method is also discussed.

1. S. Chen et al., J. Synchrotron Radiation 21, 66 (2014).
2. J. Deng et al., Proc. Natl. Acad. Sci. 112, 2314 (2015).
3. J. Deng et al., Opt. Express 23, 5438 (2015).