Quantitative biological imaging by ptychographic x-ray diffraction microscopy

Abstract

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3 · 10 5 Gy, and were shown to scale with photon fluence.bacterial nucleoid | cellular imaging | coherent x-ray diffractive imaging | x-ray microscopy

Soft X-ray spectromicroscopy using ptychography with randomly phased illumination

“…A key requirement is that the illuminated areas overlap substantially, so that iterative algorithms [1][2][3] can successfully reconstruct an image of the specimen, retrieving the amplitude and phase of both the complex sample transmission function and the illuminating probe. At X-ray wavelengths, ptychography has been used to characterize the focusing properties of X-ray optics [4][5][6] , to image interconnects within microchips 7 and to image yeast cells 8 . X-ray ptychography has recently been demonstrated using soft X-rays 9,10 , and has been used with polarized illumination to generate dichroic image contrast from magnetic thin films 11 .…”

Coherent X-ray diffractive imaging on the single-cell-level of microbial samples

“…3.29). The images of the sample regions A and B show typical shapes of D. radiodurans aggregates, as known from light and electron microscopy [77,169,342] and a previous ptychographic study [97]). Importantly, scanning di raction with the four observables transmission, phase shift DPC-x, DPC-y and dark eld allows for fast, robust visualisation of biological cells without staining and with the additional bene t of a large FOV that can be easily adapted.…”

“…Especially, in the eld of X-ray microscopy, ptychography has advanced to a powerful tool for imaging on the scale of a few nanometres of arti cial test structures in 2d [266,287,330,332] and 3d [59,133] as well as, for instance, biological specimen in 2d [62,97,172] and 3d [61,109,330]. Moreover, the ptychographic method has also been used for elemental speciation [21,135,285] and for sequential imaging of chemical reactions [142].…”

“…Due to noise in the data, it is convenient to carry out a soft projection onto the measured dataĪ [97] P n {χ n } :…”

“…Recently, ptychography could also be shown to yield information under non-ideal experimental conditions such as a low degree of spatial and temporal coherence of the illuminating wave-eld [78,299] but also vibrational modes of the object could be extracted [42]. In case of unstained biological samples, quantitative two-dimensional images of frozen-hydrated yeast cells [172], freeze-dried cells of the bacterium Deinococcus radiodurans [97] and a three-dimensional representation of a mouse femur [61] were obtained before or during the time of this thesis (more ptychographic applications are listed in section 1.3.6, p. 40).…”