Quantitative X-ray phase contrast waveguide imaging of bacterial endospores

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.

Keywords: Bacillus subtilis; Bacillus thuringiensis; contrast transfer function; endospores; holotomography; phase retrieval; waveguide imaging.

Figure 1

Schematic of the GINIX waveguide setup: downstream of the undulator source and the monochromator (not shown): (1) slits S1, (2) attenuation foils made of Al, (3) slits S2, (4) KB mirror system, (5) X-ray waveguide (entrance in the focal plane of the KB mirrors), (6) bacterial sample at a distance to the waveguide and (7) detection device at a distance to the waveguide. The flight tube between sample and detector is not shown in the image.

Figure 2

Data of one position on region (A) of the sample with B. thuringiensis cell material (cf. Table 1): (a) hologram and (b) phase of holographic reconstruction which already yields a good impression of the sample structure. For instance, the rod-shaped bacteria (or their remnant empty cell envelopes) can be identified. However, the reconstruction appears distorted owing to the twin image problem. (c) The ‘radial’ gradient of the holographic reconstruction in (b) used for estimating the support (cf. text). (d) Product of the final support and holographic phase reconstruction. Scale bars in (a) and in (b), (c), (d) denote 1 mm (real detector dimension) and 5 µm (effective geometry), respectively.

Figure 3

Optical light micrographs (differential interference contrast, 40×) of the samples of (a) freeze-dried B. thuringiensis cells and endospores and (b) freeze-dried B. subtilis endospores. The regions that were analysed with waveguide-based imaging are indicated by dashed black frames and labels (A), (B), (C) and (D) in both images. The bacteria and remnant cell material in (a) are mainly rod shaped (white arrow heads). Smaller spots are likely to be isolated endospores (black arrow head). An isolated endospore is indicated in (b) by a black arrow head.

Figure 4

(a) A TEM image of two lysing B. thuringiensis cells. The cell walls (CW) are already fragmented. One cell contains the typical protein crystal (PC) of B. thuringiensis cells, whereas the other cell is occupied by an endospore (dashed frame) that is surrounded by the exosporium (EX). Holes (H) in the TEM slice are visible. The protein crystal has dimensions of about 880 and 440 nm along the major and minor axes, respectively. The extent of the endospore including the coat is about 1190 nm along the major axis and 760 nm along the minor axis. (b) A close-up of the endospore as indicated by a dashed frame in (a), revealing details of the exosporium (EX), coat (C), cortex (CX) and core (CR). This section of the core contains mainly ribosomes (R). (c) A TEM image of an endospore of B. subtilis. Structural details including the cortex (CX) and the core (CR) with ribosomes (R) are labelled. The major and minor axes of the core including cortex extend to about 630 and 460 nm, respectively. Taking also the coat into account yields about 1030 nm along the major axis and approximately 700 nm along the minor axis. Scale bars denote 0.2 and 0.1 µm in (a) and in (b), (c), respectively.

Figure 5

Region (A): the image shows a phase map of the B. thuringiensis sample. The phase map corresponds to four single mHIO reconstructions that have been merged in the same plane. Grey arrow heads point to some of the rod-shaped remnant cell material. The black arrow heads indicate electron-dense cellular features such as endospores. The dashed black frame indicates region (B), including (C), of the sample (cf. Fig. 7 ▶). The scale bar denotes 10 µm.

Figure 6

(a) and (b) PSDs of mHIO reconstructions of B. thuringiensis [region (C), Fig. 7 ▶(b)] and B. subtilis data [region (B), Fig. 8 ▶(b)], indicating resolutions in the range of 100 nm (half-period). (c) and (d) PSDs of CTF-based reconstructions of the B. thuringiensis data [region (C), Fig. 9 ▶(b)] and B. subtilis data [region (A), Fig. 10 ▶], indicating resolutions of about 100 and 60 nm (half-period), respectively.

Figure 7

(a) Region (B): the figure shows a phase image of the B. thuringiensis sample. The phase map corresponds to a single mHIO reconstruction. The black dots indicate the boundaries of the endospores used for mass estimations whereas the white dots surround the region being defined here as ‘background’ ( fg). Endospores are labelled from (1) to (5). The average mass per single endospore is  (9) fg. The black arrow heads indicate positions of other dense bacterial features such as possible BT crystals. The grey arrow head points to an almost fully lysed cell. The scale bar denotes 2 µm. (b) Region (C) of (a): the figure shows a phase image of the B. thuringiensis sample. The phase map corresponds to a single mHIO reconstruction at higher magnification. The scale bar denotes 1 µm.

Figure 8

The figure presents mHIO phase reconstructions of endospores of B. subtilis of regions (A), (B), (C), (D) (cf. Fig. 3 ▶ b). The endospores are labelled from (1) to (8). The black dots indicate the boundaries used for mass estimations. The average mass per single endospore is  (7) fg. The colour bar is the same in (a), (b) and (c) as in (d). The scale bars in (a), (b), (c) and (d) denote 1, 0.5, 0.5 and 0.5 µm, respectively.

Figure 9

(a) Region (B): the image shows a phase reconstruction using the CTF-based reconstruction of the B. thuringiensis sample. The scale bar denotes 2 µm. (b) Region (C) of (a) but at higher magnification. Note that the density of the core of the endospore (3) appears slightly higher than the outer part. The electron-dense feature (black arrow head) on the right-hand side of the spore (3) is probably a BT crystal, as the cell already includes one endospore. The scale bar denotes 1 µm. In both images the labelling is the same as in Fig. 7 ▶.

Figure 10

(a) The CTF-based reconstruction of region (A) of the B. subtilis sample. Three isolated endospores can be seen. (b), (c) Calculated gradients (filtered with  pixel Gaussian) in the horizontal and vertical directions of the region of two endospores of (a). The black arrow heads highlight the transition between two different structural regions of the endospore that are attributed to the coat and the inner part. The repetitive structure on the gradient maps is an artefact of the reconstruction in (a). The scale bars denotes 0.5 µm.