Abstract
Quantitative waveguide-based X-ray phase contrast imaging has been carried out on the level of single, unstained, unsliced and freeze-dried bacterial cells of Bacillus thuringiensis and Bacillus subtilis using hard X-rays of 7.9 keV photon energy. The cells have been prepared in the metabolically dormant state of an endospore. The quantitative phase maps obtained by iterative phase retrieval using a modified hybrid input-output algorithm allow for mass and mass density determinations on the level of single individual endospores but include also large field of view investigations. Additionally, a direct reconstruction based on the contrast transfer function is investigated, and the two approaches are compared. Depending on the field of view and method, a resolution down to 65 nm was achieved at a maximum applied dose of below 5 × 105 Gy. Masses in the range of about ∼110-190 (20) fg for isolated endospores have been obtained.
Highlights
Coherent X-ray diffraction imaging (CDI) has reached a resolution level where internal features of single eukaryotic and prokaryotic cells can be analysed, making it a powerful tool for quantitative microscopic investigations on the scale of a few nanometres
Cryo-electron microscopy has advanced to a powerful alternative for high-resolution imaging of biological samples in their near native state, but the contrast is very weak and this technique does not allow for quantitative analyses (Dubochet, 2012; Dubochet et al, 1988; Eltsov & Dubochet, 2005; Lucicet al., 2005)
We investigate advantages and disadvantages of current waveguide-imaging schemes with respect to resolution, dose and quantitativeness, aiming at unprecedented structural analyses of bacterial spores that are not simultaneously accessible by techniques such as atomic force microscopy, transmission electron microscopy and traditional mass density measurement methods
Summary
Coherent X-ray diffraction imaging (CDI) has reached a resolution level where internal features of single eukaryotic and prokaryotic cells can be analysed, making it a powerful tool for quantitative microscopic investigations on the scale of a few nanometres. In this work the species Bacillus subtilis and Bacillus thuringiensis are imaged as unsliced, unstained, freeze-dried samples without the use of chemical fixation by quantitative phase retrieval, enabling electron density measurements and mass density determinations. Cryo-electron microscopy has advanced to a powerful alternative for high-resolution imaging of biological samples in their near native state (without staining), but the contrast is very weak and this technique does not allow for quantitative analyses (Dubochet, 2012; Dubochet et al, 1988; Eltsov & Dubochet, 2005; Lucicet al., 2005). We investigate advantages and disadvantages of current waveguide-imaging schemes with respect to resolution, dose and quantitativeness, aiming at unprecedented structural analyses of bacterial spores that are not simultaneously accessible by techniques such as atomic force microscopy, transmission electron microscopy and traditional mass density measurement methods. The work closes by summarizing the results, comparing the mass measurements with results from other work in different fields and giving an outlook
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