Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

Abstract

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolution beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ∼90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context. ptychography | X-ray fluorescence microscopy | cryogenic biological samples X -ray fluorescence microscopy (XFM) offers unparalleled sensitivity for quantitative mapping of elements, especially trace metals which play a critical role in many biological processes (1-3). It is complementary to light microscopy, which can study some elemental content in live cells (with superresolution techniques possible) but which is more difficult to quantitate because it depends on the binding affinities of fluorophores. However, XFM does not usually show much cellular ultrastructure, because the light elements (such as H, C, N, and O, which are the main constituents of biological materials) have low fluorescence yield (4). At the multi-keV X-ray energies needed to excite most X-ray fluorescence lines of interest, these light elements show little absorption contrast, but phase contrast can be used to image cellular structure (5, 6) and this can be combined with scanned-beam XFM (7-11).One can also acquire phase-contrast X-ray images with a resolution beyond X-ray lens limits by recording the diffraction pattern from a coherently illuminated, noncrystalline sample in an approach called coherent diffraction imaging (CDI) (12). This approach has been used to image isolated dried cells (13-15), and 3-nm resolution has been achieved when imaging silver nanocubes (16). The traditional CDI approach requires that samples meet a so-called "finite support" (17) requirement with no observable scattering outside of a defined region; although some limited success has been obtained (18,19), this finite support condition has proven difficult to achieve with single cells surrounded by ice layers. Ptychography (20-22) is a recently realized CDI method [with an older history (23)] that circumvents this isolated cell requirement by instead scanning a limitedsize coherent illumination spot across the sample. Ptychography has been used to image freeze-dried diatoms at 30-nm resolution (24) and bacter...

Strategies for high-throughput focused-beam ptychography

“…(iv) When smaller illumination spots d are used with smaller ptychographic gain G p , one has the option of simultaneously acquiring high-spatial-resolution scanning microscope images using other contrast modes, such as fluorescence from elemental content (Schropp et al, 2012;Deng et al, 2015bDeng et al, , 2017.…”

Investigating Cellular Nanoscale with X-rays

“…However, as explained above, each of these techniques are based on different physical properties (i.e., diffraction, absorption or emission). Hence, combining two or three of these techniques in simultaneous acquisition has proven their efficiency [47,87,95,96]. However, the most challenging issue in the field of X-ray imaging at the nanoscale remains radiation damage and X-ray optic efficiency.…”

“…However, the heat and small number of radicals that were produced by the beam were decreased by cooling the sample. This was made possible by employing a N 2 cryostream (at 100 K), which has shown great success in crystallography and the imaging of biological specimens [39,70,96]. A sample at the beamline P10 is shown as an example in Fig.…”

Practices and Standards for Data and Processing at the APS

“…As one example, X-ray fl uorescence microscopy provides unprecedented sensitivity for imaging trace element distribution of metals in cells, but the light elements that make up the main mass and ultrastructure of a cell have low fl uorescence yield. As a result, an increasing number of studies [11][12][13] have been combining X-ray fl uorescence with ptychography, providing a way to put the distribution heavier trace elements into a biological context; an example of this is shown in Figure 2. Work at the APS on this combined approach is taking advantage of the DataExchange fi le schema.…”

“…We show here a frozen hydrated Ostreococcus alga imaged at 5.2 keV, where X-ray fl uorescence maps of elemental composition (potassium, sulfur, and phosphorous) were recorded simulateously with X-ray ptychography data recorded using a pixelated array detector and reconstructed using a fast, parallelized code [10]. The lower right image shows an overlay with the psychographic reconstruction, which shows the light element ultrastructure not visible in X-ray fl uorescence [13]. Storage and processing of such data become very challenging without standardized ways to exchange data.…”